A 2-year randomised placebo-controlled trial of doxycycline for lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is characterised by lung cysts and airflow obstruction. Matrix metalloproteinases have been implicated in lung destruction in LAM. We performed a randomised, double-blind trial, comparing the matrix metalloproteinases inhibitor doxycycline with placebo on the progression of LAM. 23 females with LAM were randomised to doxycycline 100 mg daily for 3 months followed by 200 mg daily for 21 months, or matched placebo. Lung function, exercise capacity, quality of life and matrix metalloproteinases levels were measured. 21 patients completed 6 months of treatment, 17 completed 1 year of treatment and 15 completed 2 years of treatment. Eight withdrew from the trial due, four due to a pneumothorax and four because of other reasons. The mean±sd decline in FEV1, the primary endpoint, did not differ between the groups being -90±154 mL·year(-1) in the placebo group and -123±246 mL·year(-1) in the doxycycline group (difference -32.5, 95% CI -213-148; p=0.35). Doxycycline had no effect upon vital capacity, gas transfer, shuttle walk distance or quality of life. Urine matrix metalloproteinases-9 measurements were lower with doxycycline treatment (p=0.03). Although with limited numbers we cannot completely exclude an effect of doxycycline, the lack of effect on any outcome makes it unlikely that doxycycline has a useful effect in LAM.

Effects of atopy and grass pollen season on histamine H4 receptor expression in human leukocytes.

BACKGROUND: The histamine H4 receptor (H4R) is a novel therapeutic target to treat allergic inflammation. OBJECTIVE: To profile messenger RNA (mRNA) expression of H4R isoforms in human cells and evaluate the effects of atopy and grass pollen season on H4R expression in peripheral blood leukocytes ex vivo. METHODS: H4R isoform expression was assayed by quantitative polymerase chain reaction in human airway and peripheral RNA. During low and high grass pollen seasons, leukocytes were isolated from venous blood and fractionated into peripheral blood mononuclear cells and polymorphonuclear cells (PMN). H4R expression was determined and related to atopy, defined by a level of specific IgE to Timothy grass pollen of ≥0.35 kU(A)/L (n = 7 atopic patients and 9 controls). RESULTS: Expression of total and full length H4R was at the limit of detection but predominant in peripheral blood leukocytes, where truncated H4R was expressed exclusively (≤300-fold less). Suggestive evidence for total H4R in airway cells and brain indicated an expression ≤260-fold lower than in peripheral blood mononuclear cells. Total H4R mRNA expression was unaffected by atopy or grass pollen season, but truncated H4R was significantly reduced during high grass pollen season in total leukocytes, independently of atopy (P < .01). CONCLUSION: H4R mRNA is predominantly expressed in peripheral blood leukocytes, and total H4R expression levels are unrelated to atopy or grass pollen season. Atopy-independent seasonal variation in truncated H4R expression might affect putative negative regulation of full length H4R during high grass pollen season. If verified, this should be considered during the design of drugs targeting H4R to treat allergic inflammation, particularly for seasonal allergic rhinitis.

A nondepleting anti-CD19 antibody impairs B cell function and inhibits autoimmune diseases.

B cells contribute to multiple aspects of autoimmune disorders, and B cell-targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti-human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell-dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell-targeting therapies in treatment of autoimmune conditions without causing B cell depletion.

Clinical utility of diagnostic guidelines and putative biomarkers in lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis is a rare disease occurring almost exclusively in women. Diagnosis often requires surgical biopsy and the clinical course varies between patients with no predictors of progression. We evaluated recent diagnostic guidelines, clinical features and serum biomarkers as diagnostic and prognostic tools. METHODS: Serum vascular endothelial growth factor-D (VEGF-D), angiotensin converting enzyme (ACE), matrix metalloproteinases (MMP) -2 and -9, clinical phenotype, thoracic and abdominal computerised tomography, lung function and quality of life were examined in a cohort of 58 patients. 32 healthy female controls had serum biomarkers measured. RESULTS: Serum VEGF-D, ACE and total MMP-2 levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls (median = 1174 vs. 332 pg/ml p < 0.0001 with an area under the receiver operating characteristic curve of 0.967, 95% CI 0.93-1.01). Application of European Respiratory Society criteria allowed a definite diagnosis without biopsy in 69%. Adding VEGF-D measurement to ERS criteria further reduced the need for biopsy by 10%. VEGF-D was associated with lymphatic involvement (p = 0.017) but not the presence of angiomyolipomas. CONCLUSIONS: Combining ERS criteria and serum VEGF-D reduces the need for lung biopsy in LAM. VEGF-D was associated with lymphatic disease but not lung function.

Defining Target Engagement Required for Efficacy In Vivo at the Retinoic Acid Receptor-Related Orphan Receptor C2 (RORγt).

The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo. Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22, which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.

Effect of doxycycline on proliferation, MMP production, and adhesion in LAM-related cells.

Matrix metalloproteinases (MMPs) have been implicated in lung cyst formation in lymphangioleiomyomatosis (LAM). As doxycycline inhibits MMP activity in vivo, some patients take doxycycline, as one report has suggested a possible benefit in LAM. However, there have been no randomized controlled clinical trials of doxycycline for LAM, and any mechanism of action is unclear. Here, we examine previously proposed mechanisms of actions. Cell proliferation and adhesion were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and Cytomatrix cell adhesion kits. Apoptosis was examined by TdT-mediated dUTP nick end labeling (TUNEL) assay. MMP-2 expression was examined by quantitative real-time PCR and zymography in doxycycline-treated ELT3 cells and tumor growth using angiomyolipoma-derived tumor xenografts in nude mice. In ELT3 cells, >or=25 microg/ml doxycycline decreased proliferation, increased apoptosis, and caused a change in cell morphology associated with redistribution of actin stress filaments. Reduction in proliferation was also seen in human angiomyolipoma-derived cells. Cell adhesion to ECM proteins was decreased by doxycycline at 50 microg/ml and prevented detachment of already adherent cells. There was no effect of doxycycline on MMP-2 expression or activity in vitro. In the xenograft model, doxycycline (30 mg*kg(-1)*day(-1)) had no effect on tumor growth, final tumor weight, or tumor lysate MMP levels. Doxycycline at doses >or= 25 microg/ml inhibited cell proliferation and adhesion, possibly by a toxic effect. Doxycycline had no effect on MMP-2 expression or activity or tumor growth in the xenograft model. Any possible in vivo effect is unlikely to be mediated by MMP-2 or reduced cell proliferation.

Pharmacokinetics and pharmacodynamics of LGD-3303 [9-chloro-2-ethyl-1-methyl-3-(2,2,2-trifluoroethyl)-3H-pyrrolo-[3,2-f]quinolin-7(6H)-one], an orally available nonsteroidal-selective androgen receptor modulator.

Selective androgen receptor modulators (SARMs) are a new class of molecules in development to treat a variety of diseases. SARMs maintain the beneficial effects of androgens, including increased muscle mass and bone density, while having reduced activity on unwanted side effects. The mechanisms responsible for the tissue-selective activity of SARMs are not fully understood, and the pharmacokinetic (PK)/pharmacodynamic (PD) relationships are poorly described. Tissue-specific compound distribution potentially could be a mechanism responsible for apparent tissue selectivity. We examined the PK/PD relationship of a novel SARM, LGD-3303 [9-chloro-2-ethyl-1-methyl-3-(2,2,2-trifluoroethyl)-3H-pyrrolo[3,2-f]quinolin-7(6H)-one], in a castrated rat model of androgen deficiency. LGD-3303 has potent activity on levator ani muscle but is a partial agonist on the preputial gland and ventral prostate. LGD-3303 never stimulated ventral prostate above intact levels despite increasing plasma concentrations of compound. Tissue-selective activity was maintained when LGD-3303 was dosed orally or by continuous infusion, two routes of administration with markedly different time versus exposure profiles. Despite the greater muscle activity relative to prostate activity, local tissue concentrations of LGD-3303 were higher in the prostate than in the levator ani muscle. LGD-3303 has SARM properties that are independent of its pharmacokinetic profile, suggesting that the principle mechanism for tissue-selective activity is the result of altered molecular interactions at the level of the androgen receptor.

Selective androgen receptor modulators based on a series of 7H-[1,4]oxazino[3,2-g]quinolin-7-ones with improved in vivo activity.

Modification on a lead series of [1,4]oxazino[3,2-g]quinolin-7-ones at the 2-position led to selective androgen receptor modulators with improved in vivo activity. The most potent analog (-)-33a exhibited full maintenance of levator ani muscle at 3mg/kg and reduced activity on ventral prostate weight in a 2-week orally-dosed and orchidectomized rat maintenance assay.

Substituted 6-(1-pyrrolidine)quinolin-2(1H)-ones as novel selective androgen receptor modulators.

The androgen receptor is a ligand inducible transcription factor that is involved in a broad range of physiological functions. Here we describe the discovery of a new class of orally available selective androgen receptor modulators. The lead compound, 6-[(2R,5R)-2-methyl-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl]-4-trifluoromethylquinolin-2(1H)-one (6a), showed excellent anabolic activity in muscle with reduced effect on the prostate in a rat model of hypogonadism. The compound also improved bone strength in a rat model of post-menopausal osteoporosis.

Retinoic acid receptors and retinoids are up-regulated in the developing and adult rat prostate by neonatal estrogen exposure.

Exposure to estrogens during the neonatal period interrupts rat prostatic development by reducing branching morphogenesis and by blocking epithelial cells from entering a normal differentiation pathway. Upon aging, ventral prostates exhibit extensive hyperplasia, dysplasia, and massive lymphocytic infiltrate, suggesting that neonatal estrogens may predispose the prostate gland to precancerous lesions. Vitamin A (retinol) and their derivatives (retinoic acids) are known key developmental regulators that bind and activate retinoic acid receptors (RARs). To evaluate whether neonatal estrogenization alters the sensitivity of the developing rat prostate to retinoids, RARalpha, -beta, and -gamma cellular localization and protein levels were analyzed over the course of development and into adulthood by immunocytochemistry and Western analysis, whereas mRNA levels were measured using RT-PCR. In addition, intraprostatic retinol and retinoic acid levels were quantitated on d 10 and 90 using HPLC-mass spectroscopy. Male rats were given 25 micro g estradiol benzoate or oil on d 1, 3, and 5 of life, and prostatic complexes were removed on d 6, 10, 15, 30, and 90. The RARs localized to distinct cell populations: RARbeta was expressed within basal epithelial cells, RARalpha was localized to differentiated luminal epithelial cells and smooth muscle cells, and RARgamma was expressed within periductal stromal cells. Over the normal course of development, total protein and mRNA levels for the RARs declined, so that the adult prostate possessed the lowest amounts of RAR. Exposure to estrogens during the neonatal period resulted in an immediate and sustained increase in RARalpha levels and in the number of cells that expressed RARbeta, whereas RARgamma levels were unaffected. Western analysis confirmed that total prostatic RAR protein levels were significantly increased, whereas RT-PCR demonstrated that RARalpha and RARbeta mRNA levels were markedly elevated in response to estrogenic exposure. The total prostatic retinol content was tripled by estrogenic exposure on d 10 and 90, indicating that the ability to retain retinoids within the prostate was permanently increased. Intraprostatic levels of 9-cis- and all-trans-retinoic acid levels were reduced on d 10, whereas 13-cis-retinoic acid levels were increased in response to estrogens. In the adult prostates of rats exposed neonatally to estrogen, total retinoic acid levels were doubled due to significant increases in both 9-cis- and 13-cis-retinoic acids compared with those in control prostates. In summary, levels of specific RARs and their activating ligands are increased in the prostate gland after neonatal estrogenic exposure, and this effect is permanent throughout the life of the animal. Thus, we hypothesize that alterations in morphogenesis as well as dysplasia in the adult prostate may be mediated in part through augmentation of transcriptional signals in the retinoid pathway.

Sexual differentiation of the spinal nucleus of the bulbocavernosus is not mediated solely by androgen receptors in muscle fibers.

The spinal nucleus of the bulbocavernosus (SNB) neuromuscular system is a highly conserved and well-studied model of sexual differentiation of the vertebrate nervous system. Sexual differentiation of the SNB is currently thought to be mediated by the direct action of perinatal testosterone on androgen receptors (ARs) in the bulbocavernosus/levator ani muscles, with concomitant motoneuron rescue. This model has been proposed based on surgical and pharmacological manipulations of developing rats as well as from evidence that male rats with the testicular feminization mutation (Tfm), which is a loss of function AR mutation, have a feminine SNB phenotype. We examined whether genetically replacing AR in muscle fibers is sufficient to rescue the SNB phenotype of Tfm rats. Transgenic rats in which wild-type (WT) human AR is driven by a human skeletal actin promoter (HSA-AR) were crossed with Tfm rats. Resulting male HSA-AR/Tfm rats express WT AR exclusively in muscle and nonfunctional Tfm AR in other tissues. We then examined motoneuron and muscle morphology of the SNB neuromuscular system of WT and Tfm rats with and without the HSA-AR transgene. We observed feminine levator ani muscle size and SNB motoneuron number and size in Tfm males with or without the HSA-AR transgene. These results indicate that AR expression in skeletal muscle fibers is not sufficient to rescue the male phenotype of the SNB neuromuscular system and further suggest that AR in other cell types plays a critical role in sexual differentiation of this system.

Combination treatment with a selective androgen receptor modulator q(SARM) and a bisphosphonate has additive effects in osteopenic female rats.

Recent clinical trials with bisphosphonates and PTH have not supported the hypothesis that combination treatments with antiresorptive and anabolic agents would lead to synergistic activity. We hypothesized that combination treatment with a selective androgen receptor modulator (SARM), LGD-3303, and a bisphosphonate would be beneficial. In vitro competitive binding and transcriptional activity assays were used to characterize LGD-3303. LGD-3303 is a potent nonsteroidal androgen that shows little or no cross-reactivity with related nuclear receptors. Tissue selective activity of LGD-3303 was assessed in orchidectomized male rats orally administered LGD-3303 for 14 days. LGD-3303 increased the levator ani muscle weight above eugonadal levels but had greatly reduced activity on the prostate, never increasing the ventral prostate weight to >50% of eugonadal levels even at high doses. Ovariectomized female rats were treated with LGD-3303, alendronate, or combination treatment to study the effects on bone. DXA scans, histomorphometry, and biomechanics were performed. LGD-3303 increased muscle weight in females rats. In addition, LGD-3303 increased BMD and BMC at both cortical and cancellous bone sites. At cortical sites, the effects were caused in part by anabolic activity on the periosteal surface. At every measured site, combination treatment was as effective as either single agent and in some cases showed significant added benefit. LGD-3303 is a novel SARM with anabolic effects on muscle and cortical bone not observed with bisphosphonates. Combination therapy with LGD-3303 and alendronate had additive effects and may potentially be a useful therapy for osteoporosis and frailty.

Orphan nuclear receptor estrogen-related receptor alpha is essential for adaptive thermogenesis.

Survival of organisms requires the ability to adapt to changes in the environment. Adaptation of oxidative metabolism is essential for meeting increased energy demands in response to stressors, such as exposure to cold temperatures or increased physical activity. Adaptive changes in metabolism are often achieved at the level of gene expression, and nuclear receptors have prevalent roles in mediating such responses. Estrogen-related receptor alpha (ERRalpha) was the first orphan nuclear receptor to be identified, and yet its physiologic function remains unknown. Here, we show that mice lacking ERRalpha are unable to maintain body temperature when exposed to cold. Surprisingly, the inability to adapt to cold is not due to defects in the acute transcriptional induction of genes important for thermogenesis. Rather, we show that ERRalpha is needed for the high levels of mitochondrial biogenesis and oxidative capacity characteristic of brown adipose tissue (BAT), and thus for providing the energy necessary for thermogenesis. ERRalpha fulfills this role by acting directly at genes important for mitochondrial function, parallel to other factors controlling mitochondrial gene expression, such as NRF1 and NRF2/GABPA. Our findings demonstrate that ERRalpha is a key regulator of mitochondrial biogenesis and oxidative metabolism, and essential for adaptive thermogenesis.