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Cancer Therapy: Clinical |
Authors' Affiliations: 1 Endocrinology and Metabolic Medicine and Sterix Ltd., Faculty of Medicine, Imperial College, St. Mary's Hospital; 2 Cancer Research UK Laboratories, Department of Cancer Medicine, Hammersmith Hospital; 3 Drug Development Office, Cancer Research UK; 4 Statistical Advisory Service, Imperial College, London, United Kingdom; 5 Medicinal Chemistry and Sterix Ltd., Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom; 6 Centre for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom; 7 Reproductive Endocrine Research Laboratory, University of Southern California Keck School of Medicine, Women's and Children's Hospital, Los Angeles, California; and 8 Cancer Research UK Formulation Unit, Department of Pharmaceutical Sciences, University of Strathclyde, Royal College Building, Glasgow, United Kingdom
Requests for reprints: Michael J. Reed, Endocrinology and Metabolic Medicine, Imperial College, St. Mary's Hospital, London W2 1NY, United Kingdom. Phone: 44-20-7886-1738; Fax: 44-20-7886-1790; E-mail: m.reed{at}imperial.ac.uk.
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Abstract |
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Experimental Design: STX64 (667 Coumate), a tricylic coumarin-based sulfamate that irreversibly inhibits STS activity, was selected for entry into the first phase I trial of a STS inhibitor in postmenopausal women with breast cancer. STX64 was administered orally (nine patients at 5 mg and five patients at 20 mg) as an initial dose followed 1 week later by 3 x 2 weekly cycles, with each cycle comprising daily dosing for 5 days followed by 9 days off treatment. Blood and tumor tissue samples were collected for the assessment of STS activity and serum was obtained for steroid hormone measurements before and after treatment.
Results: The median inhibition of STS activity by STX64 was 98% in peripheral blood lymphocytes (PBL) and 99% in breast tumor tissue at the end of the 5-day dosing period. As expected, serum concentrations of estrone, estradiol, androstenediol, and DHEA all decreased significantly from pretreatment levels. Unexpectedly, androstenedione and testosterone concentrations also decreased. Four patients, all of whom had previously progressed on aromatase inhibitors, showed evidence of stable disease for 2.75 to 7 months. The drug was well tolerated with only minor drug-related adverse events recorded.
Conclusion: STX64 is a potent, well-tolerated STS inhibitor. It inhibits STS activity in PBLs and tumor tissues and causes significant decreases in serum concentrations of steroids with estrogenic properties.
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Expression of STS mRNA in malignant breast tissue is much higher than in the normal breast and also considerably higher than aromatase mRNA expression (15). Three studies have now confirmed that expression of STS mRNA in ER-positive breast tumors is an independent prognostic indicator in predicting relapse-free survival, with higher levels of expression being associated with a poor prognosis (16–18). This contrasts with measurements of aromatase mRNA expression in breast tumors, which is of no prognostic value (17). Although STS mRNA expression is detected in most breast tumors, only 75% of tumors from postmenopausal women express aromatase mRNA (18). The higher levels of STS mRNA expression than aromatase mRNA expression suggest that the STS pathway may be more important than the aromatase route for intratumor estrogen synthesis, as previously indicated from activity studies (19). STS is located within malignant breast epithelial cells and STS immunoreactivity is correlated with tumor size (20). Thus, inhibition of STS activity, especially in patients expressing a high level of STS mRNA, could have therapeutic potential.
A further impetus to the development of STS inhibitors was the knowledge that the production of another steroid with potent estrogenic properties, androstenediol, might be blocked by a STS inhibitor. Androstenediol, although an androgen, can bind to the ER and stimulate the in vitro growth of ER-positive breast cancer cells or carcinogen-induced mammary tumors in rodents (21, 22). Most androstenediol in postmenopausal women originates from DHEA after its formation from DHEA sulfate (DHEAS; ref. 23). As there is only one STS, which hydrolyses both aryl (E1S) and alkyl (DHEAS) steroid sulfates, inhibition of STS should lead to a reduction in both E1 and androstenediol production (24).
Evidence to support an important role for DHEAS as a substrate for androstenediol was recently obtained when it was shown that its ability to stimulate the proliferation of MCF-7 (ER+) breast cancer cells could be blocked with an antiestrogen or STS inhibitor, but not an aromatase inhibitor (25). Clinical evidence for DHEAS in having a role in supporting the growth of breast tumors in postmenopausal women has originated from a study in which serum concentrations of this steroid were measured in women receiving third-generation aromatase inhibitor therapy (26). Although estrogen concentrations were below the limit of assay detection in all patients, those progressing while receiving aromatase inhibitor therapy were found to have significantly higher serum DHEAS concentrations than those with stable disease. As aromatase inhibitors do not block androstenediol formation, this finding suggests that its production from DHEAS, while patients are receiving aromatase inhibitor therapy, may have a role in promoting tumor progression.
Research carried out over the last decade has identified a number of potent STS inhibitors most of which have an aryl sulfamate ester as their active pharmacophore (1, 27). STX64 is a tricyclic coumarin sulfamate (Fig. 2A) that was designed as an orally active, nonsteroid-based, irreversible STS inhibitor (28). STX64 causes regression of E1S-stimulated, nitrosomethylurea-induced mammary tumors in ovariectomized rats (29). STX64, in common with other aryl sulfamates, is transported in erythrocytes by binding reversibly to carbonic anhydrase II (30). After oral administration, it undergoes hepatic transit in erythrocytes without undergoing first-pass metabolism. Its oral bioavailability in rats is >90% (31). STX64 had an acceptable toxicologic profile and was selected as the first STS inhibitor to be tested in postmenopausal women with breast cancer to inhibit the production of steroids with estrogenic properties that are formed by the sulfatase pathway.
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Patients and Methods |
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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The primary end points of the study were to assess the dose of STX 64 needed for inhibition of STS activity in PBLs >90% at the cycle 1 day 5 + 8 hours time point (Fig. 2B), and to assess its tolerability profile. The secondary end points included pharmacokinetic analyses (to be reported in full elsewhere), measurement of serum steroid concentrations following STX 64 administration, determination of the dose of STX 64 needed to inhibit intratumoral STS, and establishing if there was any antitumor activity.
Full clinical assessments were done before treatment start, every 2 weeks during treatment, and at the end of the trial. In patients with radiologically assessable disease, radiology was done before treatment start and at the end of the four cycles of treatment. For patients who received extended dosing, radiological assessments were done every 3 months. All radiology was reported according to Response Evaluation Criteria in Solid Tumors (32). Adverse events were recorded daily in diaries and graded using the National Cancer Institute Common Toxicity Criteria Version 2.0.
STS activity and serum steroid concentration measurements. Blood samples were collected for the assessment of STS activity in peripheral blood lymphocytes (PBL) as a biomarker to monitor the extent and duration of STS inhibition, using CPT vacutainers (Becton Dickinson, Franklin Lakes, NJ). Blood was also collected for the measurement of serum steroid concentrations, with samples for assays being collected before treatment, 24 hours after the initial dose, before the start of cycle 1, and 8 hours after the administration of the dose on day 5 of the first cycle (Fig. 2B). These time points for blood sample collection were selected as the optimal times to monitor the effects of STX64 on STS activity and serum steroid concentrations. Blood samples were also taken for pharmacokinetic measurements, with further samples also being taken during cycles 2 and 3 to monitor patient safety.
Core biopsies (2.5-30 mg) from primary or metastatic lesions were also obtained from four patients at the 5 mg dose and three patients at the 20 mg dose before the initial dose and 8 hours after the administration of the first cycle to monitor the extent of inhibition within tumors.
PBLs isolated from 8 mL blood were used to measure STS activity for monitoring the extent and duration of enzyme inhibition. After centrifugation (1,500 x g at 22°C for 30 minutes), isolated PBLs were washed with PBS (5 mL x 2) to yield a final volume of 1.2 mL and stored at –20°C. The STS was solubilized before assaying, using Triton X-100/PBS (0.2%). Aliquots of the solubilized enzyme were used for the measurement of STS activity and also for determination of the protein concentration. STS activity of the solubilized enzyme from PBLs was assayed using a physiologic substrate concentration of [6,7-3H]E1S (2-3 nmol/L, 46-57 Ci/mmol, Perkin-Elmer Life Sciences, Wellesley, MA) over a 20-hour period, using [4-14C] E1 to monitor procedural losses (33). For some patients, STS activity was also measured in core-biopsy samples taken from the primary or metastatic lesions. For this, tissue was transported on solid carbon dioxide and stored at –20°C until assayed. Samples were homogenized in PBS sucrose (0.25 mol/L)/Triton X-100 (0.2%) to yield a supernatant containing solubilized enzyme after centrifugation (Eppendorf, 4,000 rpm, 5 minutes). Aliquots of the supernatant were used for the assay of STS activity as described for PBLs. The assay of STS activity was linear over 2 to 24 hours and 9 to 12 µg protein. Intraassay coefficient of variations were <15%. The Bradford protein assay (34) was used to measure protein concentrations in PBL and tumor preparations, with STS activity results being expressed as fmol E1/µg protein/20 hours.
Steroid concentrations. Concentrations of DHEAS and DHEA were assayed using kits obtained from DSL (Webster, TX) according to the instructions of the manufacturer. Androstenediol concentrations were measured by RIA after organic solvent extraction and Celite column partition chromatography (35). Concentrations of E1, estradiol (E2), androstenedione, testosterone, and E1S were measured using a gas chromatographic/tandem mass spectrometric assay by SFBC Taylor (Princeton, NJ; refs. 36, 37). The limits of quantitation for E1 and E2 were 6 and 2.3 pmol/L, respectively. Intraassay and interassay coefficients of variation for these assays were <15%.
Statistical analysis. Results were analyzed using two statistical software packages: SPSS version 12 (SPSS, Inc., Chicago, IL) and SAS version 8.02 (SAS Institute, Cary, NC). Nonparametric methods of analysis were used to accommodate the skewed distributions and the outlier values in analyte levels found for some patients. Descriptive statistics are reported as medians and interquartile ranges. A two-factor nonparametric approach to longitudinal analysis of changes over time with the effects of dose was used using techniques implemented in SAS macros developed by Brunner et al. (38). The exact two-sided Wilcoxon signed rank test was used for pairwise comparison of time points, whereas the exact two-sided Mann-Whitney U tests were used to compare the effects of two doses at each time point.
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Results |
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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STS inhibition. STS activity was almost completely inhibited in PBLs and tumor tissues by STX64 (Fig. 3A and B). Overall, PBL STS activity was reduced from 4.7 fmol E1/µg protein/20 h (2.0-6.6 fmol E1/µg protein/20 h) to 0.09 fmol E1/µg protein/20 h (0.03-0.17 fmol E1/µg protein/20 h) for 10 patients at the 5 and 20 mg doses for whom it was possible to measure PBL protein concentrations (P = 0.002; Table 2). For all patients, including two whose samples were contaminated with erythrocytes, STS activity was reduced by 98% (95-99%) at the day 5 + 8 hours time point. Thus, almost complete inhibition of PBL STS activity was achieved in all patients. At the 24-hour time point, median inhibition at both dose levels was 96% (94-98%) with some recovery of STS activity in PBLs (20%, 14-49%) occurring after the initial dose before the start of cycle 1 (Fig. 4A and B). For tumor samples, the median inhibition of STS activity at the 5 and 20 mg doses was 99% (P = 0.016) but only 78% inhibition was achieved in one patient at the 5 mg dose level (Fig. 3B). Three patients went on to receive extended dosing: one patient each at the 5 and 20 mg doses received six additional cycles, whereas one at the 20 mg dose had 12 cycles of extended treatment. In these patients, analyses of PBLs for STS activity showed continued reduction by >90% at the end of cycles where this was monitored.
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An important part of the first phase I trial of a STS inhibitor was to assess the effects of this novel therapy on serum E1 and E2 concentrations. As shown in Fig. 4A and B, median concentrations of E1 at the 5 and 20 mg doses decreased significantly by 55% and 42%, respectively, with E2 concentrations at these doses decreasing by 47% and 41%, respectively, at the cycle 1, day 5 + 8 hours time point. Although the precycle 0 concentrations of E1 were similar in patients recruited for the 5 and 20 mg doses, concentrations of E2 were somewhat higher in the group receiving 20 mg (35.0, 16.2-39.7 pmol/L) than for patients receiving 5 mg (11.7, 8.0-19.4 pmol/L). A similar difference in pretreatment E2 concentrations for patients receiving different doses of letrozole has been previously reported (39).
Inhibition of the hydrolysis of DHEAS resulted in significant decreases in the serum concentrations of DHEA (Fig. 4A and B) with the median value reduced by 52%. Unexpectedly, the concentrations of the precursors, androstenedione and testosterone, for E1 and E2 synthesis by aromatase, also decreased significantly by 63% and 46%, respectively. The maximum decrease in androstenedione concentrations observed was 86%. A major impetus for the development of STS inhibitors was the need to block the production of androstenediol in postmenopausal women, which is mainly derived from DHEAS. Serum androstenediol concentrations decreased by up to 90% with the median decrease at both dose levels being 72%. Pretreatment concentrations of DHEAS, androstenedione, and testosterone were higher in patients receiving the 20 mg dose of STX64, for which there is no apparent explanation apart from the lower median age of the patients receiving the 20 mg dose.
Pharmacokinetic analysis. Pharmacokinetic analysis revealed that there was good exposure to the drug as shown by the area under the plasma concentration curve after administration of the 5 mg [7,233 ± 608 (ng/mL).h, mean ± SD, n = 3) and 20 mg [16,323 ± 8,328 (ng/mL).h, mean ± SD, n = 5] doses. The apparent elimination half-lives, expressed as means ± SD after oral administration of the 5 and 20 mg doses, were 31.3 ± 6.0 and 28.5 ± 12.0 hours, respectively, indicating that it will be possible to use a once per day dosing schedule. The ratio of exposure of 667 Coumarin (the main metabolite) to 667 Coumate was very low (<0.026 in terms of ratio of area under the plasma concentration curve).
Clinical response and tolerability. Although the assessment of clinical response was not a primary end point of the study, it was assessed within 4 weeks of the end of cycle 3. Eight of the 14 patients received 4 cycles of treatment (cycles 0-3) and were evaluable for disease response according to Response Evaluation Criteria in Solid Tumors. However, one patient did not have her bone lesions examined, although her target lesions were assessed. Although the patients in this trial had been heavily pretreated with a minimum of first- and second-line therapies (Table 1), some patients, who had all previously received antiestrogen and aromatase inhibitor therapy, had stable disease as follows: Patient 1 had nonmeasurable inflammatory breast cancer that did not change for 6.25 months on STX 64 treatment. This patient had previously had a complete response to formestane and anastrozole and disease stabilization on fulvestrant. Patient 10 had lung metastases and pretracheal and subcarinal lymphadenopathy at study entry. She received six extended cycles of treatment with STX 64 after completing the trial and had stable disease for 6.5 months. She had previously progressed on anastrozole. Patient 13 had a local recurrence of her breast cancer with bone and skin metastases when enrolled on the study. Her disease stabilized for 2.5 months with STX 64 treatment. She previously had progressive disease on anastrozole. Patient 14 had lymph node metastases in the neck and mediastinum and lung nodules, which stabilized for 7 months during the phase I study. She had previously progressed on anastrozole and formestane. However, there was a further patient, number 5, who had a breast mass, lung and liver lesions stable for 2.75 months while on the study. Skeletal lesions were not assessed radiologically in this patient but she did not have worsening bone pain during treatment. This patient had previously had stable disease on exemestane and anastrozole.
The drug seemed to be well tolerated with only a few grade 1 or 2 adverse events recorded that were thought to be drug-related. The most frequent occurring event experienced by all patients was taste disturbance (maximum grade 2), which was thought to be related to the formulation of the drug (lyophilized from DMSO) and generally lasted for less that 12 hours except in one patient for whom it lasted for 8 days. Other adverse events recorded for some patients included fatigue, hot flushes, mood alterations, arthralgia, and headaches, with the maximum grade recorded for these events being 2. Blood and urine were regularly analyzed during the study but did not reveal any biochemical or hematologic changes that were thought to be drug related.
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Discussion |
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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As androstenedione has generally been considered to be secreted directly from the adrenal cortex (41, 42), the finding that levels decreased by up to 86% as a result of inhibitor therapy was unexpected. Although third-generation aromatase inhibitors reduce serum estrogen concentrations by >90%, they do not alter DHEAS or androstenedione levels (43). The reduction in serum androstenedione and testosterone levels resulting from STS inhibition raises important questions as to the origin of androgens in postmenopausal women. It suggests that, in these women, an important, if not the major, source of androstenedione is derived from the peripheral conversion of DHEAS rather than by its direct secretion from the adrenal cortex (see Fig. 1). It is known that in situ formation of E1 from androstenedione makes an important contribution to the estrogen synthesized in most breast tumors (44). It is likely that the reduction in serum androstenedione concentrations available for uptake into tumors and aromatization to E1 will contribute to the reduction in tissue levels of estrogens that should result from this new form of therapy.
STS inhibitors were developed not only to suppress the formation of E1 from E1S but also the formation of androstenediol, which in postmenopausal women is mainly derived from DHEAS. In addition to the suppression of estrogens, STS inhibitor therapy may offer the additional advantage over aromatase inhibitors of reducing androstenediol levels. Androstenediol, although an androgen, has potent estrogenic properties. Although its affinity for the ER is lower than that of E2, it has been suggested that, due to its much higher serum and tissue concentrations, it may be equipotent with E2 in postmenopausal women (45). The reduction of up to 90% in serum androstenediol concentrations indicates that STS inhibition can effectively decrease its production.
Assessment of the clinical effectiveness of the drug, although not a primary end point, revealed that four patients had stable disease according to Response Evaluation Criteria in Solid Tumors. A further patient had evidence of stable disease in target lesions. Importantly, all of the patients showing evidence of stable disease had previously received first- and second-line endocrine therapies with some also receiving chemotherapy. In conclusion, STX64 almost completely blocks STS activity in PBLs and tumor tissue in postmenopausal women with breast cancer. STS inhibition is associated with significant decreases in serum E1, E2, and androstenediol concentrations and, unexpectedly, androstenedione and testosterone. The drug was well tolerated with only minor, drug-related, adverse events noted. The drug has an acceptable pharmacokinetic profile, indicating that it is suitable for daily dosing. Further phase I/II studies are planned to fully evaluate the efficacy of this novel form of endocrine therapy.
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Acknowledgments |
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Footnotes |
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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.
Received 9/13/05; revised 11/21/05; accepted 12/19/05.
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TopAbstractPatients and MethodsResultsDiscussionReferences |
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5-steroids in the ZR-75-1 human breast cancer cell line. Cancer Res 1986;46:4933–7.This article has been cited by other articles:
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P. A. Foster, S. P. Newman, S. K. Chander, C. Stengel, R. Jhalli, L. L.W. Woo, B. V.L. Potter, M. J. Reed, and A. Purohit In vivo Efficacy of STX213, A Second-Generation Steroid Sulfatase Inhibitor, for Hormone-Dependent Breast Cancer Therapy. Clin. Cancer Res., September 15, 2006; 12(18): 5543 - 5549. [Abstract] [Full Text] [PDF]
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Deceased. 