Steven K. White

An Orally Bioavailable Synthetic Analog of an Active Dehydroepiandrosterone Metabolite Reduces Established Disease in Rodent Models of Rheumatoid Arthritis

Dehydroepiandrosterone (DHEA) treatment provides diverse anti-inflammatory benefits in rodent models of diseases, including rheumatoid arthritis (RA), but only limited benefits to patients. In rodents, DHEA is metabolized to (among others) androstene-3β,7β,17β-triol (AET), which retains potent anti-inflammatory activity. 17α-Ethynyl-5-androstene-3β,7β,17β-triol (HE3286) is a novel, metabolically stabilized, orally bioavailable derivative of AET. In the DBA mouse model of collagen-induced arthritis (CIA), once-daily oral treatments (gavage) with HE3286 (40 mg/kg), beginning at onset of disease, significantly decreased disease. Benefit was associated with reduction in joint inflammation, erosion, and synovial proliferation as measured by histological analysis and mRNA of proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-6, IL-1β, and IL-23. Significant benefit was also observed in the CIA model even when treatments were delayed until 7 days after the onset of arthritis. Furthermore, dose-dependent benefit was observed in the DBA mouse model of collagen antibody-induced arthritis, as well as reductions in IL-6 and matrix metalloproteinase-3 mRNA levels in joints at the peak of disease and at the end of the study. HE3286, in contrast to dexamethasone, was not immune-suppressive in several classic animal models of immune function. Instead, HE3286 treatment was associated with reduced nuclear factor-κB activation and in our previous studies, with increased regulatory T cells. We hypothesize that HE3286 may represent a novel, perhaps first-in-class, anti-inflammatory agent and may more fully translate the benefits of DHEA, heretofore largely limited to rodents, into treatments for human diseases, including autoimmune disorders such as RA.

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-3β,7β,16α,17β-tetrol (HE3177) and androst-5-ene-3α,7β,16α,17β-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.

HE3286, an oral synthetic steroid, treats lung inflammation in mice without immune suppression

Background17α-Ethynyl-5-androsten-3β, 7β, 17β-triol (HE3286) is a synthetic derivative of an endogenous steroid androstenetriol (β-AET), a metabolite of the abundant adrenal steroid deyhdroepiandrosterone (DHEA), with broad anti-inflammatory activities. We tested the ability of this novel synthetic steroid with improved pharmacological properties to limit non-productive lung inflammation in rodents and attempted to gauge its immunological impact.Methods and ResultsIn mice, oral treatment with HE3286 (40 mg/kg) significantly (p < 0.05) decreased neutrophil counts and exudate volumes (~50%) in carrageenan-induced pleurisy, and myeloperoxidase in lipopolysaccharide-induced lung injury. HE3286 (40 mg/kg) was not found to be profoundly immune suppressive in any of the classical animal models of immune function, including those used to evaluate antigen specific immune responses in vivo (ovalbumin immunization). When mice treated for two weeks with HE3286 were challenged with K. pneumoniae, nearly identical survival kinetics were observed in vehicle-treated, HE3286-treated and untreated groups.ConclusionsHE3286 represents a novel, first-in-class anti-inflammatory agent that may translate certain benefits of β-AET observed in rodents into treatments for chronic inflammatory pulmonary disease.

HE3286, an orally bioavailable synthetic analogue of an active DHEA metabolite suppresses spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse.

5-Androstene-3β,7β,17β-triol (AET) is a naturally occurring anti-inflammatory adrenal steroid that limits acute and chronic inflammation. HE3286 (17α-ethynyl-5-androstene-3β,7β,17β-triol) is a synthetic derivative of AET with improved pharmaceutical properties and efficacy in some animal models of autoimmunity. Here, daily oral doses of HE3286 led to a suppression of spontaneous autoimmune diabetes in the non-obese diabetic mouse model of type 1 diabetes mellitus when administered either shortly before or after the first incidence of disease onset. Efficacy was associated with reduced insulitis and a suppression of the pathogenic T helper cell type 1 and type 17 phenotypes in peripheral lymphoid organs. These results demonstrate that daily oral treatment with HE3286 administrated relatively late in the destructive autoimmune process led to a suppression of type 1 diabetes mellitus onset and of the pathological inflammatory status, supporting its clinical evaluation in type 1 diabetes mellitus subjects.

16α‐Bromoepiandrosterone (HE2000) limits non‐productive inflammation and stimulates immunity in lungs

16α‐Bromoepiandrosterone (HE2000) is a synthetic steroid that limits non‐productive inflammation, enhances protective immunity and improves survival in clinical studies of patients with human immunodeficiency virus (HIV), malaria and tuberculosis infections. We now show that HE2000 decreased nitric oxide production by lipopolysaccharide (LPS)‐stimulated RAW264·7 cells. Treatment with HE2000 also reduced non‐productive inflammation associated with carrageenan‐induced pleurisy and LPS‐induced lung injury in mice. In the hapten‐carrier reporter antigen popliteal lymph node assay, HE2000 increased absolute numbers of lymphocytes, antigen‐presenting cells, hapten‐specific immunoglobulin (Ig)M antibody‐forming cells and shifted the interferon (IFN)‐γ/interleukin (IL)‐4 balance towards IFN‐γ production. In the cystic fibrosis transmembrane conductance regulator (CFTR−/−) mouse model of acute Pseudomonas aeruginosa infection, treatment with HE2000 consistently reduced bacterial burden in lungs. All HE2000 effects were dose‐dependent. In H1N1 infection in mice, HE2000 was safe but not effective as a monotherapy, as treatment did not effect survival. HE2000 reduced mortality related to excessive inflammation and opportunistic lung infections in animals and patients, and this might extend to those with H1N1 influenza infection.

Molecular targets for 17α-ethynyl-5-androstene-3β,7β,17β-triol, an anti-inflammatory agent derived from the human metabolome.

HE3286, 17α-ethynyl-5-androstene-3β, 7β, 17β-triol, is a novel synthetic compound related to the endogenous sterol 5-androstene-3β, 7β, 17β-triol (β-AET), a metabolite of the abundant adrenal steroid dehydroepiandrosterone (DHEA). HE3286 has shown efficacy in clinical studies in impaired glucose tolerance and type 2 diabetes, and in vivo models of types 1 and 2 diabetes, autoimmunity, and inflammation. Proteomic analysis of solid-phase HE3286-bound bead affinity experiments, using extracts from RAW 264.7 mouse macrophage cells, identified 26 binding partners. Network analysis revealed associations of these HE3286 target proteins with nodes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for type 2 diabetes, insulin, adipokine, and adipocyte signaling. Binding partners included low density lipoprotein receptor-related protein (Lrp1), an endocytic receptor; mitogen activated protein kinases 1 and 3 (Mapk1, Mapk3), protein kinases involved in inflammation signaling pathways; ribosomal protein S6 kinase alpha-3 (Rsp6ka3), an intracellular regulatory protein; sirtuin-2 (Sirt2); and 17β-hydroxysteroid dehydrogenase 1 (Hsd17β4), a sterol metabolizing enzyme.

Differential metabolism of androst-5-ene-3β,17β-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-3β,17β-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.

17α-Alkynyl 3α, 17β-androstanediol non-clinical and clinical pharmacology, pharmacokinetics and metabolism

Summary17α-ethynyl-5α-androstane-3α, 17β-diol (HE3235, Apoptone) is an orally bioavailable synthetic analogue of 3β-androstanediol, that is active in rodent models of prostate and breast cancer, and is in Phase IIa clinical trials for the treatment of early- and late-stage prostate cancer. In preparation for clinical studies, nuclear hormone receptor and P450 interactions for HE3235 and major metabolites were characterized in vitro, and pharmacokinetics and metabolite profiles were studied in rodents, dogs, and monkeys. Four-week safety studies conducted in rats and dogs indicated a substantial margin of safety for clinical use, and no evidence of electrocardiographic or neurological effects, although anorexia, thrombocytopenia, and hypokalemia were identified as potentially dose-limiting toxicities at superpharmacological exposures. Pharmacokinetics and drug metabolism have been studied in prostate cancer patients.

17α-Ethynyl-5α-androstane-3α, 17β-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, 17β-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.