Preclinical and clinical perspectives on the use of estramustine as an antimitotic drug

Pharmac. Ther. Vol. 56, pp. 323-339, 1992 Printed in Great Britain. All rights reserved

0163-7258/92 $15.00 © 1993 Pergamon Press Ltd

Specialist Subject Editor: E. HAMEL

PRECLINICAL A N D CLINICAL PERSPECTIVES ON THE USE OF ESTRAMUSTINE AS AN ANTIMITOTIC D R U G KENNETH D. TEW,*II; JENNYP. GLUSKER,'~BERYL HARTLEY-ASP,~ GARY HUDES§ a n d LISA A. SPEICHER*

Departments of *Pharmacology, t Molecular Structure and §Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, U.S.A. ~Kabi Pharmacia Therapeutics AB, Scheelevagen 22, 22363 Lund, Sweden

Abstract--A variety of cell biological, pharmacological, crystallographic and clinical approaches have indicated that the antimitotic drug estramustine has interesting and unusual properties. Although designed as an alkylating agent, the marked stability of the carbamate linkage to the steroid carrier molecule prevents the formation of alkylating intermediates. The affinity of the parent molecule for microtubule associated proteins and the concomitant antimicrotubule activity have cytotoxic consequences in tumor cells. Both preclinical and clinical studies of estramustine in combination with other antimicrotubule agents have shown that this approach has great potential to achieve therapeutic advantage, especially in disease states such as hormone refractory prostate cancer.

CONTENTS 1. Introduction and Historical Perspectives 2. Pharmacological and Chemical Factors Influencing Reactivity 3. Antimicrotubule Effects 4. Resistance Mechanisms 5. Drug Combination Studies 6. Recent Clinical Trials of Estramustine/Vinblastine in Prostate Cancer 7. Conclusions Acknowledgements References

323 324 326 329 331 334 336 336 337

1. I N T R O D U C T I O N A N D H I S T O R I C A L PERSPECTIVES Sometimes a drug will be synthesized for rational reasons, but its actual mechanism of action is entirely unexpected. Such a drug is estramustine. Synthesized in the mid 1960s (Fex et al., 1967), the drug is an estradiol molecule linked to nor-nitrogen mustard through a carbamate ester group. Conceptually, the estradiol portion of this molecule was designed to facilitate uptake by steroid receptors in malignant cells. The nitrogen mustard alkylating moiety would be released intracellularly to exert cytotoxicity, following cleavage of the bonds shown in Fig. 1. In its proprietary form, Estracyt has a phosphate group at the 17fl position of the steroid D ring. This substituent makes the molecule more water soluble and thus, more suitable for clinical administration. The drug is readily absorbed following oral administration, with relatively rapid dephosphorylation by serum and intracellular phosphatases. A recently described complication, noted in early clinical studies, involves the formation of an insoluble calcium phosphate salt of estramustine. This occurs when ][Corresponding author. Abbreviations--DIC, differential interference contrast; EMR, estramustine resistant clones; GST, glutathione S-transferase; MAPs, microtubule-associated proteins; MDR, multidrug-resistant; PSA, prostate specific antigen. 323

324

K. D. TEW et al. OH

CHs e,H

CH=-CH=\ /~

/ o

FIG. 1. Structure of estramustine. Arrows show bonds that separate steroid from carbamate from mustard groups. the drug is administered with antacids or with a diet high in calcium (such as dairy products). The calcium phosphate salt that is formed is not amenable to intestinal absorption, resulting in a significant reduction in bioavailability and, potentially, a substantial reduction from the presumed administered dose of the drug (Gunnarsson et al., 1990). Such issues have served to cloud the interpretation of early clinical trials with estramustine. Initially it was thought that the steroid moiety of estramustine would facilitate its selective uptake in estrogen-receptor-positive tumors, thereby increasing the concentration of the drug in malignant tissues. Subsequent to uptake, the drug was expected to undergo cleavage by intracellular hydrolytic enzymes, forming its constitutive estradiol and nor-nitrogen moieties. Although release of estrogens does occur after estramustine administration to patients (Anderson et al., 1977; Gunnarsson et al., 1984; Gunnarsson and Forshell, 1984), the drug has been shown to be pharmacologically active in a number of cell lines that lack estrogen receptors (Tew, 1983) and causes growth inhibition of estrogen unresponsive tumors (Muntzing et al., 1979; Petrow and Padilla, 1986). In addition to showing activity in receptor-negative cells, estramustine binding was not inhibited by greater than 1000-fold excess concentrations of estradiol (Tew, 1983). The drug was shown to have no alkylating activity in vitro at concentrations that induced cytotoxicity (Tew, 1983; Tew et al., 1983). Furthermore, an absence of alkylating functionality was also indicated by observations which included: (1) noncovalent binding of the drug; (2) reversible effects of the drug; (3) the sensitivity of alkylating agent resistant cells to estramustine; (4) the absence of direct DNA damage following lethal concentrations of estramustine; (5) the absence of clastogenic potential; (6) the observation that the dose-limiting tissues were atypical of alkylating agent therapy. By 1983 it became apparent that, while estramustine possessed impressive cytotoxic efficacy at micromolar drug concentrations, the cause of such cytotoxicity did not involve either estrogens or alkylation. A clue to the possible mechanism of action of estramustine became evident when it was shown that the drug could cause the accumulation of cells in metaphase (Hartley-Asp, 1984; Tew and Hartley-Asp, 1984). Since that time, it has become apparent that the pharmacological activity of estramustine results from its effects on the cytoskeleton, specifically microtubules, rather than via covalent interactions with proteins and/or nucleic acids (Tew and Hartley-Asp, 1984; Stearns and Tew, 1985, 1988; Wallin et al., 1985; Friden et al., 1987; Wang et al., 1987). As will become apparent in the sections which follow, the precise molecular target for estramustine, microtubule associated proteins, is unique for an antitumor drug and thus firmly places the drug in the category of antimicrotubule drugs. 2. PHARMACOLOGICAL AND CHEMICAL FACTORS INFLUENCING REACTIVITY Over the last three decades, a number of steroid-alkylating agent conjugates have been synthesized and tested for antitumor potential. Drugs such as prednimustine (a prednisolone ester

Estramustine as an antimitotic drug

325

of chlorambucil) provide an example (Hartley-Asp et al., 1986). Unlike estramustine, prednimustine has an ester linkage, which is readily hydrolyzed by serum esterases. The presence of the carbamate group in estramustine creates a substrate with a biological half-life in humans that is greater than 16 hr (Gunnarsson and Forshell, 1984). This stabilization by the carbamate group has produced a drug with an unpredicted and unexpected antimicrotubule mechanism of action. An overview of the structures of many antimitotic drugs shows that an aminocarboxyalkyl group occurs in many of them. By a series of chemical substitution studies, Gupta (1986) was able to show that the antimicrotubule properties of nocodazole and other benzimidazole carbamates were diminished or abolished if the aminocarboxyalkyl group was replaced by another group. Many other antimicrotubule agents have this motif in their structure, including the simplest, isopropyl-Nphenylcarbamate. Furthermore, virtually all antimicrotubule drugs have aliphatic or aromatic ring substituents of varying degrees of complexity (Speicher and Tew, 1992), indicating that binding must involve not only hydrogen bonding but also hydrophobic interactions. This would explain the noncovalent reversible effects of estramustine on the cytoskeletons of cells in culture (Tew and Hartley-Asp, 1984). Substituents that alter the planarity and/or hydrophobicity of the ring structures can influence the binding of the drug to its target protein. The steroid ring of estramustine provides a hydrophobicity that contributes to overall binding. The antimitotic properties of diethylstilbestrol (Parry et al., 1982; Hartley-Asp et al., 1985), although certainly not its primary pharmacological property, also indicate the possible role of the planar hydrophobic aromatic groups of this agent in binding to microtubule structures. With the generalities of these structure-activity relationships in mind, hydrated and anhydrous crystals of estramustine were grown and the crystal and molecular structures of the drug determined by X-ray crystallographic techniques (Punzi et al., 1992). The mustard substituents had little effect on the overall conformation of the steroid nucleus, so that it closely resembled that found in estradiol. One chlorine atom of the mustard group was found to make a close contact (3.13 A) with the nitrogen atom. This favorable C1..... N interaction may be important for the inherent stability of estramustine, either in vitro or in vivo. It is also possible that the chlorine atoms hinder accessibility to the carbamate group by hydrolytic enzymes. A diagrammatic representation of the structural information obtained from the data on two hydrated and anhydrous crystals is shown in Fig. 2. The carbamate group, the three carbon atoms bonded directly to the nitrogen of the mustard group and the C(3) of the steroid all lie in a plane. The steroid ring system is, in each case, approximately perpendicular to this carbamate plane. The carbonyl of the carbamate group points to the ct side of the steroid. The nitrogen atom of the carbamate group is not involved in hydrogen bonding, but the oxygen of the carbonyl is capable of hydrogen bonding to target proteins (Fig. 2). The X-ray diffraction analysis has shown that the carbamate group, with a short C-N bond (Fig. 3) analogous to that found in peptides, has considerable double-bond character, thereby precluding formation of an aziridine ring. Thus,

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FIG. 3. Average geometry of carbamate group in four crystal structures of estramustine and its analogs (lower values) (Punzi et al., 1992), compared with values for a carbamate group obtained by a search of crystal structures containing this group in the Cambridge Structural Database (upper values). (a) Bond distances and (b) interbond angles. because the presence of the carbamate group has converted the mustard nitrogen atom from an amine to an amide, it is not surprising that the compound does not behave as an alkylating agent. The analogy of the aminocarbonyl linkage to a peptide bond is perhaps further emphasized by a novel group of antimitotic drugs, the dolastatins. The most potent of these is dolastatin 10. A tripeptide segment of dolastatin 10 was found to inhibit both tubulin polymerization and G T P hydrolysis but to have no effect upon nucleotide exchange (Bai et al., 1990a). It was also found that the tripeptide fragment failed to compete for vinca alkaloid binding sites on tubulin. This led to the proposal (Bai et al., 1990a) that this site, purportedly on the fl-tubulin subunit, is the same one recognized by another natural product polypeptide, phomopsin A (Lacey et al., 1987). The presence of nine asymmetrical carbon atoms in the four amino acid/complex primary amine molecule is, in this case, important for drug binding. Five of these asymmetric carbon atoms are also found in the dolastatins and the authors have determined that the correct absolute configuration at positions 18 and 19 are required for the most avid binding to tubulin (Bai et al., 1990b). The similarities between dotastatins, other natural product polypeptides, the 'standard' antimicrotubule agents and estramustine imply ring structures of various degrees of hydrophobicity and the presence of simple or modified peptide bonds for biological activity.

3. A N T I M I C R O T U B U L E E F F E C T S Microtubules are proteinaceous polymers formed by the energy-dependent assembly of heterodimer subunits, ~ and/3 tubulins (Darnell et al., 1986). Microtubules are present in all eukaryotic cells and are implicated in a diversity of functions, including cell division, cell motility, secretion and maintenance of cell polarity. Partial or complete loss of microtubule structures after treatment with colchicine or vinca alkaloids effects a dramatic inhibition of these cellular processes (Inoue, 1981). A group of proteins, collectively referred to as microtubule-associated proteins (MAPs), has been described on the basis of their ability to associate physically with microtubules, stimulate microtubule assembly, and/or stabilize microtubules under conditions that would normally lead to disassembly (Kim et al., 1979; Olmsted, 1986). The best characterized MAPs are found in brain tissue and include a group of high M W proteins, MAP1 and MAP2 (330 and 300 kDa, respectively) and a group of proteins of intermediate MW (55-68 kDa), collectively referred to as tau. The MAPs

Estramustine as an antimitotic drug

327

in tumor tissue and cell lines have been less well characterized, but appear to include MAP1 and a group of proteins with MW between 120 kDa and 210 kDa, including MAP4. The expression of these MAPs varies according to cell and tissue type (Olmsted, 1986; Wiche et al., 1986). Estramustine-induced destabilization of the structural and functional aspects of microtubules has been fairly extensively covered in the literature in the last 5-7 years. In vitro experiments showed that estramustine and estramustine phosphate inhibit microtubule assembly (Stearns et al., 1985, Wallin et al., 1985), bind MAP2 and tau (Friden et al., 1987; Stearns and Tew, 1988) and cause the dissociation of MAPI and MAP2 from taxol-stabilized isolated microtubules (Stearns and Tew, 1988). Thus, the cytotoxic effects of estramustine can be interpreted as being mediated by the dissociation of MAPs from microtubules with a subsequent disassembly of the microtubules and a resultant disruption of critical microtubule-dependent cellular processes. Video-enhanced differential interference contrast (DIC) microscopy has recently been used to examine the effects of estramustine on dividing human prostate cancer cell lines (Sheridan et al., 1991; Speicher et al., 1991). The mitotic progression of a number of individual cells was followed and estramustine was found to delay the onset of anaphase, to reduce the anaphase spindle-pole elongation (anaphase B, Table 1) and to delay cytokinesis. The integrity of the spindle apparatus was also disrupted. Infiltration of the spindle area by mitochondria was apparent in drug-treated cells (Fig. 4). Immunolocalization studies, in conjunction with DIC studies on living cells, demonstrated that specific classes of microtubules and specific mitotic stages were differentially sensitive to estramustine. Cells in metaphase, treated with estramustine at concentrations as low as 2.5 /~M, were delayed in entry to anaphase. The mitotic apparatus became reduced in size and chromosomes lost their equatorial alignment on administration of the drug. Despite the fact that estramustine disrupts spindle microtubules, immunofluorescent localization and electron microscopy demonstrated that, on administration of the drug, microtubules are present in bundles and in association with centrioles and kinetochores. Short-term drug treatment with estramustine indicated that these microtubules were resistant to disassembly if they were in the process of mitosis. Long-term drug treatment studies suggested that these classes of microtubules were able to form at the time that cells entered mitosis. It is possible that estramustine, by preventing the assembly of microtubules in cells, effectively increases free tubulin levels sufficiently to allow MAP-independent microtubule assembly in association with microtubule-organizing centers. Alternatively, classes of MAPs that do not bind estramustine may be present in cells, or certain MAPs may be post-translationally modified so that they bind drug less avidly or bind microtubules more strongly. De M e y e t al. (1987), utilizing monoclonal antibodies specific for phosphorylated forms of MAP la and MAP 1b, have provided evidence that these proteins are associated with centrioles, kinetochores and the midbody. In addition, Diaz-Nido et al. (1990) were able to phosphorylate MAPs in vivo and in vitro; this suggests that M A P l a and M A P l b may preferentially bind to assembled microtubules. Thus,

TABLE i. Estramustine Treatment During Anaphase Mean metaphase pole-to-pole distance (/~M) prior to addition of drug* Control 2.5/~M 10 #M 30/~M 60/~M

19.5 _ 19.1 + 19.1 + 18.6 _ 18.9 _

2.19 (N 1.97 (N 1.09 (N 0.94 (N 1.43 (N

= = = = =

7) 5) 8) 7) 8)

Mean anaphase spindle pole separation (/~m)t'++ 28.7 +__2.39 (N 25.0 _ 2.20 (N 22.0 _ 2.10 (N 20.1 _ 2.27 (N 18.7 _+ 3.08 (N

= = = = =

8) 8) 8) 8) 8)

Individual high/low values for anaphase chromosome separation (#m) 32.5/25.3 27.7/21.7 24.1/17.6 24.1/17.6 22.9/14.5

*The means of controls vs each treated population are not significantly different (P > 0.05). tThe means of controls vs each treated population are significantly different (P < 0.05). :~Anaphase spindle pole separation was measured as the distance from the centrosomal side of each separated chromosome set prior to the onset of cytokinesis. Reprinted from Sheridan et al. (1991), with permission of the copyright holder, Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart.

328

K.D. FEw et al.

FIG. 4. Cell treated at 00:21:00 (between (a) and (b)) with 60/~M estramustine after anaphase onset. (A) Metaphase. (B,C) Anaphase; mitochondria in spindle interzone indicated by arrowhead in (C). (D) Cytokinesis; cleavage furrow indicated by arrowhead. Bar = 10 pro. Reprinted from Sheridan et al. (1991), with permission of the copyright holder, Wissenschaftliche Verlagsgesellschafl mbH, Stuttgart. it is possible that MAPI phosphorylation may play a role in the relative insensitivity of the relevant microtubule classes to estramustine. Thus, despite the persistence in the presence of estramustine of microtubules associated with organizing centers, it is clear that the drug inhibits the assembly of microtubules that are critical to the progression to metaphase and anaphase. Apart from effects on subsequent spindle-pole elongation, estramustine treatment in early anaphase was shown by DIC microscopy to prevent stembody (and midbody) formation and to delay cytokinesis. In addition, interzonal microtubules in telophase and cytokinesis were clearly resistant to estramustine depolymerization as shown by immunofluorescence microscopy. These results suggest that mechanisms regulating interzonal microtubule stability in telophase, stembody (and midbody) formation, and, in part, cytokinesis, become insensitive to the effects of estramustine in mid to late anaphase. Interzonal microtubules in telophase are known to be resistant to a variety of microtubule depolymerizing agents (Brinkley and Cartwright, 1975; Salmon e t al., 1976). These findings suggest that there are other proteins which organize interzonal microtubules into stembodies and the midbody and serve to stabilize microtubules against both tubulin depolymerizing agents and estramustine. One of the significant issues concerning the antimitotic properties of estramustine is whether such effects could be demonstrated in t;ivo. The hormone-independent human prostatic carcinoma cell line DU145, implanted subcutaneously in nude mice, was used to demonstrate the existence of antimicrotubule effects in vivo (Eklov e t al,, 1992). Metaphase arrest was found in mice treated with estramustine intraperitoneally, 200 and 400 /~g daily for 2 weeks, 5 days a week. A significant dose-dependent decrease in the number of anaphase figures was found. A 7- to 8-fold increase in

Estramustine as an antimitotic drug

329

TABLE2. Effect o f Estramustine (EM) and Estradurin (ES) on DU 145 Heterotransplants in Nude Mice *

Treatment

Total number Normal Abnormal divisions metaphase, metaphase, Anaphase, Prophase, analyzed % % % %

No treatment EM, 0.2 mg/day EM, 0.4 mg/day ES, 3 mg/day

202 399 495 200

74.3 44.1 47.9 75.0

6.9 46.6t 47. I t 6.5

12.4 6.0t 2.8:~ 13.5

Mitotic index, %

6.4 3.3 2.2 5.0

1.6 3.1 3.6 1.9

*Mitotic index is based on 10,000 cells tChi-square test 95% significant in relation to control. ++Significant in relation to lower dose of EM. Reprinted from Eklov et al. (1992), with permission of the copyright holder, Alan R. Liss, Inc., New York.

the number of abnormal metaphases, i.e. highly contracted and unaligned chromosomes, was found (Table 2). Uptake and retention of 3H-estramustine was found in tumor tissue. No increase in the mitotic index or the number of abnormal metaphases was found in animals treated with polyestradiol phosphate. Overall, estramustine binds with different avidity to a number of different MAPs isolated from different cells and tissues. The binding constants for some of these MAPs have been estimated in the range of 10-15 //M (Stearns and Tew, 1988; Stearns et al., 1988), values not inconsistent with the concentrations required to induce a cytotoxic antimicrotubule effect.

4. R E S I S T A N C E M E C H A N I S M S Since resistance to anticancer drugs causes treatment failure, a consideration of tumor cell adaptations that result in an acquired resistance to estramustine is important. To this end, a series of estramustine-resistant DU145 human prostate carcinoma cell lines were established and characterized (Speicher et al., 1991). Most cell lines resistant to antimicrotubule drugs fall into two categories: (1) efflux mutants (i.e. cells expressing the multidrug-resistant phenotype (MDR)) (Ling, 1975; for review see Schibler and Cabral, 1985) and (2) mutants exhibiting tubulin subunit alterations (Cabral and Barlow, 1989). Frequently, the degree of resistance for the M D R phenotype is m a n y thousand-fold. For estramustine, however, resistance levels did not exceed 4- to 5-fold and

TABLE 3. Cross Resistance o f Estramust&e-resistant DU 145 Cells Toward Antimitotic / M D R Drags

ICm Values (relative degree of resistance) Wild type EMR4 EMR9 EMR 12 Estramustine (#g/mL) Adriamycin (ng/mL) Cytochalasin B (#g/mL) Taxol (ng/mL) Vinblastine (ng/mL)

2.8 (1)

9.2 (3.3)

8.1 (2.9)

8.8 (3.2)

6.9 (1)

8.7 (1.3)

6.1 (0.9)

3.0 (0.4)

t4.0 (1)

5.0 (0.4)

5.5 (0.4)

4.8 (0.3)

2.5 (1)

1.7 (0.7)

2.7 (1.1)

3.1 (1.2)

1.8 (1)

1.6 (0.9)

1.8 (1.0)

1.6 (0.9)

Colony forming assays were performed in continuous exposure to drug. IC50 values were calculated by linear regression analysis of results of at least three experiments performed in triplicate. EMR clones do not exhibit increased resistance to any of the drugs tested; however, an increased sensitivity to cytochalasin B is noted. Reprinted from Speicher et al. (1991), with permission of the copyright holder, Macmillan Journals, Ltd, London.

330

K.D. TEW et al. TABLE4. Estramustine IDso Values Jor M D R Cell Lines Cell line SKOV3 SKVLB* FCL ARN 1" ARN 2 ARN 3

Resistance to: (fold resistance) Wild type Vinblastine (100 X) Wild type Adriamycin (2.5 X) (13 X) (> 100 X)

EM IDs0 (pM) 6.4 6.2 4.3 (l) 2.7 (0.6) 2.9 (0.7) 1.4 (0.3)

SKVLB and ARN cell lines were made resistant to vinblastine and adriamycin, respectively, but demonstrate cross resistance to other anthracyclines and vinca alkaloids (Tapiero et al., 1984; Bradley et al., 1989). Values in parentheses represent fold increase in EM resistance compared to wild type cells. Reprinted from Speicher et al. (1991), with permission of the copyright holder, Macmillan Journals Ltd, London. such levels were attained only after multiple rounds of selection in increasing drug concentrations. A series of collateral resistance and biological characterization assays indicated that estramustine resistance is distinct from the MDR phenotype. Estramustine resistant clones (EMR) exhibit no cross resistance to other antimitotic/MDR agents including adriamycin, cytochalasin B, taxol and vinblastine (Table 3). In addition, two cell lines known to be part of the MDR phenotype, SKVLB (human ovarian cells) and ARN (mouse leukemia cells), do not exhibit increased resistance to estramustine (Table 4). Finally, estramustine resistant clones do not express increased mRNA or protein levels of P-glycoprotein. These results led us to conclude that the estramustine resistant clones do not display the MDR phenotype. Overexpression of glutathione S-transferase (GST) isozymes has been implicated in acquired drug resistance (Wang and Tew, 1985), including the MDR phenotype (Schisselbauer et al., 1989). Thiol-containing molecules, such as glutathione, have been shown to have a critical role in maintaining microtubule structure and organization of the mitotic spindle during cell division (Kimura, 1973; Onefelt, 1983; Tew et al., 1985). Estramustine may be a substrate for GST and has been shown previously to influence GSH levels and GST activity in wild type DUI45 cells (Tew et al., 1986). No changed expression of GST or modified intracellular GSH, however, was apparent in estramustine-resistant cells. Vinblastine and taxol are known to elicit antimicrotubule effects through their direct interaction with tubulin (Bryan, 1971; Schiff et al., 1979). The fact that the EMR clones remain sensitive to both microtubule-stabilizing as well as destabilizing agents suggests that these cells do not express modified tubulin as was previously shown for other cells (Schiff and Horwitz, 1980). Alterations in drug-transport mechanisms have frequently been correlated with the development of resistance. The possibility that EMR clones were either drug uptake or efflux mutants was examined using 3H-estramustine. The ability of estramustine to diffuse across cellular membranes indicates little role for altered mechanisms of uptake (energy-dependent or carrier-mediated, for example) in the development of resistance. This principle is confirmed by experimental data. The rapid incorporation of estramustine into wild type and EMR cells (maximum uptake at 1 hr) is consistent with reported patterns of estramustine uptake in human prostate cancer (1013L) and HeLa $3 cell lines (Kruse and Hartley-Asp, 1989). The maximum estramustine content of two of the resistant clones was only half that of the wild type line. The cellular volume of these clones was, however, also only 50% of the wild type. Thus, altered drug uptake was not responsible for the development of estramustine resistance. Results from drug efflux studies indicated that the EMR clones had altered patterns of estramustine extrusion (Fig. 5). All three resistant clones had much greater initial efflux rate constants than did the wild type cells. The exact mechanisms responsible for the enhanced estramustine efflux from resistant cells remains to be elucidated; however, the P-glycoprotein is not expressed in these cells. A number of possible mechanisms could contribute to this difference in efflux, including increased sequestration of drug into subcellular compartments, or altered quantitative expression of target proteins. Implicit in these data is the principle that reduced intracellular drug will invoke a reduced cytotoxic response.

Estramustine as an antimitotic drug

331

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Time (min) FIG. 5. The course of effluxof 3H-EM by wild type cells is presented as percent maximum drug incorporated at time zero. The efflux rate constants, determined by nonlinear regression analysis, are much greater for EMR clones than wild type cells. Each data point represents the mean of two experiments done in triplicate. Reprinted from Speicher et al. (1991), with permission of the copyright holder, Macmillan Journals Ltd, London. Population doubling times of the EMR lines were not significantly different from those of the wild type. Wild type cells had, however, nearly double the DNA content of EMR clones, suggesting that selection favors cells with reduced DNA content. Microscopic observations of mitotic EMR cells showed that they have a smaller mitotic spindle apparatus. The reduced chromosome number of the EMR clones could well be related to the smaller mitotic spindle and perhaps reflects a propensity for cells with altered spindle components to survive the estramustine challenge. Of interest, MAPs have been shown to stimulate DNA synthesis in vitro, suggesting a role for these proteins in the regulation of DNA replication (Shioda et al., 1989). Cellular DNA is not a target for estramustine (Tew et al., 1983; Hartley-Asp, 1984); however, studies have shown nuclear uptake of the drug (Tew et al., 1983; Hartley-Asp and Kruse, 1986) and that estramustine binds to the nuclear protein matrix. Thus, one may speculate that estramustine binding to nuclear matrix indirectly interferes with DNA synthesis and that resistant cells have adapted through a decreased DNA content with concomitantly fewer chromosomes. The lack of cross resistance demonstrated by the EMR lines has significant therapeutic implications and has been used as the basis for estramustine combinations with other chemotherapeutic agents. Biochemical analysis and discovery of the unique mechanism of action of estramustine serves as the rationale for its use in combination with other antimicrotubule agents. The preclinical data which support this concept are presented in the next section.

5. DRUG COMBINATION STUDIES Estramustine and the vinca alkaloids have distinct, yet complementary, molecular targets, suggesting that combinations of these microtubule inhibitors could produce greater antitumor effects than either agent administered alone. The combination of estramustine and vinblastine has been shown to lead to additive or superadditive anti-invasive activity in vitro (Mareel et al., 1988), supporting this strategy. The introduction of the estramustine/vinblastine combination into clinical trial (see next section) has circumvented extensive mechanistic studies. The question of how to improve the clinical response rates with further combinations led to a preclinical evaluation of the effects of administration of estramustine with taxol (Speicher et al., 1992). The cytotoxic effect was found statistically to be significantly greater than additive for this combination (Fig. 6). This was

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F]G. 6. Combinational effects of EM and taxol on cell survival of wild type (A), EMR4 (B) and EMR9 (C) cell lines. Data are presented as percentage of cell survival as a function of estramustine concentration in either the absence or presence of taxol (WT, 0.5 nM; EMR, 1.0 nM). Combined EM/taxol data are normalized to account for the cell survival inhibitory effect of taxol alone (approximately 20% inhibition of cell survival). Values represent results from at least two experiments done in triplicate + SE. Taxol significantly (* represent P values < 0.05) enhances the cytotoxicity of EM in both WT and EMR cell lines. (O) EM alone; (O) EM plus taxol. Reprinted from Speicher et aL (1992), with permission of the copyright holder, American Association for Cancer Research, Philadelphia.

true both for D U 1 4 5 wild type and resistant cells. When taxol/vinblastine combinations were employed, no enhancement o f the in v i t r o cytotoxicity was evident (Fig. 7), suggesting that the effect could not be generalized to any two antimicrotubule agents. It is, perhaps, counterintuitive to expect a microtubule stabilizing drug, such as taxol, to work synergistically with a destabilizing agent such as estramustine. This apparent conundrum does not

Estramustine as an antimitotic drug

333

take account of the dynamic nature of the microtubule, a structure that undergoes continual turnover (Mitchison and Kirschner, 1984). Therefore, a drug-induced inhibition of subunit turnover through either microtubule disassembly or stabilization would result in a nonfunctioning microtubule network. The greater than additive cytotoxic effects of the estramustine/taxol combination are conceivably related to the different microtubule protein targets of estramustine and taxol (MAPs and tubulin, respectively). This is not the case with vinblastine and taxol, since both drugs interact directly with tubulin. A previous report demonstrated that estramustine phosphate-induced microtubule disassembly was reversed on addition of taxol (Kanje e t al., 1985). By contrast, when isolated microtubules were disassembled with vinblastine, addition of taxol did not reinitiate microtubule assembly (B. Hartley-Asp, unpublished observation).

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MAPs may serve to stabilize and/or protect microtubules when agents that poison these structures are present. As a result, in the presence of estramustine, a drug such as taxol could be more effective than normal in disrupting the microtubule network. In addition, estramustine could remove MAPs from the microtubule and subsequently sterically increase the number of target sites available for taxol binding. The combination of estramustine and taxol also produces significant alterations in the mitotic spindles. Increased micronucleation after drug treatment indicates altered spindle function. These results are similar to those from studies that demonstrate vinca alkaloid induction of multinucleation (Jordan et al., 1991). It has been suggested that disruption of the mitotic spindle could cause reversion to an interphase state without completion of mitosis or cytokinesis. Immunofluorescent studies revealed multiple spindle asters similar, in some cases, to those previously described to occur with taxol treatment alone (Manfredi and Horwitz, 1984; Rowinsky et al., 1988). Cells with tripolar mitotic spindles and three corresponding sets of chromosomes were also observed. Examples of these tripolar spindles were observed at each phase of mitosis as well as cytokinesis. The data suggest that in the presence of EM and taxol, an individual cell is able to form a functional tripolar spindle such that the single cell completes division, producing three distinct daughter cells. The concept that one cell can divide into three is not novel. In fact, in the early part of the century Boveri (1929) proposed this to be the mechanism through which tumor cells arose. The aneuploid chromosome range in wild type cells was 80-100; therefore, one cell could conceivably produce three progeny, each carrying a functional complement of chromosomes. These results are supported by a recent study that demonstrates induction of multipolar mitotic cells by the mitotic inhibitor 2-(2-thenyl)sulfonyl-5-bromopyrimidine and tripolar cells that are able to complete cytokinesis, giving rise to three daughter cells (Holm Juul et al., 1991). One of the more significant aspects of these in vitro studies is that the concentration of taxol necessary to augment the toxic effects of estramustine is 100-fold less than those levels found in the serum of patients treated with taxol (Brown et al., 1991). The toxicity caused by taxol at standard doses may serve to limit its clinical utility; therefore, these data establish an encouraging foundation for evaluation of this combination in human malignancies. This is especially emphasized by the relative success of the clinical results with estramustine and vinblastine, the subject of the next section.

6. R E C E N T C L I N I C A L T R I A L S OF E S T R A M U S T I N E / V I N B L A S T I N E IN PROSTATE CANCER Both estramustine and vinblastine have been used as single agents in the management of prostatic carcinoma (Dexeus et al., 1985). Although vinblastine has had only limited success, estramustine has been more widely used and has realized limited efficacy (Murphy et al., 1983; Benson and Gill, 1986). Notwithstanding the problematic aspects of calcium phosphate precipitation affecting bioavailability of estramustine, each drug individually has shown only modest activity in the disease. Indeed, the majority of standard chemotherapeutic protocols have proved ineffective once androgen ablation therapy has failed and median survival is in the range of 6-9 months (Eisenberger et al., 1985). The preclinical rationale, together with the nonoverlapping host toxicities

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Estramustine as an antimitotic drug TABLE 5. Effects of Estramustine and Vinblastine on Prostate Specific Antigen No. of successive 2 week PSA measurements at given decrease from baseline /> 25 % Decrease 1 2 3 or more

No. patients (%) 28 (77.8) 25 (69.4) 18 (50.0)

~>50% Decrease 1 22 (61.1) 2 15 (41.7) 3 or more 11 (30.6) >/75% Decrease 1 8 (22.2) 2 6 (16.7) 3 or more 4 (11.1) Reprinted from Hudes et al. (1992), with permission of the copyright holder, Grune and Stratton Inc., Orlando.

TABLE 6.

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Toxicity of Estramustine Vinblastine (N = 37)

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Leukopenia 2 10 2 1 39.5 Anemia 2 10 --31.6 Thrombocytopenia . . . . 0 Nausea 15 4 1 -52.6 Edema 9 2 -1 31.5 Fatigue 10 1 1 -31.5 Breast tenderness 10 ---26.3 Paresthesias 3 2 --13.2 Leg cramps 5 ---13.2 Cardiovascular -2 1 1 10.5 Anorexia 4 ---10.5 Indigestion 3 1 --10.5 Constipation 2 1 1 -10.5 Vomiting 2 1 --7.8 Reprinted from Hudes et al. (1992), with permission of the copyright holder, Grune and Stratton Inc., Orlando.

and resistance mechanisms of estramustine and vinblastine, create an encouraging basis for evaluation of this combination in human diseases. An early phase I trial escalating vinblastine to dose-limiting toxicities produced equivocal results (van Belle et al., 1988). The administration schema shown in Fig. 8, in a recently completed phase II trial in hormone-refractory prostate cancer, has produced a 2-fold higher rate of disease stabilization (or partial response) without greater toxicity than would be expected by estramustine alone (Hudes et al., 1992). Although the methodology to judge patient response for treatment of advanced prostate cancer is far from standardized, decreases in serum prostate specific antigen (PSA) over a prolonged time period is generally a good indication of the effectiveness of the treatment. The results shown in Table 5 suggest a high proportion of responses using the PSA criteria. Almost as important, the drug combination was well tolerated by the patients with relatively mild toxicities reported (Table 6). By limiting the vinblastine and escalating the estramustine, hematologic toxicity was minimized. Once again, this serves to emphasize the lack of alkylating activity of estramustine. The overall product limit estimate of survival for patients with partial response by PSA and patients with stable disease or progression is shown in Fig. 9. In this study, the proportion of patients receiving estramustine phosphate and vinblastine that showed a sustained PSA decrease to less than 50% of the pretherapy baseline was twice that found in a recent study of the efficacy of estramustine phosphate alone at a comparable dose in which 15% of patients had this degree of PSA response associated with other evidence of clinical improvement (Yagoda et al., 1991). The published experience with vinblastine in prostate cancer is limited. In one study of vinblastine administered alone as a five day continuous infusion (1.5 m g / m 2 per day every 3-4 weeks), a 21% objective response rate (95% confidence interval, 8.5-33.5%) in 39 patients with hormonerefractory disease was reported (Dexeus et al., 1985). Two other groups have reported similar effects of estramustine phosphate/vinblastine chemotherapy on serum PSA associated with clinical improvement in a substantial proportion of men with progressive, hormone-refractory disease. Seidman et al. (1992), using a comparable schedule and doses of estramustine phosphate and vinblastine, observed sustained 50% or greater decreases in PSA in 13 of 24 (54%) patients with a median response duration of seven months. Two objective partial responses were observed in five patients with bidimensionally measurable disease. A m a t o et al. (1991) administered a higher weekly dose of vinblastine (6 m g / m 2) with a lower dose (420 mg/day) of estramustine phosphate. Eleven of 22 patients with elevated PSA had sustained > 5 0 % decreases from pretreatment measurements. As anticipated, myelosuppression was more

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severe in this study (Amato et al., 1991). The combined PSA response rate for the three published trials (percent of patients with 50% or greater decrease in PSA for a minimum of three biweekly or monthly measurements) for the 83 patients in the estramustine phosphate/vinblastine studies is 42.3% (95% confidence interval, 31.7-52.9%). A total of 19 patients with bidimensionally measurable tumor were included in these three studies; six (31.6%, 95% confidence interval, 10.7-52.7%) achieved partial response in the measurable tumor. Although a phase III trial would be required to determine conclusively if the combination is superior to either single agent, the antitumor activity observed in the three prostate cancer trials suggests that estramustine phosphate/vinblastine is more effective than estramustine phosphate alone. These results prompt further laboratory and clinical studies of antiMAPs and antitubulin agents in combination. Since there are no effective therapies for hormone refractory prostate cancer, these results from different centers are most encouraging. A cooperative group study is planned to further evaluate the activity of estramustine phosphate/vinblastine in hormonerefractory patients.

7. C O N C L U S I O N S The rational synthesis of a drug does not always guarantee a defined mechanism of action. The increasingly important role of antimicrotubule drugs in standard cancer chemotherapy would suggest a greater role for estramustine if the drug were utilized wisely. Clinical successes with single agent therapy are virtually always limited. Thus, the rationale for using combinations of antimicrotubule drugs which have distinct but overlapping targets, noncollateral resistance and nonoverlapping toxicities would appear sound. To this stage, results of this approach appear encouraging. Additional preclinical data may strengthen the arguments in favor of expanding this combinational approach. Whatever the clinical outcome, the prototype structure provided by estramustine may prove valuable as a lead compound for analog synthesis. Moreover, as a tool to dissect the intricacies o f the roles of MAPs in cytoskeletal structure and function, the potential value of estramustine has yet to be realized. Acknowledgements--Supported by NIH grants CA43783 (KDT), CAI0925 (JPG) and a grant from the Helsingborg Research Foundation (KDT).

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