Citrus Limonin Glucoside Supplementation Decreases Biomarkers of Disease and Inflammation in Humans

            It is commonly known that fresh fruits should be part of a healthy, balanced diet. Epidemiological studies reinforce this notion, revealing that increased consumption of fresh fruits is associated with reduced risk of diabetes, cardiovascular disease and cancer. Are there specific phytochemicals and biomolecules present in fruits, aside from well-known and well-studied flavonoids and vitamin C, that elicit these demonstrated health benefits in humans? Identification of fruit-derived nutraceutical components could provide access to new dietary supplements for prevention and/or treatment of various chronic diseases.

            Citrus limonoids are highly oxygenated triterpenoids that occur in a variety of citrus tissues in significant quantities (up to 900 ppm in orange juice). Limonoids exist in citrus juice and tissues as water-soluble glucosides. Citrus seeds and peel extract contain water-insoluble limonoid aglycones. Certain limonoid aglycones such as the flagship member of the limonoid natural product family, limonin, are responsible for the development of delayed bitterness in citrus. Limonin was isolated in the 1840’s but its precise chemical structure was not elucidated until 1960, when a collaborative team that included Derek Barton and E. J. Corey solved the structure using chemical derivatization and X-ray diffraction methods.

            A recent report from Darshan Kelley and co-workers at the USDA has demonstrated that consumption of a specific limonoid natural product, limonin glucoside (structure shown above), effectively reduces plasma concentrations of markers for chronic inflammatory diseases in human subjects. The authors examined the effects of limonin glucoside consumption on blood lipids, lipoproteins and liver enzymes. In this study, twelve-ounce drinks containing 250 milligrams of limonin glucoside dissolved in aqueous citrate buffer solution were consumed twice per day by subjects. The drinks were orange-flavored and contained some vitamin C and zero calories. The double blind study was also placebo controlled. Notably, at a dose of 500 mg/day, limonin glucoside had no specific adverse effects and the drinks were well-tolerated. Moreover, the drinks effectively reduced serum concentrations of several hepatic markers that are recognized to be associated with obesity and inflammation. The reduction in markers included gamma-glutamyl transferase (GGT, 34%), alanine aminotransferase (ALT, 13%) and alkaline phosphatase (ALP, 10%). Circulating concentrations of GGT, ALT and ALP are elevated in several human diseases including alcoholic and nonalcoholic fatty liver disease and metabolic syndrome. The authors note that one etiological link among these conditions is increased oxidative stress and inflammation and that future studies are warranted to examine the potential of limonoids to prevent or reverse these diseases.

            In forthcoming posts, we will examine the manner in which the authors of the study executed the deceptively challenging task of sourcing hundreds of grams of a highly complex natural product with purity in excess of 99.93%. We will also compare the USDA’s isolation method (extraction from citrus molasses) with the state-of-the-art in total chemical synthesis. The first total synthesis of racemic limonin was very recently disclosed by Shuji Yamashita’s and Masahiro Hirama’s research groups at Tohoku University (Japan). Limonin provides an outstanding case study by which to compare divergent supply chain approaches to sourcing structurally complex API’s.

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.