Differential Uptake of Estramustine Phosphate Metabolites and Its Correlation with the Levels of Estramustine Binding Protein in Prostate Tumor Tissue

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Peter H. Walz,1 Per Bj#{246}rk,Per-Olov Gunnarsson, hypothesis that the EMBP is responsible for the retention of Karin Edman, and Beryl Hartley-Asp EoM and EaM in the prostate tumor. Department of Urology, Kreiskrankenhaus LUdenscheid, D-58515 Ltidenscheid, Germany [P. H. W.J, and Pharmacia and Upjohn INTRODUCTION Oncology, 5-22007 Lund, Sweden [P. B., P-O. G., K. E., B. H-A.] EMP has been used for many years in the treatment of advanced prostate cancer. Recently, combination treatments ABSTRACT with vinblastine or etoposide have shown great promise and, in Estracyt (EMP) has been used for the treatment of addition, neoadjuvant treatment with EMP has been reported to hormone refractory prostate cancer for many years. Re- cause a reduction in the number of positive margins in T2B cently, new data from combination studies have given rise to patients (1-6). This has caused a renewed interest in an old new interest in this old drug. Explanations for the synergy drug. EMP is a prodrug that is rapidly dephosphorylated on oral administration to the two cytotoxic metabolites EaM and EoM found in the clinic are many, but one major factor may be the previous indication that the drug accumulates in the (7). EoM has been shown to be the major circulating metabolite prostate tumor. We have, therefore, examined the level of in prostate cancer patients and, thus, the proposed metabolic scheme is seen in Fig. 1 . and time-dependent the four metabolites, estromustine (E0M), estramustine EaM induces dose- metaphase arrest and breakdown of interphase microtubules in (EaM), estrone, and estradiol in the tumor and serum of 14 several different human tumor cell lines in vitro (8-10), and patients with T2 and T3 prostate cancer receiving a single i.v. causes dose-dependent metaphase arrest in hormone independ- dose of 600 mg of EMP, about 12 h before radical prosta- ent human prostate tumor cells (DU 145) implanted in nude tectomy. Because it has been suggested that the uptake into mice (11). The effect of EaM on microtubules has been shown the prostate tumor is due to binding to the estramustine to be through binding to both tubulin and the microtubule- binding protein (EMBP), we have in addition measured the associated proteins (12, 13). Similar effects have been found for level of EMBP in the prostate tumor tissue. The main serum EOM.3 In addition, EaM induced a time-dependent increase in and tissue metabolite in all patients was EoM followed by apoptosis in BT4C rat glioma cells implanted in the brain in EaM, estrone, and estradioL The levels for EoM ranged contrast to a lack of effect in normal rat brain tissue (14). from 63.8-162.8 ng/ml in the serum and from .8-1209 Apoptosis was also found in glioma tissue from patients who ng/ml in the prostate tumor, resulting in a mean ratio for had received treatment with EMP (14). serum to tumor of 1:5. The levels for EaM ranged from Norldn et a!. (15) demonstrated that at steady state, after 8.3-51.4 ng/ml in the serum and 73.9-563.4 ng/inl in the treatment with oral EMP, a preferential retention of EaM oc- tumor, giving a mean ratio for serwn to tumor of 1:13. The curred in the prostate tumor; on average, EaM was six times levels of EMBP were higher in T3 tumors than in T2 tumors, higher in the tumor tissue than in the serum. They suggested that 54.1 and 40.7 ng/g tissue, respectively. A significant corre- this could possibly be due to specific binding of EaM to a lation was found between the levels of EaM (r 0.60) and protein present in prostate tumor tissue. This protein has many the levels of EMBP in the tumor. These data demonstrate names (prostatein, 224-protein, and EMBP) and is a secretory that 12 h after a single i.v. dose of 600 mg of EMP the levels protein, the function of which is still unknown. It is a Mr 46,000 of the cytotoxic metabolites EoM and EaM are substantially protein, which in the rat ventral prostate and normal human higher in the tumor than in the serum of the same patient prostate is under androgenic control (16-19). In man, a protein and that a correlation exists between the levels of EaM in the with similar characteristics has been found in normal tissue, tumor and the levels of EMBP. Thus, this supports the benign prostatic hyperplasia, and carcinoma of the prostate, and in addition, in other tumor tissues such as pancreas, breast, lung, and glioma (20-24). In rats, after a single i.v. dose of tritium- labeled EMP, the concentration of radioactivity 24 h later in the ventral prostate was 20 times higher than in the serum (25). Received 10/21/97; revised 5/28/98; accepted 6/10/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviations used are: EMP, estracyt; EaM, estramustine; EoM, 1 To whom requests for reprints should be addressed, at Department of Urology, Kreiskrankenhaus LUdenscheid, Paulmannsh#{246}her Str. 14, estromustine; EMBP, EaM binding protein; E., estradiol; E1, estrone; D-585l5 L#{252}denscheid, Germany. Phone: 49-2351-46-36-80; Fax: 49- LOQ, limit of quantitation; PSA, prostate-specific antigen. 2351-46-36-82. 3 Unpublished data.

OH O:ILOH o OH 0

CCF2CH2Jt_#{216} ::::::NoO:5:I±:J_., ccH2cH2JOoc5:t CKH2CH2 CCH2CH-

ESTRAMUSTINE PHOSPHATE ESTRAMUSTINE ESTROMUSTINE

ESTRADIOL ESTRONE Fig. 1 Structure and metabolic scheme for EaM phosphate.

Similar results have been found in the BT4C rat glioma model, 5890) with nitrogen-phosphorus detection. The other residue where a 16: 1 tumor to serum ratio for EaM was found (20). was dissolved in toluene, and E and E1 were quantified by gas In the present study, our aim was to determine whether a chromatography with selected ion monitoring (Varian 3400- correlation exists between the level of EMBP and the levels of Finnigan Mat INCOS 500). EaM and EoM in tumor tissue. Therefore, we have carried out The LOQ was 13 ng/ml EaM, 16 ng/ml EoM, 1 ng/ml E, the present investigation on tumor tissue from radical prostate- and 3 ng/ml E1. The precision (CV) of the methods was as ctomies after a single i.v. administration of EMP. In addition, to follows: for EaM, 7% at 45 ng/ml; for EoM, 7% at 260 ng/ml; broaden our knowledge regarding the preferential uptake/reten- for E, 10% at 5 ng/ml; and for E1, 9% at 22 ng/ml. tion of these metabolites and E1 and E2 in the tumor, we have Tissues. Tissue was assayed for EaM, EoM, E, and E1 also determined the level of these four metabolites in the serum using gas chromatography according to a method by Andersson of these patients. et al. (26), modified for methanol extracts and adjusted to the capillary column instrumentation used for serum analyses. MATERIALS AND METHODS Tissue samples were homogenized with 15 volumes of methanol. The extracts were evaporated, and the residues were Study Design dissolved in 2 ml of water, extracted with hexane, and purified Fourteen patients, 52-74 years of age, with prostate cancer on an aluminum oxid column. The eluates were evaporated and 12-13 and negative bone scans, participated in the study. All derivatized with bis(trimethylsilyl)-trifluoroacetamide. The sea- patients were informed about the trial and consented to partic- gent solutions were further analyzed as described above for ipate. All patients received a single i.v. injection of 600 mg of serum samples. The LOQs were 5 ng/sample EaM, 7 ng/sample EMP, 12-15.5 h before radical retropubic prostatectomy was EoM, 1 ng/sample E, and 4 nglsample E1. A sample consisted performed. This time point was chosen to allow for full metab- typically of 10-30 ml of extract. The precision of the methods olism of EMP and distribution of the metabolites to occur. was as follows: for EaM, 18% at 45 ng/sample; for EoM, 11% Serum samples were taken at the start of the operation and at 45 ng/sample; for E, 39% at 6 ng/sample; and for E1, 54% at at the same time as the tissue samples were taken from the 6 ng/sample. prostate tumor (i.e., approximately 90 mm apart). All tissue and serum samples were immediately immersed in liquid nitrogen Radioimmunochemkal Determination of EMBP and stored at -70#{176}Cuntil analyzed. The prostatic tissue specimens were homogenized in ice- cold 50 mM Tris-HC1 (pH 7.4) containing 0.25 sucrose, 10 nmi Analysis of Metabolites KC1, 1.5 MgC12, and 1% (v/v) NP4O using an Ultra-Turrax Serum. Serum was assayed for EaM, EoM, E, and E1 homogenizer and clarified by centrifugation for 1 h at using gas chromatography. 105,000 X g. Proteins were precipitated by adding 1 volume of Briefly, before gas chromotography, the serum samples extract to 9 volumes of freeze-cold acetone and centrifugation were purified by Cl8 solid phase and solvent extraction proce- for 20,000 x g X 10 mm. The acetone was removed by dures and derivatized with bis(trimethylsilyl)-trifluoroacet- decantation, and the precipitate was washed once with 10 vol- amide. The reagent solution of each sample was divided in two umes of freeze-cold diethylether and centrifuged as above. After and evaporated. One of the residues was dissolved in xylene, decantation, the remaining ether was removed by evaporation and EaM and EoM were quantified by gas chromatography (HP under a stream of nitrogen gas, and the dry residue was dis-

Table 1 Age, tumor stage, tumor grade, and preoperative PSA Table 2 Tumo r stage and preoperative PSA values of 14 patients values of 14 patients receiving a single dose of EaM phosphate >12 receiving a sin gle dose of EaM phosphate > 12 h before radical h before radical prostatectomy prostatectomy

Patient Age Tumor stage Tumor grade’ PSA (ne/mI) Stage Number of patients PSA (ng/ml) 6 70 pT,N0 1 0.1 pT2N0 6 0.1-12 8 65 pT2,N0 1 10.23 pT3N0 4 1-75.3 9 67 pT2,,N0 2 10.6 pT3N1 1 17.6 10 61 pT2bNo 2 12 pT3N2 3 15.5-42 11 52 pT2,N0 1 0.2 12 59 pT2,N0 2 11 1 59 pT3N0 2 75.3 5 62 pT3bNo 3 4.7 13 64 PT3aN0 2 6.5 [i.e., E1 is the major estrogen ranging from 3.4-14.1 ne/mI, with 14 71 pTN0 3 1 E2 from 0.4 (LOQ) to 2.3 ne/mi]. 3 64 pTN1 1 17.6 The levels of EoM and EaM in the prostate tumor tissue 2 74 pTN2 3 42 4 68 pTN2 2 15.5 were higher than those found in the serum of the same patient 7 64 pTN2 2 20.5 except for one patient (patient 10), where the concentration of a Grade 1, highly differentiated; grade 2, poorly differentiated; EoM was very similar in both tumor and serum. The concen- grade 3, cribriform and anaplastic differentiation. trations in the prostate tumor tissue ranged from 64.8-1209 ng/g for EoM and from 73.9-563.4 ng/g for EaM (Table 3). The prostate tumor tissue levels of the two estrogens E, and E2 were also higher than those found in the serum of the solved in 50 mM Tris-HC1 (pH 7.4), 10 mi KC1, and 1 mM same patients with the tumor tissue levels ranging from 5.5-58.7 EDTA and stored at -30#{176}Cuntil analyzed for EMBP. ng/g for E1 and 4.5-252 ng/g for E2 (Table 3). The tumor to The RLA used for determination of the EMBP was as serum ratios for EoM and EaM in the individual patients are described previously (21), with some modifications. Briefly, found in Table 4. The mean values show that the concentration radioiodinated EMBP (specific activity, 50-100 p.Ci/p.g) was of EoM is 4.79 times higher and EaM 13.07 times higher in the used as tracer and unlabeled EMBP as standard in concentra- tumor tissue than in the serum (Table 4). tions from 0.04-330 ng/ml. Each incubate consisted of 0.1 ml The levels of EMBP in the prostate tissue from individual each of the radioligand (7 ng/ml), standard or sample, and patients are found in Table 4 and vary from 6.73-93.1 ng/g protein G-Sepharose 4-B purified rabbit anti-EMBP (1 p.g/ml), tissue. When the tumors are grouped according to stage, the and was allowed to react for 2 h. Each vial was rotated overnight mean value for T2 tumors is 40.69 and for T3 tumors 54.07. A with 1 ml of a Gamma Bind Plus suspension (0.2 ml slurry/lOO significant correlation was found between the level of EMBP in ml; Pharmacia Biotech, Uppsala, Sweden), and the antigen- the tumor and the concentration of EaM in the tumor sample antibody complex was recovered by repeated centrifugations [r 0.64; P = 0.0297 (least square linear regression, two- (1,500 X g X 5 mm) and aspirations. The antibody-bound tailed)]; the correlation was not statistically significant for EoM radioactivity was determined by the counting of the sediment in (r 0.29; P = 0.323; Fig. 2). a Packard COBRA gamma counter (Packard Instruments Co., Downers Grove, IL). Standards and samples were analyzed in DISCUSSION duplicates, the latter at least as three serial dilutions to allow the The present study demonstrates that a correlation exists test of congruence with the standard curve when the antigen was between the level of EMBP in prostate tumor tissue and the present in enough quantities. Values falling within 10% and level of the two major metabolites EaM and EoM after a single 90% of tracer bound in the absence of unlabeled competitor i.v. administration of 600 mg of EMP to 14 patients before were used for calculation of EMBP concentrations. radical prostatectomy. In a previous study, it was demonstrated that in 6 patients RESULTS who had received oral EMP for several years, the level of EaM In six patients, the tumor stage was pT2N0; in four patients, was on average 6.3 times higher in the prostate tumor than in the pT3N0; in one patient, pT3N1; and in three patients, pT3N2. The serum of these patients; however, no differences were found for tumor was highly differentiated in four patients, poorly differ- EoM (15). In that study, as demonstrated in other studies (27), entiated in seven patients, and cribriform or anaplastic in three a depot of the very lipophilic EoM would have built up in fat patients. The PSA values ranged from 0.1-75.3 ng/ml, the during the continuous oral dosing. The binding to fat is unspe- highest value being found in one patient with a T3N0 tumor cific as can be seen from the similarity between the quotas for (Tables 1 and 2). plasma to fat for EoM and EaM; however, as EoM is the major

The concentrations of EoM, EaM, E1 , and E2 in the serum metabolite, much more of this is released to the serum. There- and prostate tumor tissue of the individual patients are given in fore, the lack of accumulation of EoM could possibly be due to Table 3. The major serum metabolite, despite the wide variation EoM being continuously available from the fat depot for unlim- of 63.8-162.8 ng/ml, was in all patients’ EoM after a single i.v. ited binding to serum proteins, whereas the tumor binding sites dose of EMP. The serum concentrations of EaM ranged from were saturable at the concentration range found in the tumor. 8.3 (LOQ) to 51.4 ne/mI. Although the serum concentrations of This problem is overcome in the present study as a single iv. estrogens were substantially lower, a similar pattern emerges dose was given. The serum to tumor ratios were 1 :4.79 (EoM)

Table 3 Serum (ne/mi) and pr ostatic tissue (ng/g) concentrations of EoM, EaM, E1, and E in prostate cancer patients (n = 14) who received a single dose of EMP > 12 h before surgery and blood sampling Plasma” Prostate Tissue”

Patient EoM EaM E1 E EoM EaM E1 E 1 90.8 20.0 5.1 (0.6) 981.6 399.6 (7.0) (2.2) 2 78.7 19.5 7.8 1.1 895.6 457.8 (16.8) (6.0) 3 89.6 (8.3) 6.9 (0.7) 474.8 279.1 35.6c 218d 4 162.8 51.4 14.1 2.3 699.3 563.4 44.8 23.6 5 63.8 1 1.4 (4.7) (0.4) 325.2c 141.2 58.7c 252.0” 6 82.7 25.7 10.1 1.5 234.9 95.3 58.3c 36ff 7 66.0 20.9 12.3 1.9 301.3c 200.2 46.2c 19.9 8 85.8 17.1 5.7 1.0 167.4 94.4 (9.0) (10.0) 9 102.7 18.2 11.8 1.3 1,209 514.1 28.7 4.5 10 69.2 15.2 8.7 1.0 64.8 102.8 26.0 8.5 I 1 94.4 17.7 5.6 1.9 152.5 73.9 (5.5’) (7.5’) 12 147.7 23.8 11.1 1.2 163.9 251.1 (12.5c) (19.5c)

13 145.1 30.8 3.4 0.9 3344C 102.0 (26.5) (13.0’) 14 126.9 28.8 7.4 1.3 385.2c 306.0’ 440’ 10.0’

a Parentheses, one or two replicates at or below the LOQ. b Italics, median value.

C Value obtained from replicates or specimens with large variation.

d Single value.

Table 4 Prostate tissue:serum ratios for EoM and EaM and EMBP levels (ng/g prostatic tissue) in prostate cancer patients given a single r = 0.60 dose of EMP > 12 h before surgery and blood sampling p = 0.0298 U EMBP a)a Patient EoM EaM (ng/g prostate tissue) .a) 400 I 10.8 20.0 -“ 2 1 1.4 23.5 69.8 3D #{149} 3 5.30 33.6 42.6 4 4.30 11.0 93.1 5 5.10 12.4 55.8 . 6 2.84 3.71 48.5 7 4.57 9.58 34.9 100 ., 8 1.95 5.52 34.4 9 11.8 28.2 45.6 10 0.936 6.76 48.8 11 1.61 4.15 22.2 0 3 40 811 100 12 1.11 10.6 78.6 13 2.30 3.31 6.73 EMBP (nglg tumour tissue) 14 3.04 10.6 28.2 Mean ± SD 4.79 ± 3.70 13.07 ± 7.46 40.2 ± 28.7 (n 14) (n 13) (n 13) r = 0.297 Median 2.84 8.17 42.6 12! =#{176}323 #{149} (range) (0.936-1 1.8) (3.31-28.2) (6.73-93.1) a Not enough tissue sample for EMBP determination. I

and 1: 13.07 (EaM), demonstrating the retention of both metab- I olites in the prostate tumor tissues. The retention of metabolites is similar to data from exper- imental glioma models. In the rat BT4C orthotopic implanted brain tumor model, the serum to tumor ratio was 1 : 15 for EaM and 1:5 for EoM (14). Similar data have also been generated from patients with gliomas who received 280 mg of oral EaM phosphate before surgery. In 16 patients, a ratio of 16:1 between 0 40 W 80 100 tumor tissue and serum for EaM was found 2 h after drug delivery (20). EMBP (ng/g tumour tissue) The present data demonstrate a correlation between the

level of EMBP and those of EaM and EoM in samples of Fig. 2 The correlation between the levels of EoM, EaM, and the prostate tumor tissue from patients receiving EMP. This sub- EMBP found in the prostate tumor tissue of patients.

stantiates the previous hypothesis that it is this protein that is 3. Pienta, K. J., Redman, B., Hussain, M., Cummings, G., Esper, P. S., responsible for the selective uptake and retention of the two Appel, C., and Flaherty, L. E. Phase II evaluation of oral estramustine and oral etoposide in hormone-refractory adenocarcinoma of the pros- cytotoxic metabolites in the prostate tumor (15). This opens the tate. J. Clin. Oncol., 12: 2005-2012, 1994. possibility of using the presence or absence of EMBP as an 4. Hudes, G., Obasaju, C., Chapman, A., Gallo, J., McAleer, C., and indicator for treatment with EaM phosphate. The biological role Greenberg, R. Phase I study of paclitaxel and estramustine: preliminary of this protein in the normal prostate is still unknown, but in activity in hormone refractory prostate cancer. Semin. Oncol., 22 tumor tissue the highest levels of EMBP are found in hormone- (Suppl. 6): 6-11, 1995. refractory prostate cancer after androgen ablation (22). It has 5. Van Poppel, H., De Ridder, D., Elgamal, A. A., Van Dc Voorde, W., also been suggested that the tissue EMBP/dihydrotestosterone Werbrouck, P., Ackaert, P., Oyen, R., Pittomvils, G., Baert, L., and Members of the Belgian Uro-Oncological Study Group. Neoadjuvant ratio might be useful as a marker for predicting disease progres- hormonal therapy before radical prostatectomy decreases the number of sion (28). In the present study, this association with tumor positive surgical margins in stage T2 prostate cancer: interim results of progression is also indicated as higher levels of EMBP were a prospective randomized trial. J. Urol., 154: 429-434, 1995. found in T3 tumors than in T2 tumors. 6. Van Dc Voorde, W., Elgamal, A. A., Van Poppel, H. P., Verbeken, Recently, it has been demonstrated that EaM in vitro in- E. K., Baert, L. V., and Lauweryns, J. M. Morphologic and immuno- hibits the efflux of other cytostatic drugs, possibly via an inter- histochemical changes in prostate cancer after preoperative hormonal therapy: a comparative study of radical prostatectomies. Cancer (Phila.), action with the P-glycoprotein (29-31). The present data dem- 74: 3164-3175, 1994. onstrate that concentrations necessary for achieving this 7. Gunnarsson, P. 0., Plym Forshell, G., Fritjofsson, A., and Norl#{233}n, potentiating effect are present in the tumor tissue. This lends B. J. Plasma concentration of estramustine phosphate and its major credence to the hypothesis that this could be part of the mech- metabolites in patients with prostatic carcinoma treated with different anism behind the clinical results demonstrating synergy between doses ofestramustine phosphate (Estracyt). Scand. J. Urol. Nephrol., 15: EMP and various other cytotoxic agents, such as vinblastine, 201-206, 1981. etoposide, and paclitaxel in the treatment of advanced prostate 8. Hartley-Asp, B. Estramustine induced mitotic arrest in two human prostatic carcinoma cell lines DU 145 and PC-3. Prostate, 5: 93-100, cancer (1-4). 1984. The tumor levels of the estrogens were also higher in the 9. Mareel, M. M., Storme, G. A., Dragonetti, C. H., De Bruyne, G. K., tumor than the serum, which is probably a result of the metab- Hartley-Asp, B., Segers, J. L., and Rabaey, M. L. Anti-invasive activity olism of EoM and EaM in the target organ. It has recently been of estramustine on malignant MO4 mouse cells and DU 145 human demonstrated that 6 weeks of oral EMP neoadjuvant therapy not prostate carcinoma cells in vitro. Cancer Res., 48: 1842-1849, 1988. only caused a decrease in the number of positive margins in 10. Wang, M., Tew, K. D., and Stearns, M. E. Immunofluorescent studies of the anti-microtubule effects of the anti-cancer drug estramus- patients with T2B disease, but that 6 weeks of EMP therapy tine. Anticancer Res., 7: 1165-1172, 1987. produced a higher cytological regression grade than 3 months of 11. Ekl#{246}v,S.,Nilsson, S., Larson, A., Bj#{246}rk,P.,and Hartley-Asp, B. flutamide treatment (5, 6). Thus, it is not unlikely that in these Evidence for a non-estrogenic cytostatic effect of estramustine on hu- hormone-dependent tumors it is both the high levels of estro- man prostatic carcinoma cells in vivo. Prostate, 20: 43-50, 1992. gens and the cytotoxic metabolites found specifically in the 12. Dahll#{246}f,B., Hartley-Asp, B., BillstrOm, A., and Cabral, F. Estra- tumor that participate in the antitumor effect. mustine depolymerizes microtubules by binding to tubulin. Cancer Res., The present study confirms and extends previous studies 53: 4573-4581, 1993. demonstrating that the levels of EaM and EoM are significantly 13. Laing, N., Hartley-Asp, B., Ranganathan, S., and Tew, K. D. 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P H Walz, P Björk, P O Gunnarsson, et al.

Clin Cancer Res 1998;4:2079-2084.

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