Prolactin (PRL) is a protein hormone which in humans is secreted by pituitary lactotrophs as well as by many normal and malignant non-pituitary sites. Many lines of evidence demonstrate that both circulating and locally produced PRL increase breast cancer (BC) growth and metastases and confer chemoresistance. Our objective was to identify and then characterize small molecules that block the tumorigenic actions of PRL in BC. We employed three cell-based assays in high throughput screening (HTS) of 51,000 small molecules and identified two small molecule inhibitors (SMIs), named SMI-1 and SMI-6. Both compounds bound to the extracellular domain (ECD) of the PRL receptor (PRLR) at 1–3 micromolar affinity and abrogated PRL-induced breast cancer cell (BCC) invasion and malignant lymphocyte proliferation. SMI-6 effectively reduced the viability of multiple BCC types, had much lower activity against various non-malignant cells, displayed high selectivity, and showed no apparent in vitro or in vivo toxicity. In athymic nude mice, SMI-6 rapidly and dramatically suppressed the growth of PRL-expressing BC xenografts. This report represents a pre-clinical phase of developing novel anti-cancer agents with the potential to become effective therapeutics in breast cancer patients.

Background:Sirtuins are a class of proteins with important physiologic roles in metabolism and inflammation. Sirtuin (silent mating type information regulation 2 homolog) 1, or SIRT1, activation is an unexplored therapeutic approach for the treatment of ulcerative colitis (UC).Methods:Patients with mild to moderately active UC were blindly randomized to 50 mg or 500 mg daily of SRT2104, a selective activator of SIRT1, for 8 weeks. Colonic exposure and safety were assessed, as well as blinded endoscopic scoring and disease activity by Mayo score, Simple Clinical Colitis Activity Index and fecal calprotectin.Results:Across both SRT2104 groups, only 3 of 26 evaluable subjects achieved remission on blinded endoscopic assessment. Clinical remission (Mayo score <= 2, no subscore >1) was achieved in 4 patients (2 of 13 evaluable patients in each dose group). Fecal calprotectin levels declined with treatment in both groups, but after 56 days of treatment subjects were still found to have levels approximately 4-fold elevated above normal. One subject experienced an SAE requiring study withdrawal and another was withdrawn for a severe UC flare; 19 subjects (61%) across both treatment groups experienced at least 1 treatment emergent adverse event. Average drug exposure increased in a dose-dependent manner for escalating doses of SRT2104, and colonic exposure was 140 to 160 times higher than plasma exposures.Conclusions:SRT2104 did not demonstrate significant clinical activity in mild to moderately active UC. This suggests that further evaluation of SRT2104 as a therapeutic strategy for the treatment of UC is not warranted.

We previously showed that an HLA-DR variant containing arginine at position 74 of the DRβ1 chain (DRβ1-Arg74) is the specific HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides that bound to DRβ1-Arg74. We hypothesized that blocking the binding of these peptides to DRβ1-Arg74 could block the continuous T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was to identify small molecules that can block T-cell activation by Tg peptides presented within DRβ1-Arg74 pockets. We screened a large and diverse library of compounds and identified one compound, cepharanthine that was able to block peptide binding to DRβ1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT) in NOD mice expressing human DRβ1-Arg74. Furthermore, cepharanthine blocked T-cell activation by thyroglobulin peptides, in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed cepharanthine could potentially have a role in treating human AITD.

Aim SRT2104 is a novel, first-in-class, highly selective small molecule activator of the NAD + dependent deacetylase SIRT1. SRT2104 was dosed to healthy male and female volunteers in a series of phase 1 clinical studies that were designed to elucidate tolerability and pharmacokinetics associated with oral dosing to aid in dose selection for subsequent clinical trials. Methods In the first-in-human study, there was both a single dose phase and 7 day repeat dose phase. Doses used ranged from 0.03 to 3.0?g. A radioactive microtracer study was subsequently conducted to determine systemic clearance, bioavailability and preliminary metabolism, and a crossover study was conducted to determine the effect of gender, formulation and feeding state on SRT2104 pharmacokinetics. Results SRT2104 was well tolerated in all of these studies, with no serious adverse reactions observed. SRT2104 displayed a dose-dependent, but sub-proportional increase in exposure following single dose and repeated dose administration. Accumulation of three-fold or less occurs after 7 days of repeat dosing. The mean bioavailability was circa 14% and the mean clearance was circa 400?ml?min-1. Although there were no substantial effects on exposure resulting from gender or formulation differences, a notable food effect was observed, manifested as up to four-fold increase in exposure parameters. Conclusions In the absence of an optimized formulation of SRT2104, the food effect can be used to maximize exposure in future clinical studies. Combined with the good tolerability of all doses demonstrated in these studies, the favourable selectivity profile of SRT2104 allows for the use of this SIRT1 modulator for target validation in the clinic.

Type 1 diabetes (T1D) and autoimmune thyroid diseases (AITD) frequently occur together within families and in the same individual. The co-occurrence of T1D and AITD in the same patient is one of the variants of the autoimmune polyglandular syndrome type 3 [APS3 variant (APS3v)]. Epidemiological data point to a strong genetic influence on the shared susceptibility to T1D and AITD. Recently, significant progress has been made in our understanding of the genetic association between T1D and AITD. At least three genes have been confirmed as major joint susceptibility genes for T1D and AITD: human leukocyte antigen class II, cytotoxic T-lymphocyte antigen 4 (CTLA-4), and protein tyrosine phosphatase non-receptor type 22. Moreover, the first whole genome linkage study has been recently completed, and additional genes will soon be identified. Not unexpectedly, all the joint genes for T1D and AITD identified so far are involved in immune regulation, specifically in the presentation of antigenic peptides to T cells. One of the lessons learned from the analysis of the joint susceptibility genes for T1D and AITD is that subset analysis is a key to dissecting the etiology of complex diseases. One of the best demonstrations of the power of subset analysis is the CTLA-4 gene in T1D. Although CTLA-4 showed very weak association with T1D, when analyzed in the subset of patients with both T1D and AITD, the genetic effect of CTLA-4 was significantly stronger. Gene-gene and genetic-epigenetic interactions most likely play a role in the shared genetic susceptibility to T1D and AITD. Dissecting these mechanisms will lead to a better understanding of the etiology of T1D and AITD, as well as autoimmunity in general. (Endocrine Reviews 29: 697-725, 2008)

The CD40 gene, an important immune regulatory gene, is also expressed and functional on nonmyeloid-derived cells, many of which are targets for tissue-specific autoimmune diseases, including β cells in type 1 diabetes, intestinal epithelial cells in Crohn's disease, and thyroid follicular cells in Graves' disease (GD). Whether target tissue CD40 expression plays a role in autoimmune disease etiology has yet to be determined. In this study, we show that target tissue overexpression of CD40 plays a key role in the etiology of autoimmunity. Using a murine model of GD, we demonstrated that thyroidal CD40 overexpression augmented the production of thyroid-specific Abs, resulting in more severe experimental autoimmune GD (EAGD), whereas deletion of thyroidal CD40 suppressed disease. Using transcriptome and immune-pathway analyses, we showed that in both EAGD mouse thyroids and human primary thyrocytes, CD40 mediates this effect by activating downstream cytokines and chemokines, most notably IL-6. To translate these findings into therapy, we blocked IL-6 during EAGD induction in the setting of thyroidal CD40 overexpression and showed decreased levels of thyroid stimulating hormone receptor-stimulating Abs and frequency of disease. We conclude that target tissue overexpression of CD40 plays a key role in the etiology of organ-specific autoimmune disease.

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces T(H)17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70 mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of T(H)1 and T(H)17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c(+) dendritic cells and CD3(+) T cells was seen, with a greater decrease in the CD11c(+) population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of T(H)1- and T(H)17-mediated inflammatory diseases.

Background SRT2104 has been developed as a selective small molecule activator of SIRT1, a NAD+-dependent deacetylase involved in the regulation of energy homeostasis and the modulation of various metabolic pathways, including glucose metabolism, oxidative stress and lipid metabolism. SIRT1 has been suggested as putative therapeutic target in multiple age-related diseases including type 2 diabetes and dyslipidemias. We report the first clinical trial of SRT2104 in elderly volunteers. Methods Oral doses of 0.5 or 2.0 g SRT2104 or matching placebo were administered once daily for 28 days. Pharmacokinetic samples were collected through 24 hours post-dose on days 1 and 28. Multiple pharmacodynamic endpoints were explored with oral glucose tolerance tests (OGTT), serum lipid profiles, magnetic resonance imaging (MRI) for assessment of whole body visceral and subcutaneous fat, maximal aerobic capacity test and muscle 31P magnetic resonance spectroscopy (MRS) for estimation of mitochondrial oxidative capacity. Results SRT2104 was generally safe and well tolerated. Pharmacokinetic exposure increased less than dose-proportionally. Mean Tmax was 2–4 hours with elimination half-life of 15–20 hours. Serum cholesterol, LDL levels and triglycerides decreased with treatment. No significant changes in OGTT responses were observed. 31P MRS showed trends for more rapid calculated adenosine diphosphate (ADP) and phosphocreatine (PCr) recoveries after exercise, consistent with increased mitochondrial oxidative phosphorylation. Conclusions SRT2104 can be safely administered in elderly individuals and has biological effects in humans that are consistent with SIRT1 activation. The results of this study support further development of SRT2104 and may be useful in dose selection for future clinical trials in patients. Trial Registration ClinicalTrials.gov NCT00964340

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis.

Tumor resistance to chemotherapy in advanced breast cancer is a major impediment to treatment success. Resistance can be induced by the drugs themselves or result from the action of internal factors. The role of hormones in chemoresistance has received little attention. This article focuses on two classes of hormones: lactogens and estrogens. Lactogens include prolactin, growth hormone and placental lactogen, all of which can activate the prolactin receptor. Estrogens include endogenous steroids and nonsteroidal compounds from the environment termed endocrine disruptors, all of which can activate 'classical' estrogen receptors (ERα and ERβ), as well as other types of receptors. Both lactogens and estrogens antagonize cytotoxicity of multiple chemotherapeutic agents through complementary mechanisms. The implications of chemoresistance by these hormones to patients with breast cancer, and the potential benefits of developing combinatorial anti-lactogen/anti-estrogen treatment regimens, are discussed.

Autoimmune thyroid diseases (AITD) arise from complex interactions between genetic, epigenetic, and environmental factors. Whole genome linkage scans and association studies have established thyroglobulin (TG) as a major AITD susceptibility gene. However, the causative TG variants and the pathogenic mechanisms are unknown. Here, we describe a genetic/epigenetic mechanism by which a newly identified TG promoter single-nucleotide polymorphism (SNP) variant predisposes to AITD. Sequencing analyses followed by case control and family-based association studies identified an SNP (−1623A→G) that was associated with AITD in the Caucasian population (p = 0.006). We show that the nucleotide substitution introduced by SNP (−1623A/G) modified a binding site for interferon regulatory factor-1 (IRF-1), a major interferon-induced transcription factor. Using chromatin immunoprecipitation, we demonstrated that IRF-1 binds to the 5′ TG promoter motif, and the transcription factor binding correlates with active chromatin structure and is marked by enrichment of mono-methylated Lys-4 residue of histone H3, a signature of active transcriptional enhancers. Using reporter mutations and siRNA approaches, we demonstrate that the disease-associated allele (G) conferred increased TG promoter activity through IRF-1 binding. Finally, treatment of thyroid cells with interferon α, a known trigger of AITD, increased TG promoter activity only when it interacted with the disease-associated variant through IRF-1 binding. These results reveal a new mechanism of interaction between environmental (IFNα) and genetic (TG) factors to trigger AITD.

Prostate and breast cancers affect millions of men and women, respectively. Advanced forms of the disease, which can no longer be controlled by hormonal disruption or chemotherapy, have very limited treatment options. Consequently, there is a major benefit to identify new targets for therapy in both types of cancer. The prolactin (PRL) signaling cascade, by virtue of its importance to the pathology of both diseases, has emerged as a potential treatment target. To date, several methods for antagonizing the PRL receptor (PRLR) and its signaling pathways have been developed which include protein-based and small molecule antagonists. However, a better understanding of the genetic and molecular characteristics of the PRL cascade is needed for the successful therapeutic application of antagonists. At the level of genetics, it is necessary to determine the functional significance of non-synonymous single nucleotide polymorphisms of the PRLR and their association with disease prevalence and severity. At the molecular level, a comprehensive knowledge of interactions of the PRL signaling pathway with other oncogenic molecules is warranted so as to identify beneficial combinatorial strategies. This review discusses multiple features of the PRL signaling cascade and how they can be exploited in the search for effective therapies for patients with breast and prostate cancers.

An HLA-DR variant containing Arginine at position 74 of the DRbeta1 chain confers a strong genetic susceptibility to autoimmune thyroid diseases (AITD), Graves' disease (GD) and Hashimoto's thyroiditis (HT), while Glutamine at position DRbeta1-74 is protective. We hypothesized that the DRbeta1-Arg74 variant is able to present pathogenic thyroglobulin (Tg) peptides to T-cells more efficiently, thereby triggering thyroid autoimmunity. Indeed, we have previously identified 4 human Tg (hTg) peptides that bind specifically to DRbeta1-Arg74 with much weaker binding to the protective variant DRbeta1-Gln74. The aim of our study was to examine in vivo whether an hTg peptide that binds strongly and specifically to DRbeta1-Arg74 is capable of stimulating T-cells during the induction of thyroiditis in a "humanized" mouse expressing human DR3, and in patients positive for Tg antibodies. Sequencing of exon 2 of the DR transgene in the DR3 mice, null for endogenous MHC II molecules, confirmed that they expressed the disease-associated DRbeta1-Arg74 variant, thus making them an ideal in vivo model to test the presentation of hTg peptides by DRbeta1-Arg74 HLA-DR. Induction of EAT in the DR3 mice lead to T-cell stimulation and proliferation to Tg.2098, a strong and specific DRbeta1-Arg74 binder, while a non-binding control peptide, Tg.2766 did not induce this response. Moreover, Tg.2098 stimulated T-cells from 4 individuals who were positive for thyroglobulin antibodies, demonstrating that Tg.2098 is an immunogenic peptide capable of being presented in vivo and activating T-cells in EAT and AITD. Energetic analysis of the complex formed by Tg.2098 and DRbeta-Arg74 has shown that the origin of the affinity was determined by residues 1, 7 and 9 in the peptide, while the selectivity of the peptide for the MHC was determined by the Asp in position 4. The disease-protective substitution R74Q, leads to reduction in affinity due to changes in local interaction with D4 as well as non-local interaction with other residues. The electrostatic potential on the surface of the DRbeta-Arg74-Tg.2098 complex has a unique signature which may be recognized by T-cell receptors leading to autoimmune thyroiditis. Taken together these findings suggest that Tg.2098, a strong and specific binder to the disease-associated HLA-DRbeta-Arg74, is a major human T-cell epitope and participant in the pathoetiology of AITD.

Breast cancer is both a common and a significant health concern for women across the globe. Emerging evidence suggests that the prolactin (PRL) signaling cascade contributes to the pathophysiology of breast cancer as well as to chemoresistance. The importance of this pathway to breast cancer has been elucidated by in vitro studies, genetic manipulations in mice, and case control analyses in human populations. To date, a number of different strategies, none of which have yet made it to the clinical stage, have been advanced as a means for blocking the PRL receptor (PRLR). This paper presents the rationale and strategy for the development of novel small molecule competitive antagonists of the PRLR as a therapeutics in breast cancer. It is predicted that the future of breast cancer treatment will continue to evolve and will be different than what it is today. Combination therapies will be applied that will concurrently target multiple molecules of interest. Novel small molecules will be employed as a means to turn off the PRL signaling pathway in breast cancer cells. Different molecules may well be applied to different genotypic individuals on the basis of the polymorphism profile that their PRLR harbors.

Breast and prostate cancers are hormone-sensitive malignancies that afflict millions of women and men. Although prolactin (PRL) is known as a survival factor that supports tumor growth and confers chemoresistance in both cancers, its precise role in these tumors has not been studied extensively. Growth hormone and placental lactogen also bind PRL receptor (PRLR) and mimic some of the actions of PRL. Blockade of the PRLR represents a novel treatment for patients with advanced breast or prostate cancer with limited therapeutic options. This review discusses different approaches for generating PRLR antagonists. Emphasis is placed on technological advances which enable high-throughput screening for small molecule inhibitors of PRLR signaling that could serve as oral medications.

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.

Purpose Elesclomol is a novel, small-molecule, oxidative stress inducer believed to exert selective cytotoxicity by increasing intracellular concentrations of reactive oxygen species, which results in cell death via mitochondrial apoptosis. We evaluated whether the addition of elesclomol to weekly paclitaxel could improve efficacy in patients with stage IV metastatic melanoma.Patients and Methods We randomly assigned patients with metastatic melanoma, measurable disease, and one or fewer prior chemotherapy regimens to elesclomol 213 mg/m(2) plus paclitaxel 80 mg/m(2) (E + P) or to paclitaxel 80 mg/m(2) alone at a 2: 1 ratio; regimens were given as a 1-hour intravenous infusion weekly, during 3 of every 4 weeks until disease progression per Response Evaluation Criteria in Solid Tumors or death occurred. Patients who experienced progression were unblended, and patients on paclitaxel alone were permitted to cross over to E + P. The primary efficacy end point was progression-free survival (PFS); secondary end points were response rate (RR), toxicity, and overall survival (OS; analyzed post hoc).Results At 21 US sites, 53 patients were randomly assigned to E + P, and 28 patients were randomly assigned to paclitaxel. The addition of elesclomol to paclitaxel yielded a doubling of median PFS (112 v 56 days) and a 41.7% risk reduction for disease progression/death (hazard ratio, 0.583; P = .035). Respective RRs for the E + P and paclitaxel groups were 15% and 3%; median OS was 11.9 v 7.8 months. Of patients on paclitaxel alone, 19 (68%) of 28 crossed over to E + P after they experienced progression. Weekly E + P was well tolerated.Conclusion E + P resulted in a statistically significant doubling of median PFS, with an acceptable toxicity profile and encouraging OS. A multinational, phase III trial (SYMMETRY) of E + P compared with paclitaxel alone in metastatic melanoma has closed.