Discovery and development of sulfatase inhibitors via academia, biotech & mid-size pharma
Authors: Barry Potter, Michael Reed
Journal: Cancer Research (2007)

SY25-01  Breast cancer remains a major cause of death in women in most Western countries. While estrogens have an important role in supporting the development and growth of breast tumors, the highest incidence of breast cancer occurs in postmenopausal women when ovarian production of estrogens has ceased. However, the enzymes required for estrogen synthesis (aromatase, steroid sulfatase, 17β-hydroxysteroid dehydrogenase) are present in normal and malignant breast tissues and contribute to local synthesis. Steroid sulfatase (STS) is a novel therapeutic target for hormone dependent cancer, currently in clinical validation. Aromatase inhibitors, while clinically successful, do not address the large reservoir of conjugated estrone sulfate that can be converted to estrone by steroid sulfatase and on to estradiol to stimulate tumor growth, nor formation of the androgen androstenediol that, although weaker than estradiol, can still stimulate the estrogen receptor and is produced in large quantities in the post-menopausal setting. Both of these pathways represent aromatase-inhibitor-independent pathways of tumor progression with no active agents available. Our aim was to approach this challenge in a novel fashion.  While nucleoside sulfamates, such as the antitrypanosomal antibiotic nucleocidin, have been known for many years and the derivatized carbohydrate sulfamate topiramate is a marketed anticonvulsant, the discovery that an aryl sulfamate ester, as in the steroidal estrone 3-O-sulfamate, irreversibly inhibits steroid sulfatase in a time- and concentration-dependent fashion, with concomitant protein sulfamoylation, was a major advance in validating this enzyme as a novel oncology target. This was preceded by development of reversible inhibitors based upon structural surrogates of the sulfate group and early stage work was sponsored by both 3i Research Exploitation Ltd and IMPEL, the then technology transfer arm of Imperial College London. The unique activity of the aryl sulfamate pharmacophore facilitated the filing of major dominating intellectual property with broad structure per se coverage critical to future success. A licence to a major US company was achieved, to develop the steroidal estrone 3-O-sulfamate as an anticancer agent, but was terminated upon acquisition of this company and the discovery that estrone 3-O-sulfamate is highly estrogenic. Subsequent funding to develop a non-steroidal sulfatase inhibitor for breast cancer came from the UK Cancer Research Campaign (now CRUK). Fortuitously, another licence with a major European pharma company was secured for development of estradiol 3-O-sulfamate for hormone replacement therapy and this provided the seed capital for “series A” funding and incorporation of Sterix Ltd, a spin-out company from the universities of Bath and Imperial College London in 1997, that became fully functional in 1998.  Sterix had the remit to develop the potential of a non-steroidal inhibitor of steroid sulfatase in hormone-dependent breast cancer through a phase I clinical trial, to oversee the development of estradiol 3-O-sulfamate with its partner and to further exploit the potential of the aryl sulfamate pharmacophore. In addition, it supported projects in other oncology and endocrinology areas relating to hydroxysteroid dehydrogenases. Sterix raised £8M of venture capital funding in 2001 and functioned as a semi-virtual company with headquarters and biology/DMPK labs on the Oxford University Science Park with a small cohort of employees, but all discovery science funded through formal agreements with the two universities. This model functioned well for all parties and had a number of advantages that will be outlined. Issues relevant to moving from a functioning early stage company to a VC-funded company will be discussed.  Postmenopausal women with locally advanced or metastatic ER+ breast cancer were recruited to a phase I clinical trial of our “first in class” non-steroidal steroid sulfatase inhibitor STX64funded by Sterix andunder the auspicies of CRUK during 2003-5. Patients for the phase I trial were recruited from Charing Cross Hospital, London and Belfast City Hospital. Due to limited funding available laboratory measurements of STS inhibition in peripheral blood monocytes and tumor tissues were carried out in an academic laboratory. This proved to be a considerable challenge, but with the support of an MD student, a steroid biochemist and a GLP advisor these analyses were successfully carried out to a near GLP standard. The drug was well tolerated with high oral bioavailability and importantly, in this heavily pre-treated group of women (including tamoxifen and aromatase inhibitors), 5/8 evaluable patients showed evidence of stable disease for up to 7 months. While such an approach was acceptable for a preliminary phase I trial, future phase I/II trials will require considerably more support for these analyses to be carried out to an acceptable standard. Results from this trial will be discussed and examples of next generation sulfatase inhibitors and potent dual aromatase-sulfatase inhibitors presented. Further trials on STX64 (BN83495) commenced in early 2007.  The competitive landscape for sulfatase inhibition has developed during the last 15 years with a number of major companies filing patents on both irreversible and reversible sulfatase inhibitors. Most of those in the former “me-too” class have exploited the aryl sulfamate motif and are dominated by Sterix prior art, but none has yet reached a clinical trial and the reversible inhibitors have not been demonstrated to be effective beyond moderate in vitro activity. Particularly interesting is the growth of interest in other therapeutic targets for steroid sulfatase inhibition beyond oncology, ie in dermatology, immunomodulation, and aspects of cognitive function.  Sterix Ltd was acquired by Ipsen in 2004 while the STX64 trial was in progress. Ipsen is a European pharmaceutical group with over 20 products on the market and a worldwide staff of nearly 4,000. With Sterix now as a fully owned subsidiary, Ipsen has continued funding the research teams at Bath and Imperial College to maintain expertise in all major ongoing discovery and development programmes. Issues relevant to the transition from small founder-VC driven biotech to working with a mid-size major pharmaceutical company will be discussed.  As part of studies designed to reduce the estrogenicity of estrone 3-O-sulfamate we explored the chemical substitution of the A ring. This resulted in the discovery of a new class of oncology-relevant steroid sulfamates based upon 2-methoxyestradiol 3-O-sulfamate that were found to have profound effects on mitotic arrest, apoptosis and microtubule assembly in human breast cancer cells and where the sulfamate group had very significant potency- and DMPK-enhancing properties, independent of steroid sulfatase inhibition. To further exploit the aryl sulfamate pharmacophore as a non-transformable structural motif in a hormone-independent setting we have developed the steroidal bis-sulfamate derivatives STX140 and STX243 as attractive anti-cancer agents with, in addition to STS activity, a multi-targeted mechanism of action, exhibiting potent anti-proliferative and anti-angiogenic effects and disruption of microtubules with pro-apoptotic effects; G2M cell cycle arrest, p53 induction, BCL-2 phosphorylation and activation of caspases are all observed in cancer cells treated with these compounds. A full SAR study will be presented.  Steroid sulfamate esters have high oral bioavailability, atypical of estradiol derivatives, due to sequestration into red blood cells and binding to carbonic anhydrase II (CAII). We co-crystallized steroidal bis-sulfamates with CAII and obtained protein X-ray crystal data, demonstrating that an A-ring 2-substituent does not impede drug binding to this carrier, although an unexpected binding mode was observed. Interaction as the mono-anion with CAII supports the idea that the good oral bioavailability and PK properties of STX140 are due to a mechanism whereby erythrocyte drug uptake and reversible interaction with CAII provide a route to avoid first pass liver metabolism and, additionally, undesirable metabolic conjugation is blocked by hydroxyl substitution. The advantages of exploring such aspects in an academic environment will be discussed.  The potential of bis-sulfamoylated estradiol derivatives for clinical development is reinforced by their in vivo activities. In an MDA-MB-231 hormone independent breast cancer xenograft model in athymic female nude mice STX140 (40 mg/kg p.o.) caused regression and prolonged inhibition of tumor growth continuing after cessation of dosing (6/6 animals) and tumor disappearance (1/6 animals), with no observed toxicity. Moreover, in the androgen receptor negative PC-3 prostate cancer xenograft model in athymic male nude mice using STX140 (20mg/kg p.o.) impressive tumor growth inhibition was observed with 2/8 tumors disappearing and, for STX243 (40mg/kg p.o.), with 3/8 tumors disappearing. In the DU145 prostate cancer model at the same doses STX140 reduced tumor growth by 40% after 6 weeks treatment and STX243 by 60%. Studies are directed towards selection of a clinical candidate for proposed entry in 2008.  The aryl sulfamate pharmacophore is a versatile motif for anti-cancer drug design and one that that has withstood a complex development pathway from academic/technology transfer/charity-funded origins through an entrepreneurial phase to formal exploitation by mid-size pharma. Being first in class to originate the fundamental innovative science and developing a supporting intellectual property position of the highest quality are shown to be critical to this survival.