Ablation of the ATP-binding cassette transporter, Abca2 modifies response to estrogen-based therapies.

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12×100 mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100 mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism.

Simultaneous determination of estramustine phosphate and its four metabolites in human plasma by liquid chromatography-ionspray mass spectrometry.

A sensitive and selective method, using liquid chromatography-ionspray mass spectrometry, was developed and validated for the simultaneous determination of Estracyt (estramustine phosphate) and its four metabolites, estramustine, estromustine, estrone and estradiol, in human plasma. Deuterated internal standards were available for all analytes. The five compounds were extracted from plasma by protein precipitation with acetonitrile. The chromatographic separation was performed using a Zorbax SB C18, (150 x 4.6 mm i.d., 5 microm) reversed-phase column under gradient conditions with a mobile phase containing 2 mm ammonium acetate buffer (pH 6.8) and acetonitrile. MS detection was by electrospray ionization with multiple reaction monitoring in the positive ion mode for estramustine phosphate, estromustine and estramustine, and in the negative ion mode for estrone and estradiol. The limit of quantitation was 10 ng/mL for estramustine phosphate, 3 ng/mL for estromustine, estramustine and estrone and 30 ng/mL for estradiol. Linearity was verified from these LLOQs up to about 4000 ng/mL for the parent drug and 2000 ng/mL for the metabolites. Inter-day precision and accuracy values were all less than 15%. This assay was applied successfully to the routine analysis of human plasma samples collected in cancer patients administered estramustine phosphate intravenously.

Dose escalation study of intravenous estramustine phosphate in combination with Paclitaxel and Carboplatin in patients with advanced prostate cancer.

PURPOSE: The purpose is to determine a safe weekly dose of i.v. estramustine phosphate (EMP) to combine with weekly paclitaxel and monthly carboplatin in patients with advanced prostate cancer. EXPERIMENTAL DESIGN: Patients with advanced prostate cancer (castrate and noncastrate) were administered escalating doses of weekly 1-h infusion of i.v. EMP (500-1000-1500 mg/m(2)) in combination with weekly paclitaxel (100 mg/m(2) over 1 h) and i.v. carboplatin (area under the curve 6 mg/ml-min every 4 weeks). Four weeks of therapy were considered one cycle. In the first three cohorts, EMP was given i.v. 3 h before paclitaxel. Cohorts 4 and 5 reversed the administration order: EMP (doses 1000-1500 mg/m(2)) was given immediately after the end of paclitaxel infusion. Plasma levels of EMP and its metabolites, estramustine and estromustine, were monitored at time 0, at 120 min, and approximately at 20, 21, and 168 h from the start of EMP infusion. Paclitaxel concentrations were determined at basal (0), 30, 60, 90, and 120 min and 18 h after the start of paclitaxel infusion, and a concentration-time curve was estimated. Pharmacokinetic evaluation was performed in cycles 1 and 2 during the first week of therapy. RESULTS: Nineteen patients were entered on the initial three dose levels (cohorts 1-3). Dose-limiting transient hepatic toxicity was encountered in cohort 3 (EMP = 1500 mg/m(2)). An additional 13 patients were treated with paclitaxel (100 mg/m(2)) first, followed by i.v. EMP at 1000 mg/m(2) (cohort 4), and 1500 mg/m(2) (cohort 5). No dose-limiting toxicities were seen, and cohort 5 was determined safe for Phase II studies. Thromboembolic events were observed in 9% of patients (no prophylactic coumadin was used). Plasma concentrations of EMP and metabolites increased proportionally with dose. In all cohorts, there was a slight decrease in EMP and estramustine plasma concentrations between cycles 1 and 2. Although not significant, higher levels of estromustine at cycle 2 were observed in comparison to cycle 1. Decreased clearance of paclitaxel leading to higher than expected paclitaxel plasma concentrations was observed during the first cycle of therapy. Paclitaxel plasma concentrations were lower during cycle 2. In 17 patients with androgen-independent disease, 59% had >/=50% posttherapy decline in PSA and 22% showed measurable disease regression. CONCLUSIONS: The regimen of weekly i.v. EMP in combination with paclitaxel and carboplatin can be safely administered with hepatic toxicity being transient and reversible. Pharmacokinetic results suggest that EMP competitively inhibits the biotransformation of paclitaxel after the first administration. This effect is counterbalanced, after repeated administrations, by a possible induction of the metabolic system caused by EMP. Phase II testing is ongoing to evaluate the efficacy of this combination.

Determination of estramustine phosphate and its metabolites estromustine, estramustine, estrone and estradiol in human plasma by liquid chromatography with fluorescence detection and gas chromatography with nitrogen-phosphorus and mass spectrometric detection.

Bioanalytical methods for the determination of estramustine phosphate by liquid chromatography and its four main metabolites estromustine, estramustine, estrone and estradiol by gas chromatography are described. For the estramustine phosphate assay the plasma was purified by protein precipitation followed by a C18 solid-phase extraction. For the metabolite assay the plasma samples were purified by a C18 solid-phase and liquid-liquid extraction procedure and derivatised by silanization. Thereafter, estramustine and estromustine were quantified by gas chromatography with nitrogen-phosphorus detection and estradiol and estrone were quantified by gas chromatography with selected ion monitoring. The methods were validated with respect to linearity, selectivity, precision, accuracy, limit of quantitation, limit of detection, recovery and stability. The limit of quantitation was 2.3 micromol/l for estramustine phosphate, 30 nmol/l for estromustine and estramustine, 12 nmol/l for estrone and 8 nmol/l for estradiol. The results showed good precision and accuracy for estramustine phosphate and the four metabolites. The intermediate precision was 6.2-13.5% (C.V.) and the accuracy was 91.8-103.9%.

Differential uptake of estramustine phosphate metabolites and its correlation with the levels of estramustine binding protein in prostate tumor tissue.

Estracyt (EMP) has been used for the treatment of hormone refractory prostate cancer for many years. Recently, new data from combination studies have given rise to new interest in this old drug. Explanations for the synergy found in the clinic are many, but one major factor may be the previous indication that the drug accumulates in the prostate tumor. We have, therefore, examined the level of the four metabolites, estromustine (EoM), estramustine (EaM), estrone, and estradiol in the tumor and serum of 14 patients with T2 and T3 prostate cancer receiving a single i.v. dose of 600 mg of EMP, about 12 h before radical prostatectomy. Because it has been suggested that the uptake into the prostate tumor is due to binding to the estramustine binding protein (EMBP), we have in addition measured the level of EMBP in the prostate tumor tissue. The main serum and tissue metabolite in all patients was EoM followed by EaM, estrone, and estradiol. The levels for EoM ranged from 63.8-162.8 ng/ml in the serum and from 64.8-1209 ng/ml in the prostate tumor, resulting in a mean ratio for serum to tumor of 1:5. The levels for EaM ranged from 8.3-51.4 ng/ml in the serum and 73.9-563.4 ng/ml in the tumor, giving a mean ratio for serum to tumor of 1:13. The levels of EMBP were higher in T3 tumors than in T2 tumors, 54.1 and 40.7 ng/g tissue, respectively. A significant correlation was found between the levels of EaM (r = 0.60) and the levels of EMBP in the tumor. These data demonstrate that 12 h after a single i.v. dose of 600 mg of EMP the levels of the cytotoxic metabolites EoM and EaM are substantially higher in the tumor than in the serum of the same patient and that a correlation exists between the levels of EaM in the tumor and the levels of EMBP. Thus, this supports the hypothesis that the EMBP is responsible for the retention of EoM and EaM in the prostate tumor.

Uptake and retention of estramustine and the presence of estramustine binding protein in malignant brain tumours in humans.

Estraumustine phosphate (EMP), a cytotoxic drug used in the treatment of prostatic carcinoma, has been shown to exert cytotoxic effects on glioma cells in vitro. The drug uptake is assumed to depend on a specific estramustine binding protein (EMBP). One of the main difficulties in achieving cytotoxic effect in malignant brain tumours is believed to be due to the poor penetration of cytotoxic drugs into tumour tissue. In patients with malignant supratentorial brain tumours we have analysed the uptake of EMP metabolites in tumour tissue after oral administration and demonstrated EMBP in the same tissue specimens. Sixteen patients were given 280 mg EMP orally 14 h prior to surgery. Specimens from brain tumour tissue, cystic fluid, and serum were collected during surgery. Using gas chromatography the metabolites of EMP, estramustine (EaM) and estromustine (EoM), were quantified, EMBP was demonstrated by immunohistochemistry. The mean concentrations of EaM and EoM, expressed in ng g-1, were 60.3 and 38.4 in tumour tissue and 3.5 and 56.3 in serum, respectively. An accumulation of EaM in tumour tissue was found with a mean concentration gradient of 16.1 versus serum, while the gradient for EoM was 0.76. EMBP was demonstrated with a high degree of staining in all but one tumour. The high concentrations of EaM and EoM found in malignant brain tumour tissue correspond to potentially cytotoxic levels. The present results as well as the earlier in vitro demonstrated cytotoxic effects on glioma cells strengthen the possibility of a therapeutic effect of EMP in the treatment of malignant brain tumours.

Uptake and metabolism of estramustine in the Dunning R3327H tumour.

A time-dependent plasma, tumour cell and nuclear uptake was found after intraperitoneal 3H-estramustine administration, in rats bearing the Dunning H tumour. Uptake of the drug into tumour cells had not reached a maximum after 4 h, whereas no real change was seen in the nuclear fraction after 2 h. Binding to the nuclear protein matrix of Dunning H tumour cells of 3H-estramustine was also found (2.46 pmol/g protein). High performance liquid chromatography demonstrated the existence of estramustine, its oxidized metabolite estromustine, estradiol and estrone in the plasma and the tumour after intravenous administration of 100 microCi 3H-estramustine.

The hydrolysis of estramustine phosphate; in vitro studies.

The hydrolysis of estramustine phosphate by enzymes of blood, liver, intestines and prostate of man, dog and rat was studied by using specific analytical methods for the parent compound and some of its metabolites. Estramustine phosphate was stable in blood and plasma of different species but rapidly dephosphorylated to estramustine by liver, intestinal and prostatic enzymes. The prostate was the most active tissue. The ester bond by which nornitrogen mustard is linked to estradiol-17-phosphate was slowly cleaved by enzymes from the liver and prostate of the rat, dog and man. The dog tissues were more effective than those of the rat and man. Intestinal enzymes also exhibited the ability to hydrolyze the carbamic ester, but to a greater extent in the rat than in the dog. It is concluded that, with regard to the metabolism of estramustine, the rat is more similar to man than is the dog. Possible clinical implications of the biotransformation of estramustine phosphate are discussed.

Plasma concentrations of estramustine phosphate and its major metabolites in patients with prostatic carcinoma treated with different doses of estramustine phosphate (Estracyt).

Plasma concentrations of estramustine phosphate and its major metabolites were measured in patients with prostatic carcinoma treated with increasing oral doses, 70-650 mg/day, of estramustine phosphate (Estracyt). Parent drug and estradiol were measured by radioimmunoassay, and estramustine and its estrone analogue (Leo 271) utilizing gas chromatography. The concentrations of estramustine phosphate and estramustine were below or close to the limits of the methods. A linear correlation was found between the daily dose of estramustine phosphate, and plasma concentrations of Leo 271-the main metabolite-and estradiol, which suggests that no capacity limiting processes are involved in the pharmacokinetics of estramustine phosphate in man. Plasma was also analyzed during prolonged treatment with 560 mg/day. The metabolite pattern was not changed by two to three years of estramustine phosphate treatment.

Determination of estramustine and its 17-keto metabolite in plasma by high-performance liquid chromatography.

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of estramustine and its 17-keto metabolite in plasma. The assay involves extraction of the compounds into hexane from plasma buffered to pH 9.0, the residue obtained by evaporation of the hexane extract is dissolved in the mobile phase hexane-ethanol (92.5:7.5) with HPLC analysis performed on a 5-micrograms silica gel column using a fluorescence detector with excitation at 195 nm and emission at wavelengths greater than 250 nm. The overall recoveries and limits of sensitivity for estramustine and the 17-keto metabolite are 74.7% and 40 ng/ml of plasma and 85.1% and 50 ng/ml of plasma, respectively. The method was used to obtain plasma concentration-time profiles in three subjects with prostatic cancer following oral administration of a single 7 mg/kg dose of estramustine phosphate.

Estramustine phosphate: plasma concentrations of its metabolites following oral administration to man, rat and dog.

Estramustine phosphate, a nitrogen mustard derivative of estradiol used for the treatment of advanced prostatic cancer, was administered orally to man, rat and dog and the plasma concentrations of its unconjugated metabolites were determined by high performance liquid chromatography. In all species the drug was rapidly and completely dephosphorylated prior to reaching the peripheral circulation. In man and the rat, the 17-keto analogue of estramustine (estromustine) was the mamor metabolite found in plasma with considerably lesser amounts of estramustine itself. In the dog, significant concentrations of both estramustine and estromustine were present. Highly elevated levels of both estrone and estradiol, as a result of cleavage of the nitrogen mustard from the steroid, were observed in all species. Chronic administration of therapeutic doses of estramustine phosphate to man did not result in any apparent accumulation of circulating metabolites. The study suggests that the metabolite, estromustine, in addition to estramustine, may play an important role in the therapeutic efficacy of estramustine phosphate.