Breast Cancer Research and Treatment 24: 35-41, 1992. © 1992 KtuwerAcademic Publishers. Printedin the Netherlands.
Report
A bioassay for antiestrogenic activity - - potential utility in drug development and monitoring effective in vivo dosing Michael DeGregorio, Gregory Wurz, Vernon Emshoff, Steven Koester, Patrick Minor, and Valerie Wiebe Department of Medicine, Division of Oncology, University of Texas Health Science Center, San Antonio, Texas, USA
Key words: antiestrogens, bioassay, tamoxifen, toremifene
Summary Monitoring effective antiestrogenic activity of the triphenylethylenes in patients with breast cancer is usually determined by the duration of response. The pharmacokinetics of toremifene and tamoxifen have been shown to be highly variable but patient specific. In the present study, we developed a method to accurately assess the antiestrogenic activity of these agents using plasma specimens, cell culture, and cell cycle measurements. Plasma specimens (4-5mls) obtained from patients receiving toremifene (360mg/day for 5 days in a phase I trial) or tamoxifen (20mg/day) were extracted and reconstituted in tissue culture media (4-5mls), and growth inhibition was determined in estrogen responsive MCF-7 cells. Additionally, plasma specimens were quantified for toremifene or tamoxifen concentrations using HPLC. Growth inhibition of plasma specimens containing either toremifene or tamoxifen and their metabolites was also examined. Cell cycle measurements were determined following in vitro exposure with flow cytometric techniques. Our results show that a dose-response relationship exists between cell growth inhibition and cell cycle measurements for human plasma with added toremifene or tamoxifen, and also for human plasma specimens containing drug and its metabolites after treatment. Our antiestrogenic bioassay can address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance.
Introduction Toremifene, tamoxifen, and their metabolites inhibit cell growth in breast tumors primarily by estrogen receptor blockade. Both estrogen receptor agonist and antagonist properties have
been described for these agents [1]. However, they are generally considered to be antiestrogens in human breast tumors due to their inhibitory effects on estrogen-stimulated cell growth. The major metabolic pathways for toremifene and tamoxifen include N-desmethylation and 4-hy-
Addressfor offprints: Dr. Michael DeGregorio,Medicine/Oncology,Universityof Texas Health Science Center, 7703 Floyd Curl Drive, San AntonioTX 78284-7884, USA
36
M DeGregorio et al
droxylation [2,3]. Although the 4-hydroxy metabolite is a minor metabolite, it binds estrogen receptors with high affinity and may contribute significantly to antiestrogenic activity [4]. The pharmacokinetics of toremifene and tamoxifen in patients with breast cancer are well described. Fabian et al. examined tamoxifen plasma kinetics after continuous oral dosing and found that most patients achieved steady-state levels within 16 weeks of therapy, although a wide range of concentrations were reported [5]. Similar pharmacokinetic results were reported for toremifene following 8 weeks of therapy [3]. Both studies show a wide variation in steady-state concentrations. In addition, it has been suggested that reduced plasma antiestrogen concentrations either from altered bioavailability or from noncompliance may contribute to clinical failure [1]. Although tamoxifen resistance has not been correlated with plasma antiestrogen levels [6], alterations in the cellular pharmacology of tamoxifen may be an early hallmark of acquired resistance to tamoxifen [7]. In the present study, we developed a bioassay that allows for the assessment of effective antiestrogen dosing on a patient-specific basis. In addition, this assay can address clinical research problems such as patient-specific pharmacokinetics, patient compliance, and mechanisms of acquired antiestrogen resistance such as metabolic tolerance and production of estrogenic metabolites.
Methods
Cell culture MCF-7 cells were grown in Coming T25 flasks containing 7.0ml IMEM + 10% fetal bovine serum and maintained in 5% CO 2 and 95% air. Cells were plated at a concentration of 10,000 cells/ml, and incubated at 37°C for 12-24 hours prior to treatment.
Sample extraction All patient plasma or spiked plasma specimens were extracted with 6.0 ml of 2% butanol in hexane and vortexed for 1 minute. If necessary, samples were then centrifuged for 10 minutes at 1,000 x g. The organic phase was dried under nitrogen gas at 37°C. All samples were immediately capped with sterile caps and stored at -20°C until used. A 1 ml aliquot of each patient sample was also quantified by HPLC (see below).
Growth inhibition All specimens were reconstituted in sterile medium and placed on MCF-7 cells yielding the same gg/ml concentrations seen following therapy. MCF-7 cells were incubated with drug for 4-5 days or harvested at 75-80% confluency of controls. All medium was aspirated, and cells were washed with 2ml normal saline, trypsinized, and resuspended in 5ml of fresh medium. All cell counts were performed using Coulter Counter technique. Cell viability was estimated by trypan blue dye exclusion. Cells were then processed for flow cytometric analysis (see below).
HPLC analysis Following extraction, all 1 ml plasma extracts were reconstituted in 200 gl methanol before analysis. The reconstituted samples were transferred to an Infrasil quartz cuvette (Fisher Scientific, Fair Lawn, NJ), exposed to high-intensity ultraviolet light for 1 minute using a 15 W mercury vapor lamp, and then injected onto the column. The HPLC assay was performed and validated as previously described [8]. The system consisted of a Beckman model 320 gradient liquid chromatograph, two model 110A pumps, and a model 420 controller. The HPLC was equipped with a reverse phase Axxion C18 ultrasphere
A bioassay for antiestrogenic activity
propidium iodide in a hypotonic sodium citrate solution with 0.3% NP-40 and 1.0 m g / m l RNAse-A), vortexed, and stained for 30 minutes at r o o m temperature in the dark. Prior to flow cytometfic measurements, samples were filtered through a 37 micron nylon mesh into 12 x 75 m m tubes and stored at 4°C until analysis within 24 hours. Flow cytometric measurements were performed on an EPICS 753 instrument (Coulter Cytometry, Hialeah, FL) with an argon-ion laser tuned to 488 nm and 400 m W of power. Red fluorescence from the propidium iodide (peak fluorescence at 610nm) was collected through 515nm long pass and 610nm long pass filters. Compartmental analysis of D N A histograms was accomplished with M O D F I T software (Verity Software House, Topsham, ME).
column and a 100 gl injection loop. The mobile phase consisted of 7% water and 0.18% triethylamine in methanol° The flow rate of the mobile phase was set at 1.00 ml/min. The fluorescence of photochemically activated compounds was detected with an Applied Biosystems 980 fluorom e t e r with excitation wavelength set at 266 nm. Retention times and peak heights were recorded with a Spectraphysics integrator (Menlo Park, CA).
Flow cytometry Cells were stained with a modified Krishan technique [9]. Samples were spun at 1000 x g for 7 minutes at 4 ° C , cell pellets were resuspended in 1.0 ml of Krishan staining solution (50 g g / m l
Table 1. Cell cycle measurements of MCF-7 cells following exposure to spiked Toremifene or Tamoxifen and their metabolites. Drug
~tM
% Inhibition a %GOG1b
%SPF
%G2+M
Cell control
-
0
58.0
34.0
8.0
TOR TOR TOR
1.0 3.3 6.6
65 82 89
76.2 84.4 88.1
17.4 10.7 6.8
6.4 4.9 5.0
4-OH TOR 4-OH TOR 4-OH TOR
1.0 3.3 6.6
69 72 79
79.8 81.1 81.3
14.1 13.2 12.8
6.1 5.7 5.9
n-des TOR n-des TOR n-des TOR
1.0 3.3 6.6
44 71 82
68.8 77.2 83.6
23.5 16.3 11.3
7.7 6.5 5.1
Ceil control
--
0
61.2
30.0
8.8
TAM TAM TAM
1.0 3.3 6.6
52 53 63
78.4 81.6 83.7
15.4 13.2 t 1.0
6.2 5.2 5.3
4-OH TAM 4-OH TAM 4-OH TAM
1.0 3.3 6.6
52 61 69
83.6 84.6 84.5
12.0 11.0 10.7
4.4 4.5 4.9
n-des TAM n-des TAM n-des TAM
1.0 3.3 6.6
49 61 75
79.4 82.6 85.3
14.9 12.4 10.5
5.8 4.9 4.2
a
37
Based on cell control. Each % inhibition value represents the mean value of triplicate samples. Ranges were within 5% of the mean.
38
M DeGregorio et al
Results
their metabolites had similar potency. Table 2 shows similar results on cell cycle measurements when each drug was spiked into plasma and extracted with hexane/butanol (>80% extraction efficiency). Growth inhibition was essentially equivalent to that seen with direct administration of drug in the tamoxifen group and slightly less than that observed in the toremifenetreated cells, although not significantly, Figure 1 shows the dose-response growth inhibition and cell cycle measurements for the extracted tamoxifen and toremifene groups. Table 3 shows the results o f patient plasma specimens containing either toremifene or tamoxifen and their metabolites. Cell growth was
Table 1 shows the effects of toremifene and tamoxifen and their two major metabolites on MCF-7 cell growth and cell cycle kinetics. A dose-response inhibition o f cell growth was seen for each compound. The range of dosing was from antiestrogenic concentrations (l~tM) to doses necessary to inhibit critical biologic pathways such as protein kinases, calcium channels, and others (6.6~tM). Similar to other reports, all compounds appeared to block cells in G0/G1, while decreasing S and G 2 + M in a dosedependent manner. Toremifene was slightly more active in blocking in G0/G1 than tamoxifen, while
Table 2. Cell cycle measurements of MCF-7 cells following exposure to extracted Toremifene or Tamoxifen and their metabolites.
Drug
].tM
% Inhibition a
%GOGIb
%SPF
%G2+M
Plasma alone
-
0
70.8
22.5
6.7
TOR TOR TOR
1.0 3.3 6.6
28 50 58
75.0 80.0 81.0
18,4 14.4 13.3
6.6 6.1 5.6
4-OH TOR 4-OH TOR 4-OH TOR
1.0 3.3 6.6
53 59 76
83.4 83.5 81.0
11.1 11.2 13,9
5.5 5.3 5,2
n-des TOR n-des TOR n-des TOR
1.0 3.3 6.6
12 38 47
74.4 78.8 80.5
18.5 15.2 14.1
7.1 6.0 5.4
TOR IV TOR IV TOR IV
1.0 3.3 6.6
48 56 59
83.2 83.2 84.1
11.6 12.0 11,3
5.1 4.9 4.7
Plasma
-
0
66.8
25.8
7.4
TAM TAM TAM
1.0 3.3 6.6
53 61 64
75.0 78.4 78.3
18.4 15.6 15,6
6,6 6.0 6.3
4-OH TAM 4-OH TAM 4-OH TAM
1.0 3.3 6.6
52 63 69
83.7 82.7 80.2
11.1 12,1 14,6
5.2 5.2 5.2
n-des TAM n-des TAM n-des TAM
1.0 3.3 6.6
43 54 69
76,2 79.4 78.8
17.5 15.3 15.4
6.3 5.4 5.8
a Each inhibition represents the mean value of triplicate samples. Ranges were within 5% of the mean. b Triplicates were pooled for cell cycle measurements.
A bioassay for antiestrogenic activity
39
90
100 " 90"
85 80~o
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7060"
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50"
r, 0
40"
75 70
30"
65 20" 10
, 1.0
, 3.3
, 6.6
.
60
. ctl
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Drug Concentration (uM)
Drug Concentration (uM)
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¢u
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6.6
Drug Concentration (uM)
4
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Figure 1. Survivaland cell cycle measurementsof MCF-7 cells followingexposure to extractedTOR and TAM and their metabolites. Legend: + TOR; C) 4-OH-TOR; • n-des TOR; [] TAM; • 4-OH-TAM; II n-des TAM. significantly inhibited in all toremifene specimens. Tamoxifen produced similar inhibitory effects, with only one patient specimen producing minimal effects. This patient specimen had the lowest concentration of tamoxifen and its metabolites,
suggesting a potential dose-response relationship similar to spiked plasma specimens. Clearly, patient plasma, specimens containing various concentrations of the triphenylethylenes can be monitored for drug-induced effects using cell
40
M DeGregorio et al
Table 3. Cell cycle measurements (MCF-7) o f extracted h u m a n plasma samples following toremifene or tamoxifen therapy. Patient
g M Drug a
% Inhibition
%GOG1
%SPF
%G2+M
--
0
61,8 _+ 2.0 b (58.3 - 65.4) c
29.8 _+ 2.1 (25.9 - 32.8)
8.4 + 1,1 (6.6 - 11.3)
1
8.0 + 3.4 (2.6 - 13.7) b
45 + 10.3 (55 - 18)
83.1 _+ 1.6 (80.7 - 85.7)
1t.5 + 1.2 (9.5 - 13.5)
5.4 _+ 0.6 (4.6 - 6.9)
2
6.5 + 2.9 (2.6 - 12.7)
48 + 21.4 (79 - 0)
68.7 + 4.5 (58.7 - 76.1)
24.1 _+ 3.3 (18.4 - 26.9)
7.2 _+ 1.3 (5.5 - 10.6)
3
4.8 + 2.0 (2.5 - 8.4)
42 + 4.7 (47 - 33)
73.7 + 1.5 (71.6 - 75.7)
19.5 _+ 1.2 (18.2 - 21.6)
6.8 + 0.4 (6.0 - 7.6)
4
8.0 + 3.5 (2.6 - 15.2)
42 + 9.8 (65 - 20)
74.7 + 2.6 (68.4 - 79.6)
18.5 + 2.2 (14.8 - 23.7)
6.8 + 0.6 (5.6 - 7.9)
--
0
66.9 + 0.8 (66.1 - 68.4)
24.6 ± 0.9 (23.0 - 25.4)
8.5 + 0.2 (8.3 - 8.7)
Toremi~ene Control
Tamoxifen Control 1
1.09
20
72.0
21.0
7.0
2
0.57
08
66.9
24.7
8.4
3
1.38
35
70.5
21.4
8.1
4
1.54
14
70.0
22.2
7.8
Concentrations the totals o f 4-OH, desmethyl, and parent drug. b Mean _+ population SD. c N u m b e r s in parentheses represent value ranges. Total number o f patient plasma specimens: TOR: control n=24, Pt 1 n=13, Pt 2 n = l l , Pt 3 n=7, Pt 4 n=18; TAM: control n=4, Pts 1-4 n = l a
cycle measurements. These effects can then be correlated to in vivo drug levels, and potentially to clinical response.
Discussion
Monitoring effective antiestrogen dosing is largely based on patient response. Approximately 50% of patients with metastatic, estrogen receptor positive breast cancer wilt respond to tamoxifen therapy. The reason why some patients fail to respond to antiestrogen therapy is unknown. Based on the highly variable pharmacokinetics of tamoxifen and toremifene, a method of assessing the antiestrogenic effects of tamoxifen in plasma would be a step towards individually designed antiestrogen therapy. The obvious limitation of
such a measurement would be that it measures drug present in plasma, which may not accurately predict activity at the tumor level. Whether plasma can be used as a surrogate measurement remains to be tested; however, any assessment of in vivo dosing would be an improvement on current methods. Our results demonstrate that plasma samples obtained from patients receiving antiestrogen therapy can be used to assess whether the parent drug and its metabolites are present in sufficient concentration to inhibit tumor cell growth and block cells in G0/G1. This bioassay may also be useful to monitor patient compliance and pharmacokinetic variation. An obvious potential application of our bioassay would be in drug development. Assessment of new drugs by this methods would examine the
A bioassay for antiestrogenic activity activity not only of the parent drug but also of its metabolites. For example, it is well known that tamoxifen and some o f its metabolites have both estrogenic and antiestrogenic activity. It is conceivable that the estrogen activity is more pronounced in some patients because of metabolic variation. If this were true, then non-metabolizable antiestrogens could be designed. Acquired tamoxifen resistance is yet a further research area in which our bioassay could be employed. The isomerization of trans-4-hydroxytamoxifen to cis-4-hydroxytamoxifen may significantly reduce the antiestrogenic contribution of 4-hydroxytamoxifen. We recently reported that both trans-4-hydroxytamoxifen and cis-4-hydroxytamoxifen could be measured in a tamoxifen resistant MCF-7 tumor mouse model [7]. Acquired tamoxifen resistance was correlated to tumor tamoxifen concentrations and increased cis/trans 4-hydroxytamoxifen ratios. Tumors that had developed resistance to tamoxifen therapy were predictable based on the cis/trans ratios of 4-hydroxytamoxifen present in each tumor. Unfortunately, tumor biopsies are often difficult to obtain clinically. Therefore, a potential use for our bioassay is to serially monitor plasma specimens or patients' tumors obtained by needle biopsy (since very few cells are needed, on the order of 50,000) and determine whether resistance to antiestrogen therapy can be detected on a patient specific basis.
Acknowledgements This work was supported by a grant from Orion Corporation-Farmos. W e would like to thank Dr. C. Kent Osborne and the Phase 1 team for the clinical specimens.
References 1. Jordan VC, Robinson SP, Wetshons WV: Resistance to
2.
3.
4.
5.
6.
7.
8.
9.
41
antiestrogen therapy. In Kessle D (ed) Drug Resistance. CRC Press, Boca Raton, 1987. Fromson JM, Pearson S, Bramah S: The metabolism of tamoxifen (IC146,474) in laboratory animals. Xenobiotica 3: 693, 1973. Wiebe VJ, Benz CC, Shemano I, Cadman TB, DeGregorio MW: Pharmacokinetics of toremifene and its metabolites in patients with advanced breast cancer. Cancer Chemother Pharmacol 25: 247-251, 1990. Buckley MM-T, Goa KL: Tamoxifen: A reappraisal of its pharmacodynamic and pharmacokinetic properties and therapeutic use. Drugs 37: 451-490: 1989. Fabian C, Sternson L, Barnett M: Clinical pharmacology of tamoxifen in patients with breast cancer: comparison of traditional and loading dose schedules. Cancer Treat Rep 64: 765-773, 1980. Langan-Fahey SM, Tormey DC, Jordan VC: Tamoxifen metabolites in patients on long-term adjuvant therapy for breast cancer. Eur J Cancer 26: 883-888, 1990. Osborne CK, Coronado E, Atlred DC, Wiebe V, DeGregorio MW: Acquired tamoxifen resistance: Correlation with reduced breast tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen. J Natl Cancer Inst 83: 1477-1482, 1991. Holleran WM, Gharbo SA, DeGregorio MW: Quantitation of toremifene and its major metabolites in human plasma by high-performance liquid chromatography following fluorescent activation. Anal Letters 20: 871-879, 1987. Krishan A: Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66: 188-195, 1975.
Report
A bioassay for antiestrogenic activity - - potential utility in drug development and monitoring effective in vivo dosing Michael DeGregorio, Gregory Wurz, Vernon Emshoff, Steven Koester, Patrick Minor, and Valerie Wiebe Department of Medicine, Division of Oncology, University of Texas Health Science Center, San Antonio, Texas, USA
Key words: antiestrogens, bioassay, tamoxifen, toremifene
Summary Monitoring effective antiestrogenic activity of the triphenylethylenes in patients with breast cancer is usually determined by the duration of response. The pharmacokinetics of toremifene and tamoxifen have been shown to be highly variable but patient specific. In the present study, we developed a method to accurately assess the antiestrogenic activity of these agents using plasma specimens, cell culture, and cell cycle measurements. Plasma specimens (4-5mls) obtained from patients receiving toremifene (360mg/day for 5 days in a phase I trial) or tamoxifen (20mg/day) were extracted and reconstituted in tissue culture media (4-5mls), and growth inhibition was determined in estrogen responsive MCF-7 cells. Additionally, plasma specimens were quantified for toremifene or tamoxifen concentrations using HPLC. Growth inhibition of plasma specimens containing either toremifene or tamoxifen and their metabolites was also examined. Cell cycle measurements were determined following in vitro exposure with flow cytometric techniques. Our results show that a dose-response relationship exists between cell growth inhibition and cell cycle measurements for human plasma with added toremifene or tamoxifen, and also for human plasma specimens containing drug and its metabolites after treatment. Our antiestrogenic bioassay can address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance.
Introduction Toremifene, tamoxifen, and their metabolites inhibit cell growth in breast tumors primarily by estrogen receptor blockade. Both estrogen receptor agonist and antagonist properties have
been described for these agents [1]. However, they are generally considered to be antiestrogens in human breast tumors due to their inhibitory effects on estrogen-stimulated cell growth. The major metabolic pathways for toremifene and tamoxifen include N-desmethylation and 4-hy-
Addressfor offprints: Dr. Michael DeGregorio,Medicine/Oncology,Universityof Texas Health Science Center, 7703 Floyd Curl Drive, San AntonioTX 78284-7884, USA
36
M DeGregorio et al
droxylation [2,3]. Although the 4-hydroxy metabolite is a minor metabolite, it binds estrogen receptors with high affinity and may contribute significantly to antiestrogenic activity [4]. The pharmacokinetics of toremifene and tamoxifen in patients with breast cancer are well described. Fabian et al. examined tamoxifen plasma kinetics after continuous oral dosing and found that most patients achieved steady-state levels within 16 weeks of therapy, although a wide range of concentrations were reported [5]. Similar pharmacokinetic results were reported for toremifene following 8 weeks of therapy [3]. Both studies show a wide variation in steady-state concentrations. In addition, it has been suggested that reduced plasma antiestrogen concentrations either from altered bioavailability or from noncompliance may contribute to clinical failure [1]. Although tamoxifen resistance has not been correlated with plasma antiestrogen levels [6], alterations in the cellular pharmacology of tamoxifen may be an early hallmark of acquired resistance to tamoxifen [7]. In the present study, we developed a bioassay that allows for the assessment of effective antiestrogen dosing on a patient-specific basis. In addition, this assay can address clinical research problems such as patient-specific pharmacokinetics, patient compliance, and mechanisms of acquired antiestrogen resistance such as metabolic tolerance and production of estrogenic metabolites.
Methods
Cell culture MCF-7 cells were grown in Coming T25 flasks containing 7.0ml IMEM + 10% fetal bovine serum and maintained in 5% CO 2 and 95% air. Cells were plated at a concentration of 10,000 cells/ml, and incubated at 37°C for 12-24 hours prior to treatment.
Sample extraction All patient plasma or spiked plasma specimens were extracted with 6.0 ml of 2% butanol in hexane and vortexed for 1 minute. If necessary, samples were then centrifuged for 10 minutes at 1,000 x g. The organic phase was dried under nitrogen gas at 37°C. All samples were immediately capped with sterile caps and stored at -20°C until used. A 1 ml aliquot of each patient sample was also quantified by HPLC (see below).
Growth inhibition All specimens were reconstituted in sterile medium and placed on MCF-7 cells yielding the same gg/ml concentrations seen following therapy. MCF-7 cells were incubated with drug for 4-5 days or harvested at 75-80% confluency of controls. All medium was aspirated, and cells were washed with 2ml normal saline, trypsinized, and resuspended in 5ml of fresh medium. All cell counts were performed using Coulter Counter technique. Cell viability was estimated by trypan blue dye exclusion. Cells were then processed for flow cytometric analysis (see below).
HPLC analysis Following extraction, all 1 ml plasma extracts were reconstituted in 200 gl methanol before analysis. The reconstituted samples were transferred to an Infrasil quartz cuvette (Fisher Scientific, Fair Lawn, NJ), exposed to high-intensity ultraviolet light for 1 minute using a 15 W mercury vapor lamp, and then injected onto the column. The HPLC assay was performed and validated as previously described [8]. The system consisted of a Beckman model 320 gradient liquid chromatograph, two model 110A pumps, and a model 420 controller. The HPLC was equipped with a reverse phase Axxion C18 ultrasphere
A bioassay for antiestrogenic activity
propidium iodide in a hypotonic sodium citrate solution with 0.3% NP-40 and 1.0 m g / m l RNAse-A), vortexed, and stained for 30 minutes at r o o m temperature in the dark. Prior to flow cytometfic measurements, samples were filtered through a 37 micron nylon mesh into 12 x 75 m m tubes and stored at 4°C until analysis within 24 hours. Flow cytometric measurements were performed on an EPICS 753 instrument (Coulter Cytometry, Hialeah, FL) with an argon-ion laser tuned to 488 nm and 400 m W of power. Red fluorescence from the propidium iodide (peak fluorescence at 610nm) was collected through 515nm long pass and 610nm long pass filters. Compartmental analysis of D N A histograms was accomplished with M O D F I T software (Verity Software House, Topsham, ME).
column and a 100 gl injection loop. The mobile phase consisted of 7% water and 0.18% triethylamine in methanol° The flow rate of the mobile phase was set at 1.00 ml/min. The fluorescence of photochemically activated compounds was detected with an Applied Biosystems 980 fluorom e t e r with excitation wavelength set at 266 nm. Retention times and peak heights were recorded with a Spectraphysics integrator (Menlo Park, CA).
Flow cytometry Cells were stained with a modified Krishan technique [9]. Samples were spun at 1000 x g for 7 minutes at 4 ° C , cell pellets were resuspended in 1.0 ml of Krishan staining solution (50 g g / m l
Table 1. Cell cycle measurements of MCF-7 cells following exposure to spiked Toremifene or Tamoxifen and their metabolites. Drug
~tM
% Inhibition a %GOG1b
%SPF
%G2+M
Cell control
-
0
58.0
34.0
8.0
TOR TOR TOR
1.0 3.3 6.6
65 82 89
76.2 84.4 88.1
17.4 10.7 6.8
6.4 4.9 5.0
4-OH TOR 4-OH TOR 4-OH TOR
1.0 3.3 6.6
69 72 79
79.8 81.1 81.3
14.1 13.2 12.8
6.1 5.7 5.9
n-des TOR n-des TOR n-des TOR
1.0 3.3 6.6
44 71 82
68.8 77.2 83.6
23.5 16.3 11.3
7.7 6.5 5.1
Ceil control
--
0
61.2
30.0
8.8
TAM TAM TAM
1.0 3.3 6.6
52 53 63
78.4 81.6 83.7
15.4 13.2 t 1.0
6.2 5.2 5.3
4-OH TAM 4-OH TAM 4-OH TAM
1.0 3.3 6.6
52 61 69
83.6 84.6 84.5
12.0 11.0 10.7
4.4 4.5 4.9
n-des TAM n-des TAM n-des TAM
1.0 3.3 6.6
49 61 75
79.4 82.6 85.3
14.9 12.4 10.5
5.8 4.9 4.2
a
37
Based on cell control. Each % inhibition value represents the mean value of triplicate samples. Ranges were within 5% of the mean.
38
M DeGregorio et al
Results
their metabolites had similar potency. Table 2 shows similar results on cell cycle measurements when each drug was spiked into plasma and extracted with hexane/butanol (>80% extraction efficiency). Growth inhibition was essentially equivalent to that seen with direct administration of drug in the tamoxifen group and slightly less than that observed in the toremifenetreated cells, although not significantly, Figure 1 shows the dose-response growth inhibition and cell cycle measurements for the extracted tamoxifen and toremifene groups. Table 3 shows the results o f patient plasma specimens containing either toremifene or tamoxifen and their metabolites. Cell growth was
Table 1 shows the effects of toremifene and tamoxifen and their two major metabolites on MCF-7 cell growth and cell cycle kinetics. A dose-response inhibition o f cell growth was seen for each compound. The range of dosing was from antiestrogenic concentrations (l~tM) to doses necessary to inhibit critical biologic pathways such as protein kinases, calcium channels, and others (6.6~tM). Similar to other reports, all compounds appeared to block cells in G0/G1, while decreasing S and G 2 + M in a dosedependent manner. Toremifene was slightly more active in blocking in G0/G1 than tamoxifen, while
Table 2. Cell cycle measurements of MCF-7 cells following exposure to extracted Toremifene or Tamoxifen and their metabolites.
Drug
].tM
% Inhibition a
%GOGIb
%SPF
%G2+M
Plasma alone
-
0
70.8
22.5
6.7
TOR TOR TOR
1.0 3.3 6.6
28 50 58
75.0 80.0 81.0
18,4 14.4 13.3
6.6 6.1 5.6
4-OH TOR 4-OH TOR 4-OH TOR
1.0 3.3 6.6
53 59 76
83.4 83.5 81.0
11.1 11.2 13,9
5.5 5.3 5,2
n-des TOR n-des TOR n-des TOR
1.0 3.3 6.6
12 38 47
74.4 78.8 80.5
18.5 15.2 14.1
7.1 6.0 5.4
TOR IV TOR IV TOR IV
1.0 3.3 6.6
48 56 59
83.2 83.2 84.1
11.6 12.0 11,3
5.1 4.9 4.7
Plasma
-
0
66.8
25.8
7.4
TAM TAM TAM
1.0 3.3 6.6
53 61 64
75.0 78.4 78.3
18.4 15.6 15,6
6,6 6.0 6.3
4-OH TAM 4-OH TAM 4-OH TAM
1.0 3.3 6.6
52 63 69
83.7 82.7 80.2
11.1 12,1 14,6
5.2 5.2 5.2
n-des TAM n-des TAM n-des TAM
1.0 3.3 6.6
43 54 69
76,2 79.4 78.8
17.5 15.3 15.4
6.3 5.4 5.8
a Each inhibition represents the mean value of triplicate samples. Ranges were within 5% of the mean. b Triplicates were pooled for cell cycle measurements.
A bioassay for antiestrogenic activity
39
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Figure 1. Survivaland cell cycle measurementsof MCF-7 cells followingexposure to extractedTOR and TAM and their metabolites. Legend: + TOR; C) 4-OH-TOR; • n-des TOR; [] TAM; • 4-OH-TAM; II n-des TAM. significantly inhibited in all toremifene specimens. Tamoxifen produced similar inhibitory effects, with only one patient specimen producing minimal effects. This patient specimen had the lowest concentration of tamoxifen and its metabolites,
suggesting a potential dose-response relationship similar to spiked plasma specimens. Clearly, patient plasma, specimens containing various concentrations of the triphenylethylenes can be monitored for drug-induced effects using cell
40
M DeGregorio et al
Table 3. Cell cycle measurements (MCF-7) o f extracted h u m a n plasma samples following toremifene or tamoxifen therapy. Patient
g M Drug a
% Inhibition
%GOG1
%SPF
%G2+M
--
0
61,8 _+ 2.0 b (58.3 - 65.4) c
29.8 _+ 2.1 (25.9 - 32.8)
8.4 + 1,1 (6.6 - 11.3)
1
8.0 + 3.4 (2.6 - 13.7) b
45 + 10.3 (55 - 18)
83.1 _+ 1.6 (80.7 - 85.7)
1t.5 + 1.2 (9.5 - 13.5)
5.4 _+ 0.6 (4.6 - 6.9)
2
6.5 + 2.9 (2.6 - 12.7)
48 + 21.4 (79 - 0)
68.7 + 4.5 (58.7 - 76.1)
24.1 _+ 3.3 (18.4 - 26.9)
7.2 _+ 1.3 (5.5 - 10.6)
3
4.8 + 2.0 (2.5 - 8.4)
42 + 4.7 (47 - 33)
73.7 + 1.5 (71.6 - 75.7)
19.5 _+ 1.2 (18.2 - 21.6)
6.8 + 0.4 (6.0 - 7.6)
4
8.0 + 3.5 (2.6 - 15.2)
42 + 9.8 (65 - 20)
74.7 + 2.6 (68.4 - 79.6)
18.5 + 2.2 (14.8 - 23.7)
6.8 + 0.6 (5.6 - 7.9)
--
0
66.9 + 0.8 (66.1 - 68.4)
24.6 ± 0.9 (23.0 - 25.4)
8.5 + 0.2 (8.3 - 8.7)
Toremi~ene Control
Tamoxifen Control 1
1.09
20
72.0
21.0
7.0
2
0.57
08
66.9
24.7
8.4
3
1.38
35
70.5
21.4
8.1
4
1.54
14
70.0
22.2
7.8
Concentrations the totals o f 4-OH, desmethyl, and parent drug. b Mean _+ population SD. c N u m b e r s in parentheses represent value ranges. Total number o f patient plasma specimens: TOR: control n=24, Pt 1 n=13, Pt 2 n = l l , Pt 3 n=7, Pt 4 n=18; TAM: control n=4, Pts 1-4 n = l a
cycle measurements. These effects can then be correlated to in vivo drug levels, and potentially to clinical response.
Discussion
Monitoring effective antiestrogen dosing is largely based on patient response. Approximately 50% of patients with metastatic, estrogen receptor positive breast cancer wilt respond to tamoxifen therapy. The reason why some patients fail to respond to antiestrogen therapy is unknown. Based on the highly variable pharmacokinetics of tamoxifen and toremifene, a method of assessing the antiestrogenic effects of tamoxifen in plasma would be a step towards individually designed antiestrogen therapy. The obvious limitation of
such a measurement would be that it measures drug present in plasma, which may not accurately predict activity at the tumor level. Whether plasma can be used as a surrogate measurement remains to be tested; however, any assessment of in vivo dosing would be an improvement on current methods. Our results demonstrate that plasma samples obtained from patients receiving antiestrogen therapy can be used to assess whether the parent drug and its metabolites are present in sufficient concentration to inhibit tumor cell growth and block cells in G0/G1. This bioassay may also be useful to monitor patient compliance and pharmacokinetic variation. An obvious potential application of our bioassay would be in drug development. Assessment of new drugs by this methods would examine the
A bioassay for antiestrogenic activity activity not only of the parent drug but also of its metabolites. For example, it is well known that tamoxifen and some o f its metabolites have both estrogenic and antiestrogenic activity. It is conceivable that the estrogen activity is more pronounced in some patients because of metabolic variation. If this were true, then non-metabolizable antiestrogens could be designed. Acquired tamoxifen resistance is yet a further research area in which our bioassay could be employed. The isomerization of trans-4-hydroxytamoxifen to cis-4-hydroxytamoxifen may significantly reduce the antiestrogenic contribution of 4-hydroxytamoxifen. We recently reported that both trans-4-hydroxytamoxifen and cis-4-hydroxytamoxifen could be measured in a tamoxifen resistant MCF-7 tumor mouse model [7]. Acquired tamoxifen resistance was correlated to tumor tamoxifen concentrations and increased cis/trans 4-hydroxytamoxifen ratios. Tumors that had developed resistance to tamoxifen therapy were predictable based on the cis/trans ratios of 4-hydroxytamoxifen present in each tumor. Unfortunately, tumor biopsies are often difficult to obtain clinically. Therefore, a potential use for our bioassay is to serially monitor plasma specimens or patients' tumors obtained by needle biopsy (since very few cells are needed, on the order of 50,000) and determine whether resistance to antiestrogen therapy can be detected on a patient specific basis.
Acknowledgements This work was supported by a grant from Orion Corporation-Farmos. W e would like to thank Dr. C. Kent Osborne and the Phase 1 team for the clinical specimens.
References 1. Jordan VC, Robinson SP, Wetshons WV: Resistance to
2.
3.
4.
5.
6.
7.
8.
9.
41
antiestrogen therapy. In Kessle D (ed) Drug Resistance. CRC Press, Boca Raton, 1987. Fromson JM, Pearson S, Bramah S: The metabolism of tamoxifen (IC146,474) in laboratory animals. Xenobiotica 3: 693, 1973. Wiebe VJ, Benz CC, Shemano I, Cadman TB, DeGregorio MW: Pharmacokinetics of toremifene and its metabolites in patients with advanced breast cancer. Cancer Chemother Pharmacol 25: 247-251, 1990. Buckley MM-T, Goa KL: Tamoxifen: A reappraisal of its pharmacodynamic and pharmacokinetic properties and therapeutic use. Drugs 37: 451-490: 1989. Fabian C, Sternson L, Barnett M: Clinical pharmacology of tamoxifen in patients with breast cancer: comparison of traditional and loading dose schedules. Cancer Treat Rep 64: 765-773, 1980. Langan-Fahey SM, Tormey DC, Jordan VC: Tamoxifen metabolites in patients on long-term adjuvant therapy for breast cancer. Eur J Cancer 26: 883-888, 1990. Osborne CK, Coronado E, Atlred DC, Wiebe V, DeGregorio MW: Acquired tamoxifen resistance: Correlation with reduced breast tumor levels of tamoxifen and isomerization of trans-4-hydroxytamoxifen. J Natl Cancer Inst 83: 1477-1482, 1991. Holleran WM, Gharbo SA, DeGregorio MW: Quantitation of toremifene and its major metabolites in human plasma by high-performance liquid chromatography following fluorescent activation. Anal Letters 20: 871-879, 1987. Krishan A: Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66: 188-195, 1975.
