NM101 Phase III study of NE3107 in Alzheimer's disease: rationale, design and therapeutic modulation of neuroinflammation and insulin resistance.

Recently, the roles of inflammation and insulin resistance in neurodegeneration have become better appreciated. NE3107, an oral small molecule, blood-brain permeable anti-inflammatory insulin sensitizer that binds extracellular signal-regulated kinase, has been shown to selectively inhibit inflammation-driven ERK- and NF-κB-stimulated inflammatory mediators, including TNF-α, without inhibiting their homeostatic functions. We describe the rationale and design of NM101, the first randomized, multicenter Phase III clinical study to examine the safety and efficacy of 30 week treatment with NE3107 versus placebo in elderly adults with mild-to-moderate Alzheimer's disease. Patients (316) will be randomized in a 1:1 ratio. The co-primary end points measure cognitive function (ADAS Cog12), and functional and behavioral characteristics (ADCS CGIC). Trial registration number: NCT04669028 (Clinicaltrials.gov).

Lay abstract Diabetes and other inflammatory diseases increase the risk of Alzheimer's disease. Insulin controls energy use in both the body and the brain. NE3107 is an oral pharmaceutical that has been shown to decrease inflammation and to improve insulin function in animals and in human subjects. We have designed a Phase III clinical trial to test the safety and activity of NE3107 in 316 elderly adults with Alzheimer's disease, compared with a placebo capsule. After 30 weeks, assessments will be made to look for benefits in cognition, function and behavior compared with the control group.

A synthetic anti-inflammatory sterol improves insulin sensitivity in insulin-resistant obese impaired glucose tolerance subjects.

OBJECTIVE: To study the activity of HE3286 (17α-ethynylandrost-5-ene-3ß,7ß,17ß-triol), an anti-inflammatory sterol that is active in models of obesity-induced inflammation and insulin resistance in high body mass index (BMI) subjects with impaired glucose tolerance (IGT). DESIGN AND METHODS: HE3286 was explored in high BMI IGT subjects using hyperinsulinemic, euglycemic clamp studies. RESULTS: In insulin-resistant subjects, HE3286 significantly increased day 29 insulin-stimulated glucose disposal and HDL cholesterol, and decreased C-reactive protein (CRP) compared to placebo. For HE3286, change in M value showed a significant negative correlation with baseline M value. Subjects with baseline M value below the median (4.2 mg/kg/min) had significantly lower adiponectin and higher lipopolysaccharide-stimulated peripheral blood mononuclear cell cytokine secretion. After 28 days of HE3286 treatment, adiponectin levels were significantly increased in insulin-resistant (baseline M < 4.2), but not insulin-sensitive (baseline M > 4.2) subjects, compared to placebo. CONCLUSIONS: HE3286 significantly increased the frequency of subjects with increased insulin-stimulated glucose disposal and HDL, and decreased CRP compared to placebo, in insulin-resistant, but not insulin-sensitive subjects. Thus, HE3286 may preferentially benefit insulin-resistant, inflamed, high BMI IGT subjects.

17α-Ethynyl-androst-5-ene-3ß,7ß,17ß-triol (HE3286) Is Neuroprotective and Reduces Motor Impairment and Neuroinflammation in a Murine MPTP Model of Parkinson's Disease.

17α-Ethynyl-androst-5-ene-3ß,7ß,17ß-triol (HE3286) is a synthetic androstenetriol in Phase II clinical development for the treatment of inflammatory diseases. HE3286 was evaluated for blood-brain barrier (BBB) permeability in mice, and efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) murine model of Parkinson's disease (PD). We found that HE3286 freely penetrated the BBB. HE3286 treatment significantly improved motor function compared to vehicle in the rotarod test (mean 58.2 sec versus 90.9 sec, P < 0.0001), and reduced inflammatory mediator gene expression in the brain (inducible nitric oxide synthase, 20%, P = 0.002; tumor necrosis factor α, 40%, P = 0.038, and interleukin-1ß, 33%, P = 0.02) measured by reverse-transcriptase polymerase chain reaction. Brain tissue histopathology and immunohistochemistry showed that HE3286 treatment increased the numbers of tyrosine hydroxylase-positive cells by 17% compared to vehicle (P = 0.003), and decreased the numbers of damaged neurons by 38% relative to vehicle (P = 0.029). L-3,4-dihydroxyphenylalanine (L-DOPA) efficacy was not enhanced by concurrent administration of HE3286. HE3286 administration prior to MPTP did not enhance efficacy. Our data suggest a potential role for HE3286 in PD treatment, and provides incentive for further investigation.

Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein corticosteroids before and after adrenocorticotropic hormone stimulation.

CONTEXT: Although steroid hormones produced by the adrenal gland play critical roles in human physiology, a detailed quantitative analysis of the steroid products has not been reported. The current study uses a single methodology (liquid chromatography-tandem mass spectrometry, LC-MS/MS) to quantify ten corticosteroids in adrenal vein (AV) samples pre- and post-adrenocorticotropic hormone (ACTH) stimulation. DESIGN/METHODS: Three men and six women with a diagnosis of an adrenal aldosterone-producing adenoma (APA) were included in the study. Serum was collected from the iliac vein (IV) and the AV contralateral to the diseased adrenal. Samples were collected, before and after administration of ACTH. LC-MS/MS was then used to quantify serum concentrations of unconjugated corticosteroids and their precursors. RESULTS: Prior to ACTH stimulation, the four most abundant steroids in AV were cortisol (90%), cortisone (4%), corticosterone (3%) and 11-deoxycortisol (0.8%). Post-ACTH administration, cortisol remained the major adrenal product (79%); however, corticosterone became the second most abundantly produced adrenal steroid (11%) followed by pregnenolone (2.5%) and 17α-hydroxypregnenolone (2%). ACTH significantly increased the absolute adrenal output of all ten corticosteroids measured (P < 0.05). The four largest post-ACTH increases were pregnenolone (300-fold), progesterone (199-fold), 17α-hydroxypregnenolone (187-fold) and deoxycorticosterone (82-fold). CONCLUSION: Using LC-MS/MS, we successfully measured 10 corticosteroids in peripheral and AV serum samples under pre- and post-ACTH stimulation. This study demonstrates the primary adrenal steroid products and their response to ACTH.

Studies of the pharmacology of 17α-ethynyl-androst-5-ene-3ß,7ß,17ß-triol, a synthetic anti-inflammatory androstene.

17α-Ethynyl-androst-5ene-3ß, 7ß, 17ß-triol (HE3286) is an orally bioavailable analogue of androst-5-ene-3ß,7ß,17ß-triol, a non-glucocorticoid anti-inflammatory metabolite of the adrenal steroid, dehydroepiandrosterone. The pharmacology of HE3286 was characterized in preparation for clinical trials in type 2 diabetes mellitus and other diseases of inflammation. Interactions with nuclear hormone receptors and P450 enzymes were measured in vitro. Drug metabolism was studied preclinically in mice, rats, dogs, and monkeys. Neurological and cardiopulmonary safety and dose-ranging and chronic toxicity studies were conducted in rats and dogs in accordance with FDA guidelines. Pharmacokinetics and metabolites were measured in Phase I clinical trials. HE3286 was differentially metabolized between species. HE3286 and metabolites did not bind or transactivate steroid binding nuclear hormone receptors or inhibit P450 enzymes. There were no adverse effects in safety pharmacology and canine toxicology studies. Although HE3286 did not elicit systemic toxicity in rats, mild estrogenic effects were observed, but without apparent association to hormonal changes. Safety margins were greater than 20-fold in rats and dogs with respect to the most commonly used clinical dose of 10 mg/day. The terminal half-life in humans was 8 hours in males and 5.5 hours in females. HE3286 is the first derivative of the DHEA metabolome to undergo a comprehensive pharmacological and safety evaluation. The results of these investigations have shown that HE3286 has a low potential for toxicity and possesses pharmacological properties generally suitable for use in human medicine. The favorable profile of HE3286 warrants further exploration of this new class of anti-inflammatory agents.

Phase I and Phase II clinical trials of androst-5-ene-3ß,7ß,17ß-triol.

UNLABELLED: The immune regulating DHEA metabolite, androst-5-ene-3ß,7ß,17ß-triol (ßAET), was evaluated for safety, cholesterol lowering, and vaccine enhancement in phase I and phase II clinical trials. Safety and pharmacokinetics were evaluated in one study of normal subjects that received ßAET or placebo transmucosally (buccal tablets) for 4 days. In a second study ßAET was given by daily subcutaneous injection for 3 days. ßAET was subsequently evaluated in placebo-controlled trials for cholesterol lowering in hyperlipidemic subjects and for potentiation of hepatitis B surface antigen (HBsAg) vaccine in elderly subjects. Adverse events were primarily associated with injection site reactions. Pharmacokinetics indicated that ßAET was rapidly cleared after either route of administration in both normal and elderly subjects. Plasma ßAET concentrations typically declined below the limit of detection within a few hours of administration. ßAET pharmacokinetics was similar in males and females and in normal and elderly subjects. ßAET significantly lowered cholesterol in normal adult, but not in elderly or hyperlipidemic subjects. HBsAg titers were not increased in elderly ßAET treated subjects relative to placebo. CONCLUSIONS: Short-term administration of ßAET is safe in humans. ßAET has a cholesterol lowering effect in healthy humans, but not hyperlipidemics. Exogenous ßAET appeared to be rapidly metabolized, which may be consequential to the lack of pharmacological activity. A longer duration of ßAET treatment with higher doses or chemical derivatives that are resistant to metabolic inactivation are likely necessary to treat human disease. The utility of ßAET in humans may be limited to maintenance of homeostasis in healthy adults.

Pharmacology and immune modulating properties of 5-androstene-3ß,7ß,17ß-triol, a DHEA metabolite in the human metabolome.

Androst-5-ene-3ß,7ß,17ß-triol (ßAET) is an anti-inflammatory metabolite of DHEA that is found naturally in humans, but in rodents only after exogenous DHEA administration. Unlike DHEA, C-7-oxidized DHEA metabolites cannot be metabolized into potent androgens or estrogens, and are not peroxisome proliferators in rodents. The objective of our current studies was to characterize the pharmacology of ßAET to enable clinical trials in humans. The pharmacology of ßAET was characterized by pharmacokinetics, drug metabolism, nuclear hormone receptor interactions, androgenicity, estrogenicity, and systemic toxicity studies. ßAET's acute anti-inflammatory activity and immune modulating characteristics were measured in vitro in RAW264.7 cells and in vivo in murine models with parenteral administration. ßAET was rapidly metabolized and cleared from circulation in mice and monkeys. ßAET was weakly androgenic and estrogenic in immature rodents, but not bound by androgen, estrogen, progesterone, or glucocorticoid nuclear hormone receptors. ßAET did not induce peroxisome proliferation, nor was it systemically toxic or trophic for sex hormone responsive tissues in mature rats and monkeys. ßAET significantly attenuated acute inflammation both in vitro and in vivo, augmented immune responses in adult mice, and reversed immune senescence in aged mice. ßAET may contribute to the anti-inflammatory activity in rodents attributed to DHEA. Unlike DHEA, ßAET's anti-inflammatory activity cannot be ascribed to activation of PPARs, androgen, or estrogen nuclear hormone receptors. Exogenous ßAET is unlikely to produce untoward toxicity or hormonal perturbations in humans.

Differential metabolism of androst-5-ene-3ß,17ß-diol between rats, canines, monkeys and humans.

The potent anti-inflammatory activity of exogenous dehydroepiandrosterone (DHEA) in rodents has not translated to humans. This disparity in pharmacological effects has been attributed to factors such as differences in expression and function of molecular targets and differential metabolism. Hepatocytes from rats, dogs, monkeys, and humans were used to measure species-specific metabolism of a related compound, androst-5-ene-3ß,17ß-diol (5-AED) using reversed-phase radio-HPLC, to explore the metabolic contribution to this interspecies disparity. We found that rat hepatocytes transformed 5-AED predominantly into an array of highly oxidized metabolites. Canine metabolites overlapped with rat, but contained a greater abundance of less hydrophilic species. Monkey and human metabolites were strikingly less hydrophilic, dominated by 5-AED and DHEA conjugates. From the accumulating evidence indicating that the DHEA anti-inflammatory activity may actually reside in its more highly oxidized metabolites, we advance a hypothesis that the virtual absence of these metabolites in humans is central to the failure of exogenous DHEA to produce a potent pharmacological effect in clinical investigations. Accordingly, emulation of its anti-inflammatory activity in humans will require administration of an active native metabolite or a synthetic pharmaceutical derivative.

A potential role for 5-androstene-3ß,7ß,17ß-triol in obesity and metabolic syndrome.

Metabolic syndrome is marked by perturbed glucocorticoid (GC) signaling, systemic inflammation, and altered immune status. Dehydroepiandrosterone (DHEA), a major circulating adrenal steroid and dietary supplement, demonstrates antiobesity, anti-inflammatory, GC-opposing and immune-modulating activity when administered to rodents. However, plasma DHEA levels failed to correlate with metabolic syndrome and oral replacement therapy provided only mild benefits to patients. Androstene-3ß,7ß,17ß-triol (ß-AET) an anti-inflammatory metabolite of DHEA, also exhibits GC-opposing and immune-modulating activity when administered to rodents. We hypothesized a role for ß-AET in obesity. We now report that plasma levels of ß-AET positively correlate with BMI in healthy men and women. Together with previous studies, the observations reported here may suggest a compensatory role for ß-AET in preventing the development of metabolic syndrome. The ß-AET structural core may provide the basis for novel pharmaceuticals to treat this disease.

Novel components of the human metabolome: the identification, characterization and anti-inflammatory activity of two 5-androstene tetrols.

Two natural 5-androstene steroid tetrols, androst-5-ene-3ß,7ß,16α,17ß-tetrol (HE3177) and androst-5-ene-3α,7ß,16α,17ß-tetrol (HE3413), were discovered in human plasma and urine. These compounds had significant aqueous solubility, did not bind or transactivate steroid-binding nuclear hormone receptors, and were not immunosuppressive in murine mixed-lymphocyte studies. Both compounds appear to be metabolic end products, as they were resistant to primary and secondary metabolism. Both were orally bioavailable, and were very well tolerated in a two-week dose-intensive toxicity study in mice. Anti-inflammatory properties were found with exogenous administration of these compounds in rodent disease models of multiple sclerosis, lung injury, chronic prostatitis, and colitis.

17α-ethynyl-5α-androstane-3α, 17ß-diol treatment of MNU-induced mammary cancer in rats.

N-methyl-N-nitrosourea (MNU) induces estrogen-dependent mammary tumors in female Lewis rats. We explored the antineoplastic activity of a synthetic androstane derivative, 17α-ethynyl-5α-androstane-3α, 17ß-diol (HE3235), as a single agent or in combination with docetaxel compared to tamoxifen, anastrazole, and docetaxel monotherapies against MNU-induced mammary tumors in female Lewis rats. Treatment with HE3235 alone rapidly reduced tumor burden, similar in effect to tamoxifen and anastrozole. The combination of HE3235 with docetaxel was more effective than any single agent, although without apparent toxicity. Only HE3235 or HE3235 plus docetaxel continued to suppress tumor growth after cessation of treatment. HE3235 treatment increased immunohistochemical markers of apoptosis and expression of proapoptotic genes and estrogen receptor beta and decreased expression of antiapoptotic genes, androgen receptor, and estrogen receptor alpha. These data warrant clinical investigation of HE3235 for breast cancer treatment.

5-Androstene-3ß,7ß,17ß-triol (ß-AET) slows thermal injury induced osteopenia in mice: relation to aging and osteoporosis.

5-Androstene-3ß,7ß,17ß-triol (ß-AET), an active metabolite of dehydroepiandrosterone (DHEA), reversed glucocorticoid (GC)-induced suppression of IL-6, IL-8 and osteoprotegerin production by human osteoblast-like MG-63 cells and promoted osteoblast differentiation of human mesenchymal stem cells (MSCs). In a murine thermal injury model that includes glucocorticoid-induced osteopenia, ß-AET significantly (p<0.05) preserved bone mineral content, restored whole body bone mineral content and endochondral growth, suggesting reversal of GC-mediated decreases in chondrocyte proliferation, maturation and osteogenesis in the growth plate. In men and women, levels of ß-AET decline with age, consistent with a role for ß-AET relevant to diseases associated with aging. ß-AET, related compounds or synthetic derivatives may be part of effective therapeutic strategies to accelerate tissue regeneration and prevent or treat diseases associated with aging such as osteoporosis.