A Bioprecursor Prodrug Approach to Neuroselective Delivery of Estradiol to the Brain

            Estrogen deficiency in the human brain is observed as a rather serious side effect of preventative oophorectomy, the surgical removal of ovaries, which is increasingly performed today in gynecological oncology. The deficiency arises because women who have had bilateral oophorectomy surgeries lose most of their ability to produce the hormones estrogen and progesterone. The sudden inability to produce estrogen initiates what is referred to as ‘surgical menopause,’ which is generally accompanied by an abrupt onset of neurological and psychiatric maladies triggered by the hormonal deficiencies. Those menopausal indications are commonly addressed through equine estrogen-based hormone replacement therapy. Unfortunately, hormone replacement therapy is not desirable for all symptomatic women due to the peripheral side effects and tumor-promoting properties of estrogen. Therefore, the invention and commercialization of a safe and effective treatment of the adverse consequences of estrogen deficiency in the brain, which include depression and impaired sexuality, is a major unmet medical need.

            The development of brain-selective estrogen therapies has been a formidable medical challenge. Recent efforts to generate neuroselective estrogen receptor modulators have been achieved through GLP-1 receptor-mediated cellular targeting and intracellular delivery. This strategy uses a covalently attached peptide carrier, the glucagon-like peptide-1 (GLP-1), that delivers estrogen selectively to specific tissues in order to improve the therapeutic index of estrogen. Tissue specific delivery of estrogen is limited to cells that co-express both estrogen as well as GLP-1 receptors. The (GLP-1)-estrogen conjugates (Figure above, lower panel) that were discovered contain plasma-stable linkages and are reported to exhibit improved sex-independent efficacy over either of the individual hormones alone for the treatment of diabetes and obesity.

            In order to develop an orally bioavailable treatment option for brain-selective estrogen therapy, an alternate small-molecule strategy is desired. A research group led by Laszlo Prokai has initiated preclinical evaluation of a unique synthetic steroid that was designed for the treatment of estrogen-responsive central disorders. His approach involves development of a ‘bioprecursor prodrug’ that undergoes enzymatic bioactivation to 17b-estradiol (E₂) by a reductive process catalyzed by an enzyme that is selectively expressed in the brain. The specific bioprecursor, 10b,17b-dihydroxyestra-1,4-dien-3-one (or DHED), unlike a conventional prodrug, does not contain any auxiliary promoieties that require enzymatic or chemical cleavage. Instead, a short-chain NADPH-dependent reductase (SDR) promotes the reductive bioactivation of DHED through hydride transfer from the coenzyme NADPH (mechanism shown above, top panel) to the C1 position of the A-ring dienone followed by elimination of water to furnish E₂. In vitro metabolism studies using tissue homogenates indicated that DHED was converted to E₂ in the brain, but not in peripheral tissues such as the uterus. In vivo experiments using deuterated-DHED demonstrated that d₃-E₂ was produced exclusively in the brain. Estrogen was not detected in peripheral tissues, nor was DHED detected in the brain. Oral, intravenous and subcutaneous administration of the prodrug to ovariectomized rodents resulted in rapid, brain-selective bioconversion to E₂.

            In order to evaluate DHED in a preclinical model of an estrogen-responsive human CNS disorder, a Forced Swim Test (FST) study was conducted. The Porsolt Forced Swim Test is a widely accepted animal model of the human condition of depression, used to screen for antidepressant-like pharmacological activity. The test is centered on a rodent’s response to the threat of drowning and the result of the test, a quantitation of reduced behavioral immobility, is interpreted as measuring susceptibility to negative mood. In an FST study, animals are subjected to two trials during which they are forced to swim in a glass cylinder filled with water, from which they cannot escape. The second trial is performed 24 hours after the first. The time that the animal spends in the second trial without making movements beyond those required to keep its head above water, referred to as immobility time, is measured and is known to be decreased by antidepressant drugs. The FST is also referred to as the “behavioral despair test.”

Reproduced from: Science Translational Medicine 2015, 7, pp. 297ra113.

            When subcutaneously administered to ovariectomized rodents at identical doses, DHED treatments engendered decreased FST immobility times as compared to direct administration of E₂ (FST data shown above, reproduced from Science Translational Medicine 2015, 7, pp. 297ra113). Importantly, co-injection of a high-affinity estrogen receptor antagonist (ICI 182,780, structure depicted above) blocked the antidepressant-like effect in both treatment groups, suggestive of an estrogen receptor-mediated mechanism of action. The profoundly reduced behavioral immobility induced by DHED in the FST suggests that this unique bioprecursor prodrug holds promise as a potentially safe therapy to alleviate hypoestrogenic depression resulting from surgical menopause. The treatment should be devoid of adverse peripheral side effects associated with the use of systemic estrogens. Moreover, the physicochemical properties of DHED, such as lipophilicity and intrinsic aqueous solubility, are significantly improved relative to the corresponding properties of 17b-estradiol. The attractive biopharmaceutical properties of the small-molecule DHED, in comparison to those of E₂ or estrogen-peptide conjugates, could facilitate applications involving oral administration, which is a much-coveted feature of a drug candidate.

            DHED is a synthetic molecule obtained from chemical oxidation of estrogen derivatives. Upon exposure of an estrogen to an initiator (benzoyl peroxide) and an oxidant (mCPBA) with simultaneous irradiation from a 60-Watt light bulb, the A-ring phenol is converted to its corresponding p-quinol in moderate yield via the radical mechanism depicted in the scheme above. It’s interesting to note that subsequent treatment of quinols related to DHED with a strong Brönsted acid under thermally forcing conditions promotes a fascinating skeletal rearrangement reaction that ultimately yields an A-ring quinone through the intermediacy of its corresponding hydroquinone. Estrane-type A-ring p-quinones have been reported to exhibit moderate cytotoxicity against certain cell lines. It remains to be seen as to whether or not this unique mode of reactivity will preclude the use of DHED in applications involving intestinal drug absorption from the gut, given that gastric pH is, of course, strongly acidic.