[Toxicity and efficacy of intermittent docetaxel chemotherapy for hormone refractory prostate cancer]. / Toxizität und Effektivität wiederholter Docetaxel-therapie des hormonrefraktären Prostatakarzinoms.

BACKGROUND: Until today, docetaxel is the only EMEA and FDA approved active agent in hormone refractory prostate cancer (HRPC). In the absence of other effective and approved drugs we evaluated the toxicity and efficacy of intermittent-docetaxel-chemotherapy in patients whose cancers progressed after successful first-line docetaxel therapy. METHODS: 46, 18, and 5 patients with HRPC received 1, 2, or 3 cycles of docetaxel based chemotherapy. Toxicity, PSA response and general condition were evaluated systematically. SPSS 15.0 was applied for statistic analysis. RESULTS: 26 (56 %) patients achieved a PSA response of > 50 %, another 10 (22 %) patients of up to 50 %; 10 (22 %) patients were progressive under docetaxel. The median overall survival of the whole cohort calculated from the first docetaxel application was 16 (3-60 +) months. Tolerance, toxicity and general condition were crucial for the administration of a second cycle (n = 18); in contrast, age or the degree of the PSA decline in cycle 1 did not seem to be of importance. The -median overall survival of all patients who -received at least two blocks was 35 months; more-over, 13 / 18 patients achieved a biochemical response in cycle 2. Toxicity did not rise significantly. Five patients were given a third docetaxel cycle, three of whom responded. Higher frequencies of -grade 3 / 4 stomatitis, skin toxicity and leukocytopaenia were observed. CONCLUSION: Intermittent docetaxel therapy is well tolerated and shows high response rates in the sec-ond and third sequences of treatment in select-ed HRPC patients who presented with low docetaxel toxicity, good clinical condition and responded to prior docetaxel-based treatment.

Hoosier Oncology Group randomized phase II study of docetaxel, vinorelbine, and estramustine in combination in hormone-refractory prostate cancer with pharmacogenetic survival analysis.

PURPOSE: To determine the safety and efficacy of two docetaxel doublets in hormone-refractory prostate cancer (HRPC) patients and to examine the prognostic role of polymorphisms in host genes important to docetaxel metabolism and transport. EXPERIMENTAL DESIGN: Sixty-four chemotherapy-naive patients with HRPC were randomized to docetaxel and vinorelbine (D, 20 mg/m2 i.v. days 1 and 8; V, 25 mg/m2 i.v. days 1 and 8) or docetaxel and estramustine phosphate (D, 60-70 mg/m2 i.v. day 1; E, 280 mg oral thrice daily days 1-5) administered q21d. Primary end point was clinically significant toxicity. A pharmacogenetic analysis of host genes was done in patients who received at least one cycle of docetaxel therapy. RESULTS: Grade 3/4 toxicity occurred in 15.6% of DV patients and in 28.6% DE patients. Neither arm exceeded the threshold of clinically significant toxicity. In the DV arm, objective response rate was 33%, prostate-specific antigen response rate was 20%, and median survival was 16.2 months. In the DE arm, objective response rate was 67%, prostate-specific antigen response rate was 43%, and median survival was 19.7 months. Pharmacogenetic analyses showed a significant association between survival beyond 15 months and the ABCG2 421 C > A (Q141K) polymorphism compared with the wild-type (C/C) genotype (66% versus 27%; P = 0.05). CONCLUSIONS: DV and DE doublets are active with a tolerable toxicity profile in patients with HRPC; however, efficacy does not seem superior to standard single-agent docetaxel. The ABCG2 421 C > A (Q141K) polymorphism may be an important predictor of response and survival in HRPC patients treated with docetaxel-based chemotherapy.

High dose pulse calcitriol, docetaxel and estramustine for androgen independent prostate cancer: a phase I/II study.

PURPOSE: We determined the safety and preliminary efficacy of the combination of high dose pulse calcitriol (1,25-dihydroxycholecalciferol) with a standard regimen of docetaxel plus estramustine in patients with metastatic androgen independent prostate cancer. MATERIALS AND METHODS: Patients were treated with 60 microg calcitriol orally on day 1, 280 mg estramustine orally 3 times daily on days 1 to 5 and 60 mg/m docetaxel on day 2 (70 mg/m after cycle 1) every 21 days for up to 12 cycles. Patients also received 325 mg aspirin and 1 or 2 mg warfarin orally daily. Regimen safety was assessed in the first 6 patients and a dose de-escalation scheme for calcitriol was planned if dose limiting toxicities were noted during treatment cycle 1 in greater than a third of patients. RESULTS: A total of 24 patients, including 11 who were chemotherapy naïve and 13 who had previously been treated with docetaxel, were evaluable for toxicity and 22 for prostate specific antigen decrease data. The regimen was generally well tolerated. Treatment related grades 3 or greater toxicity seen in more than 1 patient included hypophosphatemia in 16.7% and neutropenia in 12.5%. Four patients had thromboembolic complications. Asymptomatic hypercalcemia was seen in 4 patients, including grades 2 and 1 in 1 and 3, respectively. Six of 11 evaluable, chemotherapy naïve patients (55%) met prostate specific antigen response criteria. One of 11 patients (9%) treated with prior docetaxel met these criteria. CONCLUSIONS: High dose calcitriol may be safely added to docetaxel and estramustine administered on a 21-day schedule.

[Significance of docetaxel in the chemotherapy of hormone-refractory prostate cancer]. / Bedeutung von Docetaxel für die Chemotherapie des hormonrefraktären Prostatakarzinoms.

Docetaxel (Taxotere) is a taxoid derived from the European yew tree, taxus baccata. In 4 phase-II studies docetaxel has important single agent activity with an overall prostate-specific antigen response rate of 42% in hormone refractory prostate cancer. Other phase-II studies suggest that the addition of estramustine to docetaxel results in a higher response rate but also in an increased toxicity. At present Docetaxel with and without estramustine is being evaluated in phase-III studies that will provide definitive information about its role in hormone refractory prostate cancer.

Phase II study of combination taxol and estramustine phosphate in the treatment of recurrent glioblastoma multiforme.

Taxol has activity in the treatment of high grade gliomas but estramustine phosphate (EMP) has not been used in this setting. In vitro data demonstrates that EMP is cytotoxic to glioma cell lines and estramustine binding proteins are expressed by glioma cells. The combination of Taxol and EMP is reported to be active in the treatment of hormone-refractory prostate cancer and in taxane-resistant breast and ovarian cancer. We therefore performed a phase II study to assess the activity and toxicity of this combination in high grade gliomas. Taxol was given at a dose of 225 mg/m2 intravenously over three hours on day 1 and EMP was given at a dose of 900 mg/m2 orally on days 1 through 3. Cycles were repeated every three weeks. Twenty patients with recurrent glioblastoma multiforme (GBM) were enrolled: 11 male, median age 45 years. All patients received anti-epileptic medications and 17 (80%) had received prior chemotherapy. Of 18 evaluable patients, two had partial responses (11) and six had stable disease (33%) for a minimum of eight weeks. Treatment was well tolerated with grade 3 neutropenia occurring in only three patients. There were no other grade 3 or 4 toxicities. The median time to progression for the cohort was only six weeks (range 3-60+ weeks). The median overall survival was 12 weeks (range 3-60+ weeks). In conclusion, the combination of Taxol and EMP is well tolerated and has modest activity in the treatment of recurrent GBM.

The study of gemcitabine in combination with other chemotherapeutic agents as an effective treatment for prostate cancer.

BACKGROUND: Gemcitabine has demonstrated clinical activity against several common cancers. Our studies examine the ability of gemcitabine, both alone and in combination with other chemotherapeutic agents, to inhibit the in vitro and in vivo growth of several prostate cancer cell lines. MATERIALS AND METHODS: Cultures of LNCaP, PC-3 or MLL cells were exposed to either gemcitabine or other appropriate agents for specified amounts of time. Cells were lysed and nuclei counted utilizing a Coulter Counter. For in vivo experiments, animals were injected with 1 x 10(5) MLL cells subcutaneously into the right flank. Animals were treated as indicated for 14 days. Tumors were then excised, weighed and measured. RESULTS: In both human (PC-3 and LNCaP) and rat prostate (MLL) cancer cell lines our studies demonstrated gemcitabine had a strong effect in vitro, with an IC50 of approximately 500 nM in the human lines and 10 nM in MLL cells. In vivo, studies using the Dunning prostate cancer model in Copenhagen rats resulted in a dose response inhibition of tumor growth, with an 80% decrease in tumor size in rats treated with gemcitabine at 10 mg/kg. CONCLUSIONS: Our results demonstrated the potent activity of gemcitabine against prostate cancer in the Dunning rat model and suggest the addition of paclitaxel may not aid in this activity.

Induction of aneuploidy by the antineoplastic drug estramustine in human lymphocytes.

Estramustine (EM) is an antineoplastic drug used in the therapy of human prostatic carcinoma. The aim of our work was to evaluate the potential aneuploidogenic activity of estramustine, by analysing its cytogenetic effects induced in human lymphocytes. To estimate the ability of EM to induce mitotic spindle disturbances, two parameters were used: the presence of c-mitoses (according to the degree of chromatid spreading and contraction) and mitotic index evaluation (increase after exposure indicating the accumulation of mitoses). EM induced c-mitoses and mitotic index increases starting from the 4 microM dose: statistically significant increases were observed up to the highest dose (40 microM). A strong correlation between c-mitoses and mitotic index increase was found. The micronucleus (MN) assay combined with the fluorescence in situ hybridization technique with a pancentromeric DNA probe was also carried out. Compared to the control, EM induced significant MN increases in binucleated lymphocytes at two doses (8-16 microM). Moreover, we found that estramustine induced significant percentages of MN with positive hybridization signal at the same doses, confirming the presence of entire chromosomes in micronuclei. Additional experiments included induction of numerical and structural chromosome aberrations, and evaluation of sister chromatid exchanges (SCE) and satellited (D- and G-group chromosomes) chromosome associations. The results of numerical chromosome aberration analysis indicated that EM was positive in inducing a statistically significant increase in aneuploid cells and/or polyploid cells at all doses tested. On the basis of these observations, EM may be defined as a typical aneuploidy inducer, whereas it was not found to increase the frequency of structural chromosome aberrations and SCE frequency.

Taxol and estramustine-induced modulation of human prostate cancer cell apoptosis via alteration in bcl-xL and bak expression.

bcl-xL is an antiapoptotic protein that shares sequence homology with bcl-2 and seems to convey chemoresistance in many human tumor cell lines. bcl-xL protein is expressed at a 3-fold higher level in PC-3 cells than it is in LNCaP cells. Taxol causes apoptosis in both these cell lines, as measured by the formation of DNA ladders and by the observation of typical cellular morphological changes (chromatin condensation and nuclear fragmentation) after 4', 6-diamidino-2-phenylindole staining. Overexpression of bcl-2 in LNCaP cells did not prevent Taxol-induced apoptosis. Treatment of LNCaP cells with 10 nm Taxol led, after 24 h, to relatively specific and almost total down-regulation of bcl-xL protein in the absence of alteration of bax, bak, or bcl-2 levels. This change was paralleled by a similar decrease in the level of bcl-xL mRNA, as demonstrated by reverse transcription-PCR. The level of glyceraldehyde-3-phosphate dehydrogenase mRNA was not changed. In PC-3 cells, 48 h were required for both maximal bcl-xL protein down-regulation and cellular apoptosis. In contrast, treatment of LNCaP cells with estramustine induced apoptosis, but this was not associated with any change in the intracellular level of bcl-xL or bax protein. Instead, relatively specific 2-fold up-regulation of the proapoptotic protein bak was observed. In PC-3 cells, cellular apoptosis induced by estramustine was bak independent. These results augment our understanding of the importance of bcl-xL in prostate cancer and suggest that appropriate manipulation of cytotoxic chemotherapeutic agents may favorably alter the balance between pro- and antiapoptotic proteins in this tumor.

Estramustine in malignant glioma.

Estramustine, a carbamate ester combining 17 beta-estradiol and nornitrogen mustard, has primarily been employed in the treatment of advanced prostatic carcinoma. However, a significant amount of preclinical investigation has been directed toward estramustine's activity against human malignant glioma. These studies have demonstrated that estramustine has potent antiproliferative effects against malignant glioma both in vitro and in vivo. Similar antimitotic effects also have been demonstrated for other carbamate esters. Estramustine does not impair proliferation of nonneoplastic astrocytes at concentrations that inhibit glioma cells. Although the reasons for this selective activity remain to be determined, it has been shown that malignant gliomas expresses an estramustine-specific binding site, estramustine-binding protein, more than brain tissue. In the clinical situation, an uptake and accumulation of estramustine in human glioma tissue have been demonstrated. Estramustine has been shown to enhance the cytotoxic effects of irradiation in relatively radioresistant glioma cells both in cell culture and in a rat glioma model. Estramustine has been regarded as mainly an anti-mitotic drug but recently other effects such as inhibition of DNA synthesis, induction of apoptosis, and membrane alterations have been shown. This report summarizes the preclinical observations concerning the effects of estramustine and related compounds on human malignant gliomas. These findings form the basis for proposing further laboratory and clinical investigation regarding estramustine and human malignant gliomas.

Estramustine: a novel radiation enhancer in human carcinoma cells.

PURPOSE: Estramustine (EM), an antimicrotubule agent, binds microtubule-associated proteins, causes spindle disassembly, and arrests cells at the late G2/M phase of the cell cycle. Since cells in the G2/M phase are the most radiosensitive and some human cancer cells contain high level of EM-binding protein, experiments were carried out to determine whether radiation sensitization could be obtained in human carcinoma cells. METHODS AND MATERIALS: Cells containing a high level of EM-binding protein such as prostate carcinoma (DU-145), breast carcinoma (MCF-7), and malignant glioma (U-251) were used to demonstrate radiosensitization. Cervical carcinoma (HeLa-S3) and colon carcinoma (HT-29) cells which are not known to contain EM-binding protein were also employed. Cell survival was assayed by the colony forming ability of single plated cells in culture to obtain dose-survival curves. RESULTS: Pretreatment of DU-145, MCF-7, and U-251 cells to a nontoxic concentration (5 microM) of EM for more than one cell cycle time, substantially enhanced the radiation-induced cytotoxicity. The sensitizer enhancement ratio of these cells ranged from 1.35-1.52. The magnitude of the enhancement was dependent on the drug concentration and exposure time. The rate of cell accumulation in G2/M phase, as determined by flow cytometry, increased with longer treatment time in the cell lines which showed radiosensitization. Other antimicrotubule agents such as taxol and vinblastine caused minimal or no radiosensitization at nontoxic concentrations. CONCLUSION: The data provide a radiobiological basis for using EM as a novel radiation enhancer, with the property of tissue selectivity.

Estramustine depolymerizes microtubules by binding to tubulin.

To investigate the mechanism of action of the antineoplastic drug estramustine, we compared its effects on human prostate cancer cells with those of vinblastine. At their respective concentrations that result in 50% inhibition of clonogenic growth, both drugs caused an accumulation of cells blocked at mitosis and similar dose- and time-dependent depolymerization of interphase microtubules. Also, colcemid-resistant and colcemid-hypersensitive Chinese hamster ovary cells with tubulin mutations were collaterally cross-resistant or -sensitive to estramustine. Thus, the cytotoxicity of estramustine is due to its microtubule depolymerization properties. This could be caused by interaction with tubulin and/or with microtubule-associated proteins (MAPs). Previous investigations have shown that high concentrations of estramustine phosphate can inhibit microtubule polymerization in vitro by binding to MAPs. However, estramustine phosphate is the clinical prodrug to estramustine, the intracellular active compound. In this study, we investigated the effects of estramustine on the binding of MAPs to taxol-stabilized microtubules in vivo. In contrast to previous reports, no effect of estramustine on the binding of MAPs to microtubules was found. Furthermore, we found that polymerization of purified tubulin could be inhibited by estramustine in vitro. Taken together, these results demonstrate that estramustine causes depolymerization of microtubules by direct interaction with tubulin.

Early morphological detection of estramustine cytotoxicity measured as alteration in cell size and shape by a new technique of microperifusion.

The present study describes a new microscopic perifusion technique for detecting momentary alterations in cell volume and shape. The method has been applied for evaluating early signs of cytotoxicity following chemotherapeutic treatments. The effects of estramustine phosphate (EMP) have been evaluated. EMP is a complex between oestradiol-17 beta and the alkylating agent nor-nitrogen mustard and has recently demonstrated a marked cytotoxicity against malignant glioma cells. The results showed a concentration-dependent increase in cell size and a concomitant decrease in shape factor following EMP-treatment of glioma cells. These changes correlated with cytotoxicity evaluated as cell proliferation and cell membrane alterations shown by 86Rb fluxes and ultrastructural visible membrane damage. The colon cancer line HT-29 displayed no reactions at all following EMP treatment. It is suggested that acute alterations in cell morphology and shape display a strong correlation to the cytotoxicity of EMP encountered by traditional cell culture systems. The findings are discussed with respect to cell membrane disturbances caused by EMP and its potential role as an early test of cytotoxicity.

Hormone-independent, non-alkylating mechanism of cytotoxicity for estramustine.

Over two decades, experience with estramustine has provided limited data which support an estrogenic mechanism of action and no data which indicate the nitrogen mustard involvement in the cytotoxic properties of the drug. Consideration of the carbamate-ester portion of estramustine supports the pharmacokinetic evidence that estramustine has a long half life since enzymatic hydrolysis of the carbamate is an uncommon event. Using a variety of immunocytochemical and cellular morphology procedures, estramustine per se has been found to express anti-cytoskeletal properties through non-covalent binding to microtubule associated proteins (MAP's). In both fish erythrophores and in dividing human prostatic carcinoma cells, estramustine exerts an antimicrotubule effect at micromolar concentrations. Thus, estramustine possesses unique pharmacology and protein binding specificity. As such, it should not be classified as an alkylating agent. The estrogenic effects, while possibly of relevance to clinical administration, are not the primary mechanism by which the drug exerts cytotoxicity.

Design of cytotoxic steroids for prostate cancer.

Our object was to determine if the aromatic nucleus of estramustine (I) is optimal for binding affinity to prostate cytosolic proteins, and if C3 is the preferred position for the N-mustard carbamate moiety. To this end we have submitted 34 steroids for in vitro assay of binding affinity to total prostate cytosolic proteins. Our structures included aromatic and hydroaromatic steroids containing N-mustard carbamate and other substituents at C3, C6, C11, C16, C17, C20, and C21. Our results show that binding affinity to prostate proteins is optimally present in C3-nitrogen mustard carbamates attached directly to a totally planar aromatic ring as in (IV). Partial deviation from total planarity as in enol-carbamates (V) leads to some loss of binding affinity, which largely disappears in hydroaromatic structures (VI). Thus, our data lead to the Ring A aromatic structure (X) as a basis for the design of steroidal N-mustard carbamates with prostate selectivity. Preliminary in vivo studies using the Dunning R3327AT prostatic adenocarcinoma implanted in the Copenhagen rat generally support our in vitro data.

Dansylated estramustine, a fluorescent probe for studies of estramustine uptake and identification of intracellular targets.

Fluorescence-microscopic studies with dansylated estramustine (DnsEM) has permitted investigation of the mechanism of estramustine (EM) uptake in live human prostatic tumor cells (DU 145). DnsEM appeared to enter cells rapidly at the peripheral cell margins. A progressive increase in fluorescence was observed until the perinuclear material and cytoplasm were labeled brightly and the nucleoplasm was labeled faintly. Light microscopy showed that DnsEM is assimilated first in preexisting vesicles and then in numerous newly created vesicles that accumulate in the cytoplasm and around the nucleus. Colony-forming assays showed EM and DnsEM to be equally cytotoxic to cultured DU 145 cells. Cellular uptake and subsequent manifestation of cytotoxicity are presumably dependent upon these vesicles. However, after uptake of DnsEM, its diffusion into the cytoplasm was observed.

Cytotoxic properties of estramustine unrelated to alkylating and steroid constituents.

Using cultured HeLa S3 cells an ID50 of 2.5 micrograms/ml was found after a twenty-four-hour incubation with estradiol-17 beta- 3N -bis-(2-chloroethyl) carbamate (estramustine). Similar ID90 values were found in two Walker 256 rat carcinoma cell lines which were either sensitive or resistant to nitrogen mustards. Alkaline elution methodology revealed the complete absence of DNA strand breaks or cross-links in cells receiving up to 10 micrograms/ml estramustine for twenty-four hours. Nuclear uptake was 1.34 per cent of the available drug, one third of which was hydrophobically associated with the protein/phospholipid components of the nuclear matrix. In the human prostatic cell lines DU145 and PC3 , estramustine caused a drastic dose-dependent increase in the mitotic index. This increase resulted from an arrest of cells in metaphase, with highly contracted disoriented chromosomes present. Rapid reverse of the arrest on removal of drug resulted in cell death. Neither nor-nitrogen mustard nor estradiol demonstrated antimitotic properties. The lack of macromolecular alkylation together with the observed antimitotic effects predict a mechanism of action for estramustine which is distinct from either of its constituent components.

Estramustine-induced mitotic arrest in two human prostatic carcinoma cell lines DU 145 and PC-3.

In growth proliferation experiments on two human prostatic carcinoma cell lines, DU 145 cells were found to be more sensitive to the cytotoxic effect of estramustine and nor-nitrogen mustard than PC-3 cells. Estramustine was, however, much more cytotoxic in both cell lines than nor-nitrogen mustard. Cytogenetic experiments revealed that estramustine produced a drastic increase of the mitotic index in both these cell lines. This increase could be accounted for by the arrest of cells in their first treatment-metaphase. The arrested metaphases exhibited all the characteristics commonly found for stathmokinetic agents such as colchicine and vinca-analogues. No mitotic arrest was found for nor-nitrogen mustard but chromosomal aberrations were found at toxic concentrations. Estradiol exhibited minimal toxicity and caused no mitotic arrest in these cell lines. The mitotic arrest induced by estramustine was found to be reversible on removal of the drug.