ANALYTICAL
Rapid
BIOCHEMISTRY
94,
112- 116 (1979)
Determination of Doxorubicin and its Fluorescent by High Pressure Liquid Chromatography R. BAURAIN,
International
Institute
D. DEPREZ-DE
of Cellular and Molecular 74, Avenue Hippocrate,
CAMPENEERE, Pathology B-1200
AND A. TROUET
and UniversitC Bruxelles,
Metabolites
Catholique
de
Louvain,
Belgium
Received July 6, 1978 An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, nondestructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by Ruorometry down to a concentration of 1.5 &ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBAz and NMRI mice.
Doxorubicin (DOX)] is an anthracycline widely used in the treatment of neoplastic diseases (1). In view of establishing the pharmacologic properties of DOX in experimental animals and in humans, many efforts have been devoted to developing quantitative assay procedures for this drug and its metabolites. In particular, two methods have been described for determination of DOX by high-pressure liquid chromatography (hplc). The first one (2) involves the separation of DOX, doxorubicinone (DOX-ONE), and doxorubicinol (DOX-OL) (Fig. 1) by hplc and quantitation of DOX by radioimmunoassay. The second method (3) has a low efficiency and is time consuming since the retention time of DOX on the hplc column exceeds 20 min. We report here a rapid and nondestructive extraction technique of DOX from biological samples with a 100% recovery and an improved hplc procedure to separate and quantify in less than 5 min very small
amounts of DOX tabolites. MATERIALS
and its fluorescent
me-
AND METHODS
Materials
Doxorubicin hydrochloride (DOX; adriamycin) was provided by Rhone-Poulenc S.A. (Paris, France). Doxorubicinol was supplied by R. Hulhoven (Laboratoire de Pharmacotherapie, Universite Catholique de Louvain). Solvents were of analytical grade from E. Merck (Darmstadt, Germany) and used without further purification. Methods Extraction procedure. To 0.1 ml of a biological sample, 0.1 ml of internal standard (daunorubicin at 10 pg/ml borate buffer pH 9.8) is added and the drugs extracted by 1.8 ml of a chloroform:methanol mixture (4: 1 by volume). After sonication during 20 s at 50 W (Branson Sonicator) an aliquot of the organic layer is collected and injected into the chromatograph. High-pressure liquid chromatography. A Gilson Spectra-Glo fluorometer (Model
1 Abbreviations used: DOX, Doxorubicin; hplc, highpressure liquid chromatography; DOX-ONE, doxorubicinone; DOX-OL, doxorubicinol; DNR, daunorubicin. 112
DOXORUBICIN
AND ITS FLUORESCENT
Q$$p OCH, 0
OH H R2
COMPOUND
DOXORUBlClN
+0tl -c=o
DOXOR”BlClNOL
F”*W -CH-OH
d 0
c + NH* OH
d
C” e OH NH2
FIG. 1. Structure of doxorubicinone, and doxorubicinol.
doxorubicin,
FL-l A/B, Gilson, Middleton, Wis.) with narrow band width interference filters at 480 and 560 nm for the excitation and emission wavelengths, respectively, and a flow cell of 50 ~1 is connected to a Hewlett-Packard Model 1084 high-pressure liquid chromatograph. The fluorescent peaks detected are analyzed by the H-P software which include an integrator and a plotter. A column (250 x 3 mm) prepacked with 7-pm silica gel particles (Hibar Lichrosorb Si-60 from E. Merck, Darmstadt, Germany) is used and the elution performed with a mixture of chloroform, methanol, glacial acetic acid, and a 0.3 mM MgCl, water solution (720: 210:40:30 by volume). The samples are injected into the 20-~1 loop valve of the chromatograph (Rheodyne six-port rotary valve, Model 7120, Rheodyne, Calif.). RESULTS
113
METABOLITES
water (720:210:40:30 by volume) as eluant. Using this eluant system at a flow rate of 1.0 ml/min, DOX and DOX-OL are eluted more slowly from the column (respective t,: 10 and about 15 min). The increased fR of DOX and its metabolite DOX-OL can be explained by their greater polarity and the stronger interaction of their distinctive hydroxyl groups with the silica gel. It is possible to decrease this interaction and thus to reduce the retention times by adding magnesium salts to the eluant system. Magnesium chloride was chosen because it can form complexes with DNR (5) and promote the differentiation of tetracyclines during thin layer chromatography on cellulose plates (6,7).
c” 0
5 .-C
.-c u .-13 2
0 s u
U
CD ‘32 .a: 20
OU
s
0 II
LAW-
AND DISCUSSION
As described elsewhere (4) daunorubicin (DNR) and its metabolites can be extracted from biological fluids by a lo-fold excess of chlorofotmmethanol (4: 1 by volume) and determined rapidly (retention time on the column: fR of 6.8 min) using a mixture of chloroform:methanol:glacial acetic acid:
min.
af ter
injection
FIG. 2. Separation of doxorubicinone, doxorubicin, and doxorubicinol by hplc. DOX, DOX-OL, and DOXONE were separated from a mixture by hplc and estimated by fluorometry as described under Materials and Methods. At a flow rate of 1.0 mYmin, the following ts were obtained: 1.5 min (DOX-ONE); 3.4 min (DOX); 4.7 min (DOX-OL).
114
BAURAIN,
DEPREZ-DE
ng
CAMPENEERE,
DOX
AND TROUET
/ml
FIG. 3. Calibration curve of doxorubicin. Samples obtained from DOX in a chloroform:methanol mixture (4:l by volume) were injected into the chromatograph and analyzed as described under Materials and Methods. The results are expressed as the relative integrated areas of the fluorescent peaks.
As shown in Fig. 2, the addition of 0.3 mM MgC& solution to the eluant decreases the retention time of DOX by a factor of 3. At a flow rate of 1.0 ml/min the tR are, respectively, 1.5, 3.4, and 4.7 min for DOXONE, DOX, and DOX-OL. The peaks are sharp and well resolved and the analysis time is reduced to less than 5 min. Figure 3 shows the linear relationship found between the integrated area of the DOX peak and the concentration of DOX injected, in the range of 10 to 70 @ml. This linearity has been observed up to 1000 n&ml (8). The lowest amount of DOX detectable, corresponding to threefold the noise level, is 5 x lo-l4 mol (25 pg), i.e., an injected solution of 2.5 x lo-l2 mol of DOX/ml (1.5 r&ml). When DOX is added in increasing concentrations to blood samples obtained from NMRI mice, the drug is extracted and measured by hplc with a mean percentage recovery of 99.7 * 3.1% (Table 1). Using this technique, we have followed
DOX and its metabolites in plasma and urine, after an iv injection into NMRI or DBAz mice of DOX at 7 mg/kg. TABLE RECOVERY
Doxorubicin added b&-a 5.0 10.0 25.0 50.0 75.0 100.0
1
OF DOXORUBICIN
IN BLOODY
Doxorubicin measured0 (&ml) 5.2 9.5 24.5 50.7 74.6 100.3
-c c ? 2 f 2
0.3 0.5 0.8 1.9 3.2 3.0
Percentage recovery 104.0 95.0 98.0 101.4 99.5 100.3
D DOX is added to blood samples obtained from NMRI mice in order to obtain drug concentrations of 5, 10, 25, 50, 75, and 100 &ml. To 0.1 ml of the blood sample, 0.1 ml of the internal standard is added (DNR at 10 &ml, pH 9.8 borate buffer). The drugs are extracted with 1.8 ml chlorofotmmethanol(4: 1 by volume) and an aliquot of the organic phase is injected into the chromatograph. b Mean -t SD of four determinations.
DOXORUBICIN
0
AND ITS FLUORESCENT
20 MINUTES
METABOLITES
40 AFTER
115
60 INJECTION
FIG. 4. Plasma levels of doxorubicin after iv injection into mice. DOX was injected intravenously at 7 mg/kg into DBAz and NMRI mice. After various times, blood samples were collected, during sacrifice, from the femoral vein. DOX concentrations in the corresponding individual plasma were determined as described under Materials and Methods. Mean % SD of three separate assays are given. (m) DBA, mice; (A) NMRI mice.
The main compound found in the plasma is DOX with trace amounts of DOX-ONE and DOX-OL (less than 2% of the total fluorescence). The disappearance of DOX from the plasma (Fig. 4) is at least biphasic with a first rapid phase characterized by a half-life of 1.2 and 0.9 min in DBA, and NMRI mice, respectively. In urine samples, nine minor metabolites could be detected besides the parent drug which represents at least 85% of the total fluorescence (Fig. 5), and among the metabolites we identified DOX-ONE and DOX-OL on the basis of their retention times. Nevertheless as the unidentified metabolites could have quantum yields of fluorescence different from the one of the parent drug, the percentage of fluorescence could differ somewhat from the mole fraction of DOX present in the urine samples. As illustrated in Fig. 6, we found different
percentages of DOX-OL in the urine of both mice strains studied. In DBAz mice, DOXOL represents about 4% of the total fluorescence between 40 and 60 min after the iv injection of DOX, while in NMRI mice the percentage remains below 0.4%. Comparative metabolic studies among various mice strains injected iv with DOX have not been done previously. The differences observed in the DOX-OL levels in urine could reflect differences in the intracellular level and activity of the ketoreductase in both strains. By comparison, DOX-OL represents about 14% of total fluorescence of the urine in man (9), about 8% in rabbit (lo), and 0 to 8% in rat (11). Our data are also in agreement with the results of Bachur (9) who found and characterized nine fluorescent metabolites of DOX in human urine, DOX-OL being the prominent one.
BAURAIN,
DEPREZ-DE
CAMPENEERE,
AND
TROUET
MINUTES
AFTER
INJECTION
FIG. 6. Doxorubicinol in urine samples after iv injection of doxorubicin into mice. DOX was injected intravenously at 7 m&kg into DBA, and NMRI mice. After various times, urine aliquots were analyzed as described under Materials and Methods. The results are expressed as a percentage of the total fluorescent metabolites. (m) DBA, mice; (A) NMRI mice.
ACKNOWLEDGMENTS This work was supported by Rh8ne-Poulenc, Paris (France) and by a grant from “Caisse G&&ale d’Epargne et de Retraite,” Belgium.
REFERENCES 1. Carter, S. K. (1975) J. Nat. Cancer Inst. 55, 126% 1274. 2. Langone, J. J., Van Vunakis, H., and Bachur, N. R. (1975) Eiochem. Med. l&283-289. 3. Barth, H. G., and Conner, A. Z. (1977) J. Chromatogr.
131, 375-381.
4. Baurain, R., Zenebergh, A., and Trouet, A. (1978) .I. Chromatogr.,
157, 331-336.
5. Calendi, E., Di Marco, A., Reggiani, M., Scarpinato, S., and Valentini, L. (1965) Biachim.
Biophys.
Acta
103, 25-49.
6. Poiger, H., and Chlatter, C. (1976) Analysf 101, 808-814. 7. Ragazzi, E., and Veronese, G. (1977) J. Chromatogr.
II
11
11
11
11
0
2
4
6
8
I
10 min.
FIG. 5. Separation of doxorubicin and its metabolites in the mine, after iv injection into mice. DOX was injected intravenously at 7 mg/kg into NMRI mice. After 2 h, an urine aliquot was analyzed as described under Materials and‘Methods. Flow rate, 0.6 ml/min. The following compounds were identified: DOX-ONE (tR = 1.28 min); internal standard: DNR (tR = 3.50 min); DOX (tR = 5.53 min); DOX-OL (tR = 8 min).
132, 105- 114.
8. Baurain, R., Masquelier, M., Peterson, C., Deprez-De Campeneere, D., and Trouet, A. (1978) in Current Chemotherapy (Siegenthaler, W., and Lilthy, R., eds.), Proceedings 10th International Congress of Chemotherapy, Vol. II, pp. 1130- 1132, American Society of Microbiologists, Washington, D. C. 9. Takanashi, S., and Bachur, N. R. (1976) Drug Metabol.
Dispos.
4, 79-87.
10. Bachur, N. R., R. C. Hildebrand, and R. S. Jaenke (1974). J. Pbarmacol. Exp. Ther. 191, 331-350. 11. Yesair, D. W., Schwartzbach E, Schuck, D., Denine, E. P., and Asbell, M. A., (1972) Cancer Res. 32, 1177- 1183.
Rapid
BIOCHEMISTRY
94,
112- 116 (1979)
Determination of Doxorubicin and its Fluorescent by High Pressure Liquid Chromatography R. BAURAIN,
International
Institute
D. DEPREZ-DE
of Cellular and Molecular 74, Avenue Hippocrate,
CAMPENEERE, Pathology B-1200
AND A. TROUET
and UniversitC Bruxelles,
Metabolites
Catholique
de
Louvain,
Belgium
Received July 6, 1978 An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, nondestructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by Ruorometry down to a concentration of 1.5 &ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBAz and NMRI mice.
Doxorubicin (DOX)] is an anthracycline widely used in the treatment of neoplastic diseases (1). In view of establishing the pharmacologic properties of DOX in experimental animals and in humans, many efforts have been devoted to developing quantitative assay procedures for this drug and its metabolites. In particular, two methods have been described for determination of DOX by high-pressure liquid chromatography (hplc). The first one (2) involves the separation of DOX, doxorubicinone (DOX-ONE), and doxorubicinol (DOX-OL) (Fig. 1) by hplc and quantitation of DOX by radioimmunoassay. The second method (3) has a low efficiency and is time consuming since the retention time of DOX on the hplc column exceeds 20 min. We report here a rapid and nondestructive extraction technique of DOX from biological samples with a 100% recovery and an improved hplc procedure to separate and quantify in less than 5 min very small
amounts of DOX tabolites. MATERIALS
and its fluorescent
me-
AND METHODS
Materials
Doxorubicin hydrochloride (DOX; adriamycin) was provided by Rhone-Poulenc S.A. (Paris, France). Doxorubicinol was supplied by R. Hulhoven (Laboratoire de Pharmacotherapie, Universite Catholique de Louvain). Solvents were of analytical grade from E. Merck (Darmstadt, Germany) and used without further purification. Methods Extraction procedure. To 0.1 ml of a biological sample, 0.1 ml of internal standard (daunorubicin at 10 pg/ml borate buffer pH 9.8) is added and the drugs extracted by 1.8 ml of a chloroform:methanol mixture (4: 1 by volume). After sonication during 20 s at 50 W (Branson Sonicator) an aliquot of the organic layer is collected and injected into the chromatograph. High-pressure liquid chromatography. A Gilson Spectra-Glo fluorometer (Model
1 Abbreviations used: DOX, Doxorubicin; hplc, highpressure liquid chromatography; DOX-ONE, doxorubicinone; DOX-OL, doxorubicinol; DNR, daunorubicin. 112
DOXORUBICIN
AND ITS FLUORESCENT
Q$$p OCH, 0
OH H R2
COMPOUND
DOXORUBlClN
+0tl -c=o
DOXOR”BlClNOL
F”*W -CH-OH
d 0
c + NH* OH
d
C” e OH NH2
FIG. 1. Structure of doxorubicinone, and doxorubicinol.
doxorubicin,
FL-l A/B, Gilson, Middleton, Wis.) with narrow band width interference filters at 480 and 560 nm for the excitation and emission wavelengths, respectively, and a flow cell of 50 ~1 is connected to a Hewlett-Packard Model 1084 high-pressure liquid chromatograph. The fluorescent peaks detected are analyzed by the H-P software which include an integrator and a plotter. A column (250 x 3 mm) prepacked with 7-pm silica gel particles (Hibar Lichrosorb Si-60 from E. Merck, Darmstadt, Germany) is used and the elution performed with a mixture of chloroform, methanol, glacial acetic acid, and a 0.3 mM MgCl, water solution (720: 210:40:30 by volume). The samples are injected into the 20-~1 loop valve of the chromatograph (Rheodyne six-port rotary valve, Model 7120, Rheodyne, Calif.). RESULTS
113
METABOLITES
water (720:210:40:30 by volume) as eluant. Using this eluant system at a flow rate of 1.0 ml/min, DOX and DOX-OL are eluted more slowly from the column (respective t,: 10 and about 15 min). The increased fR of DOX and its metabolite DOX-OL can be explained by their greater polarity and the stronger interaction of their distinctive hydroxyl groups with the silica gel. It is possible to decrease this interaction and thus to reduce the retention times by adding magnesium salts to the eluant system. Magnesium chloride was chosen because it can form complexes with DNR (5) and promote the differentiation of tetracyclines during thin layer chromatography on cellulose plates (6,7).
c” 0
5 .-C
.-c u .-13 2
0 s u
U
CD ‘32 .a: 20
OU
s
0 II
LAW-
AND DISCUSSION
As described elsewhere (4) daunorubicin (DNR) and its metabolites can be extracted from biological fluids by a lo-fold excess of chlorofotmmethanol (4: 1 by volume) and determined rapidly (retention time on the column: fR of 6.8 min) using a mixture of chloroform:methanol:glacial acetic acid:
min.
af ter
injection
FIG. 2. Separation of doxorubicinone, doxorubicin, and doxorubicinol by hplc. DOX, DOX-OL, and DOXONE were separated from a mixture by hplc and estimated by fluorometry as described under Materials and Methods. At a flow rate of 1.0 mYmin, the following ts were obtained: 1.5 min (DOX-ONE); 3.4 min (DOX); 4.7 min (DOX-OL).
114
BAURAIN,
DEPREZ-DE
ng
CAMPENEERE,
DOX
AND TROUET
/ml
FIG. 3. Calibration curve of doxorubicin. Samples obtained from DOX in a chloroform:methanol mixture (4:l by volume) were injected into the chromatograph and analyzed as described under Materials and Methods. The results are expressed as the relative integrated areas of the fluorescent peaks.
As shown in Fig. 2, the addition of 0.3 mM MgC& solution to the eluant decreases the retention time of DOX by a factor of 3. At a flow rate of 1.0 ml/min the tR are, respectively, 1.5, 3.4, and 4.7 min for DOXONE, DOX, and DOX-OL. The peaks are sharp and well resolved and the analysis time is reduced to less than 5 min. Figure 3 shows the linear relationship found between the integrated area of the DOX peak and the concentration of DOX injected, in the range of 10 to 70 @ml. This linearity has been observed up to 1000 n&ml (8). The lowest amount of DOX detectable, corresponding to threefold the noise level, is 5 x lo-l4 mol (25 pg), i.e., an injected solution of 2.5 x lo-l2 mol of DOX/ml (1.5 r&ml). When DOX is added in increasing concentrations to blood samples obtained from NMRI mice, the drug is extracted and measured by hplc with a mean percentage recovery of 99.7 * 3.1% (Table 1). Using this technique, we have followed
DOX and its metabolites in plasma and urine, after an iv injection into NMRI or DBAz mice of DOX at 7 mg/kg. TABLE RECOVERY
Doxorubicin added b&-a 5.0 10.0 25.0 50.0 75.0 100.0
1
OF DOXORUBICIN
IN BLOODY
Doxorubicin measured0 (&ml) 5.2 9.5 24.5 50.7 74.6 100.3
-c c ? 2 f 2
0.3 0.5 0.8 1.9 3.2 3.0
Percentage recovery 104.0 95.0 98.0 101.4 99.5 100.3
D DOX is added to blood samples obtained from NMRI mice in order to obtain drug concentrations of 5, 10, 25, 50, 75, and 100 &ml. To 0.1 ml of the blood sample, 0.1 ml of the internal standard is added (DNR at 10 &ml, pH 9.8 borate buffer). The drugs are extracted with 1.8 ml chlorofotmmethanol(4: 1 by volume) and an aliquot of the organic phase is injected into the chromatograph. b Mean -t SD of four determinations.
DOXORUBICIN
0
AND ITS FLUORESCENT
20 MINUTES
METABOLITES
40 AFTER
115
60 INJECTION
FIG. 4. Plasma levels of doxorubicin after iv injection into mice. DOX was injected intravenously at 7 mg/kg into DBAz and NMRI mice. After various times, blood samples were collected, during sacrifice, from the femoral vein. DOX concentrations in the corresponding individual plasma were determined as described under Materials and Methods. Mean % SD of three separate assays are given. (m) DBA, mice; (A) NMRI mice.
The main compound found in the plasma is DOX with trace amounts of DOX-ONE and DOX-OL (less than 2% of the total fluorescence). The disappearance of DOX from the plasma (Fig. 4) is at least biphasic with a first rapid phase characterized by a half-life of 1.2 and 0.9 min in DBA, and NMRI mice, respectively. In urine samples, nine minor metabolites could be detected besides the parent drug which represents at least 85% of the total fluorescence (Fig. 5), and among the metabolites we identified DOX-ONE and DOX-OL on the basis of their retention times. Nevertheless as the unidentified metabolites could have quantum yields of fluorescence different from the one of the parent drug, the percentage of fluorescence could differ somewhat from the mole fraction of DOX present in the urine samples. As illustrated in Fig. 6, we found different
percentages of DOX-OL in the urine of both mice strains studied. In DBAz mice, DOXOL represents about 4% of the total fluorescence between 40 and 60 min after the iv injection of DOX, while in NMRI mice the percentage remains below 0.4%. Comparative metabolic studies among various mice strains injected iv with DOX have not been done previously. The differences observed in the DOX-OL levels in urine could reflect differences in the intracellular level and activity of the ketoreductase in both strains. By comparison, DOX-OL represents about 14% of total fluorescence of the urine in man (9), about 8% in rabbit (lo), and 0 to 8% in rat (11). Our data are also in agreement with the results of Bachur (9) who found and characterized nine fluorescent metabolites of DOX in human urine, DOX-OL being the prominent one.
BAURAIN,
DEPREZ-DE
CAMPENEERE,
AND
TROUET
MINUTES
AFTER
INJECTION
FIG. 6. Doxorubicinol in urine samples after iv injection of doxorubicin into mice. DOX was injected intravenously at 7 m&kg into DBA, and NMRI mice. After various times, urine aliquots were analyzed as described under Materials and Methods. The results are expressed as a percentage of the total fluorescent metabolites. (m) DBA, mice; (A) NMRI mice.
ACKNOWLEDGMENTS This work was supported by Rh8ne-Poulenc, Paris (France) and by a grant from “Caisse G&&ale d’Epargne et de Retraite,” Belgium.
REFERENCES 1. Carter, S. K. (1975) J. Nat. Cancer Inst. 55, 126% 1274. 2. Langone, J. J., Van Vunakis, H., and Bachur, N. R. (1975) Eiochem. Med. l&283-289. 3. Barth, H. G., and Conner, A. Z. (1977) J. Chromatogr.
131, 375-381.
4. Baurain, R., Zenebergh, A., and Trouet, A. (1978) .I. Chromatogr.,
157, 331-336.
5. Calendi, E., Di Marco, A., Reggiani, M., Scarpinato, S., and Valentini, L. (1965) Biachim.
Biophys.
Acta
103, 25-49.
6. Poiger, H., and Chlatter, C. (1976) Analysf 101, 808-814. 7. Ragazzi, E., and Veronese, G. (1977) J. Chromatogr.
II
11
11
11
11
0
2
4
6
8
I
10 min.
FIG. 5. Separation of doxorubicin and its metabolites in the mine, after iv injection into mice. DOX was injected intravenously at 7 mg/kg into NMRI mice. After 2 h, an urine aliquot was analyzed as described under Materials and‘Methods. Flow rate, 0.6 ml/min. The following compounds were identified: DOX-ONE (tR = 1.28 min); internal standard: DNR (tR = 3.50 min); DOX (tR = 5.53 min); DOX-OL (tR = 8 min).
132, 105- 114.
8. Baurain, R., Masquelier, M., Peterson, C., Deprez-De Campeneere, D., and Trouet, A. (1978) in Current Chemotherapy (Siegenthaler, W., and Lilthy, R., eds.), Proceedings 10th International Congress of Chemotherapy, Vol. II, pp. 1130- 1132, American Society of Microbiologists, Washington, D. C. 9. Takanashi, S., and Bachur, N. R. (1976) Drug Metabol.
Dispos.
4, 79-87.
10. Bachur, N. R., R. C. Hildebrand, and R. S. Jaenke (1974). J. Pbarmacol. Exp. Ther. 191, 331-350. 11. Yesair, D. W., Schwartzbach E, Schuck, D., Denine, E. P., and Asbell, M. A., (1972) Cancer Res. 32, 1177- 1183.
