Pharmacological properties of A-204176, a novel and selective alpha1A adrenergic agonist, in in vitro and in vivo models of urethral function.

A-204176 (N-[5-(1H-imidazol-4-y1)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide) is a potent and selective alpha1A adrenoceptor agonist that binds with 17-fold and 9-fold greater affinity to the alpha1A (Ki=176 nM) than the alpha1b and alpha1d subtypes, respectively. In functional studies A-204176 is potent (pD2=6.4) and efficacious (83% of maximum control phenylephrine response) at rabbit urethra alpha1A receptors, with weaker potency and greatly reduced efficacy at rat spleen alpha1B (pD2=5.3, 11%) and rat aorta alpha1D (pD2=4.4, 10%) subtypes. In anesthetized female dogs, A-204176 is more potent than the non-selective alpha1 adrenoceptor agonist phenylpropanolamine (PPA) to increase measures of urethral tone and is more efficacious to increase pressure in the proximal region of the urethra. Significant increases on parameters of the urethral pressure profilometry were induced at 100 and 300 nmol/kg, i.v., by A-204176 and PPA, respectively. A-204176 was more potent than PPA to increase the abdominal pressure required to produce leakage. In the simultaneous measurement of intraurethral pressure and mean arterial blood pressure, A-204176 displays enhanced urethral selectivity relative to PPA. However, despite its selectivity for alpha1A versus alpha1B and alpha1D adrenoceptors in vitro, A-204176 did not display the degree of urethral selectivity in vivo that would have been expected. The observed effect of A-204176 on blood pressure may be due to the presence of extra-synaptic alpha1A adrenoceptors in the vasculature or to activation of spinal and supraspinal alpha1A adrenoceptors. These data indicate that A-204176 may represent a useful pharmacological tool to investigate the functional role of the alpha1A adrenoceptor in the urethra and to elucidate the lack of uroselectivity observed in vivo.     

A peroxisome proliferator-activated receptor alpha/gamma dual agonist with a unique in vitro profile and potent glucose and lipid effects in rodent models of type 2 diabetes and dyslipidemia

LSN862 is a novel peroxisome proliferator-activated receptor (PPAR)alpha/gamma dual agonist with a unique in vitro profile that shows improvements on glucose and lipid levels in rodent models of type 2 diabetes and dyslipidemia. Data from in vitro binding, cotransfection, and cofactor recruitment assays characterize LSN862 as a high-affinity PPARgamma partial agonist with relatively less but significant PPARalpha agonist activity. Using these same assays, rosiglitazone was characterized as a high-affinity PPARgamma full agonist with no PPARalpha activity. When administered to Zucker diabetic fatty rats, LSN862 displayed significant glucose and triglyceride lowering and a significantly greater increase in adiponectin levels compared with rosiglitazone. Expression of genes involved in metabolic pathways in the liver and in two fat depots from compound-treated Zucker diabetic fatty rats was evaluated. Only LSN862 significantly elevated mRNA levels of pyruvate dehydrogenase kinase isozyme 4 and bifunctional enzyme in the liver and lipoprotein lipase in both fat depots. In contrast, both LSN862 and rosiglitazone decreased phosphoenol pyruvate carboxykinase in the liver and increased malic enzyme mRNA levels in the fat. In addition, LSN862 was examined in a second rodent model of type 2 diabetes, db/db mice. In this study, LSN862 demonstrated statistically better antidiabetic efficacy compared with rosiglitazone with an equivalent side effect profile. LSN862, rosiglitazone, and fenofibrate were each evaluated in the humanized apoA1 transgenic mouse. At the highest dose administered, LSN862 and fenofibrate reduced very low-density lipoprotein cholesterol, whereas, rosiglitazone increased very low-density lipoprotein cholesterol. LSN862, fenofibrate, and rosiglitazone produced maximal increases in high-density lipoprotein cholesterol of 65, 54, and 30%, respectively. These findings show that PPARgamma full agonist activity is not necessary to achieve potent and efficacious insulin-sensitizing benefits and demonstrate the therapeutic advantages of a PPARalpha/gamma dual agonist.     

Mycophenolic acid as a latent agonist of PPARgamma

Mycophenolic acid (MPA), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), was found to inhibit the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes. Although the effect of MPA was attributed to inhibition of IMPDH, we uncovered a hidden biological property of MPA as an agonist of peroxisome proliferator activated receptor gamma (PPARgamma).     

Design and synthesis of a novel class of dual PPARgamma/delta agonists

The design and synthesis of dual PPAR gamma/delta agonist (R)-3-{2-ethyl-4-[3-(4-ethyl-2-pyridin-2-yl-phenoxy)-butoxy]-phenyl}propionic acid is described. This compound dose-dependently lowered plasma glucose in hyperglycemic male Zucker diabetic fatty (ZDF) rats and produced less weight gain relative to rosiglitazone at an equivalent level of glucose control.     

The rexinoid LG100754 is a novel RXR:PPARgamma agonist and decreases glucose levels in vivo.

The RXR serves as a heterodimer partner for the PPARgamma and the dimer is a molecular target for insulin sensitizers such as the thiazolidinediones. Ligands for either receptor can activate PPAR-dependent pathways via PPAR response elements. Unlike PPARgamma agonists, however, RXR agonists like LG100268 are promiscuous and activate multiple RXR heterodimers. Here, we demonstrate that LG100754, a RXR:RXR antagonist and RXR:PPARalpha agonist, also functions as a RXR:PPARgamma agonist. It does not activate other LG100268 responsive heterodimers like RXR:liver X receptoralpha, RXR:liver X receptorbeta, RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor induced gene B. This unique RXR ligand triggers cellular RXR:PPARgamma-dependent pathways including adipocyte differentiation and inhibition of TNFalpha-mediated hypophosphorylation of the insulin receptor, but does not activate key farnesoid X receptor and liver X receptor target genes. Also, LG100754 treatment of db/db animals leads to an improvement in insulin resistance in vivo. Interestingly, activation of RXR:PPARgamma by LG100268 and LG100754 occurs through different mechanisms. Therefore, LG100754 represents a novel class of insulin sensitizers that functions through RXR but exhibits greater heterodimer selectivity compared with LG100268. These results establish an approach to the design of novel RXR-based insulin sensitizers with greater specificity.     

Thermogenically competent nonadrenergic recruitment in brown preadipocytes by a PPARgamma agonist

Most physiologically induced examples of recruitment of brown adipose tissue (BAT) occur as a consequence of chronic sympathetic stimulation (norepinephrine release within the tissue). However, in some physiological contexts (e.g., prenatal and prehibernation recruitment), this pathway is functionally contraindicated. Thus a nonsympathetically mediated mechanism of BAT recruitment must exist. Here we have tested whether a PPARgamma activation pathway could competently recruit BAT, independently of sympathetic stimulation. We continuously treated primary cultures of mouse brown (pre)adipocytes with the potent peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist rosiglitazone. In rosiglitazone-treated cultures, morphological signs of adipose differentiation and expression levels of the general adipogenic marker aP2 were manifested much earlier than in control cultures. Importantly, in the presence of the PPARgamma agonist the brown adipocyte phenotype was significantly enhanced: UCP1 was expressed even in the absence of norepinephrine, and PPARalpha expression and norepinephrine-induced PGC-1alpha mRNA levels were significantly increased. However, the augmented levels of PPARalpha could not explain the brown-fat promoting effect of rosiglitazone, as this effect was still evident in PPARalpha-null cells. In continuously rosiglitazone-treated brown adipocytes, mitochondriogenesis, an essential part of BAT recruitment, was significantly enhanced. Most importantly, these mitochondria were capable of thermogenesis, as rosiglitazone-treated brown adipocytes responded to the addition of norepinephrine with a large increase in oxygen consumption. This thermogenic response was not observable in rosiglitazone-treated brown adipocytes originating from UCP1-ablated mice; hence, it was UCP1 dependent. Thus the PPARgamma pathway represents an alternative, potent, and fully competent mechanism for BAT recruitment, which may be the cellular explanation for the enigmatic recruitment in prehibernation and prenatal states.     

A novel partial agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma) recruits PPARgamma-coactivator-1alpha, prevents triglyceride accumulation, and potentiates insulin signaling in vitro

Partial agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), also termed selective PPARgamma modulators, are expected to uncouple insulin sensitization from triglyceride (TG) storage in patients with type 2 diabetes mellitus. These agents shall thus avoid adverse effects, such as body weight gain, exerted by full agonists such as thiazolidinediones. In this context, we describe the identification and characterization of the isoquinoline derivative PA-082, a prototype of a novel class of non-thiazolidinedione partial PPARgamma ligands. In a cocrystal with PPARgamma it was bound within the ligand-binding pocket without direct contact to helix 12. The compound displayed partial agonism in biochemical and cell-based transactivation assays and caused preferential recruitment of PPARgamma-coactivator-1alpha (PGC1alpha) to the receptor, a feature shared with other selective PPARgamma modulators. It antagonized rosiglitazone-driven transactivation and TG accumulation during de novo adipogenic differentiation of murine C3H10T1/2 mesenchymal stem cells. The latter effect was mimicked by overexpression of wild-type PGC1alpha but not its LXXLL-deficient mutant. Despite failing to promote TG loading, PA-082 induced mRNAs of genes encoding components of insulin signaling and adipogenic differentiation pathways. It potentiated glucose uptake and inhibited the negative cross-talk of TNFalpha on protein kinase B (AKT) phosphorylation in mature adipocytes and HepG2 human hepatoma cells. PGC1alpha is a key regulator of energy expenditure and down-regulated in diabetics. We thus propose that selective recruitment of PGC1alpha to favorable PPARgamma-target genes provides a possible molecular mechanism whereby partial PPARgamma agonists dissociate TG accumulation from insulin signaling.     

Pharmacology of PPARalpha, PPARgamma and dual PPARalpha/gamma agonists in clinical development

Cardiovascular diseases (CVD) remain the leading cause of mortality in the western societies. Several risk factors predispose to CVD including diabetes, obesity, insulin resistance, dyslipidemia and hypertension. Various pharmacological therapies have been developed to control the risk factors associated to CVD. Fibrates are able to correct dyslipidemia, therefore decreasing CVD risk. Thiazolidinediones (TZD) or glitazones by increasing insulin sensitivity decrease plasma glucose levels in diabetic patients. Both fibrates and TZD activate the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors that play a central role in the control of lipid and glucose metabolism. In this review, we will discuss the mode of action of fibrates and TZD and we will present an overview on PPAR ligands under development.     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用.结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用.体内实验中0.1 g/kg,0.5g/kg,1.2g/kg 3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间;降低肺炎小鼠的肺指数和血凝滴度(P<0.01).其作用与病毒唑相当.结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂.     

Design and synthesis of novel and potent amide linked PPARgamma/delta dual agonists

A series of potent amide linked PPARγ/δ dual agonists (1a) has been discovered through rational design. In the ZDF rat model of type 2 diabetes, compound (R)-3-[4-(3-{1-[(5-chloro-1,3-dimethyl-1H-indole-2-carbonyl)-amino]-ethyl}-5-fluoro-phenoxy)-2-ethyl-phenyl]-propionic acid (42) from this series has demonstrated glucose lowering efficacy comparable to the marketed PPARγ agonist rosiglitazone with less weight gain.     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用。结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用。体内实验中0.1g/kg,0.5g/kg,1.2g/kg3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间:降低肺炎小鼠的肺指数和血凝滴度(P<0.01)。其作用与病毒唑相当。结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂。     

Quantitative determination of a novel dual PPAR alpha/gamma agonist using on-line turbulent flow extraction with liquid chromatography-tandem mass spectrometry

Turbulent flow chromatograph (TFC) is a technique for the direct and efficient analysis of drugs and metabolites in biological matrices. We report here TFC on-line with an HPLC-MS/MS assay for the determination of 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, MK-0767, KRP297, Fig. 1) in plasma. Samples were transferred using an automated system followed by the addition of internal standard (II), prepared in 0.1 M ammonium acetate (pH 4.0). The plasma samples were directly injected onto a C18 turbulent flow column on-line with an HPLC-MS/MS system, and the analytical column used was a ThermoHypersil Keystone C18. Detection was achieved by MS/MS, using positive ionization on a TurboIonSpray probe, operated in multiple reaction monitoring (MRM) mode. The linear range was 4-2000 ng/mL for I when using 50 microL of plasma. The method exhibited good linearity and reproducibility. The method also showed good selectivity and ruggedness when applied to clinical samples, and was successfully cross-validated with a conventional off-line SPE, LC-MS/MS method.     

Collagen-based cell migration models in vitro and in vivo

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.     

Biochemical mechanism of insulin sensitization, lipid modulation and anti-atherogenic potential of PPAR alpha/gamma dual agonist: Ragaglitazar

The current goal in the treatment of diabetes is not only to enhance the glycemic control but also to improve the associated cardiovascular risk factors. Among many of the strategies available, a co-ligand of PPARalpha and gamma in a single molecule which combines the insulin sensitizing potential of PPARgamma and the beneficial lipid modulating properties of PPARalpha agonism, has gained attention in the recent past. Here we report the biochemical mechanism by which a dual PPAR alpha/gamma agonist Ragaglitazar (Raga) achieves this goal. The PPARalpha component of Raga appears to contribute to a significant increase in beta oxidation, ApoA1 secretion and inhibition of TG biosynthesis in HepG2 cells. These effects of Raga at 60 microM were similar to that shown by Fenofibrate (Feno) at 250 microM. The PPARgamma component of Raga showed significant G3PDH activity and TG accumulation with a corresponding increase in aP2 expression in 3T3L1 cells. Significantly reduced levels of IL-6 and TNFalpha were observed in the culture supernatants of Raga treated 3T3L1 cells. Raga resulted in significant insulin dependent glucose uptake in 3T3L1 with a corresponding increase in GLUT4 expression. Further, Raga showed a significant cholesterol efflux with a corresponding increase in ABCA1 protein expression in THP-1 macrophages. In conclusion, Raga activates both PPARalpha and gamma regulated pathway in adipocytes as well as in hepatocytes which together contributes for its insulin sensitizing and lipid lowering activity. In addition the dual activation of PPAR alpha/gamma also shows an athero-protective potential by inducing reverse cholesterol efflux and inhibiting the pro-inflammatory cytokines.     

稀有人参皂苷IH901在体内外的生物转化及其生物活性

人参皂苷IH901是近年人参代谢组学研究中新发现的一种稀有人参皂苷。IH901在天然人参中并不存在,系口服人参后通过系列肠道微生物在体内代谢转化,最终入血的主要代谢产物之一。最新药理学研究表明,IH901在抗肿瘤、抗炎、抗糖尿病和抗衰老等方面均表现出良好的生物活性,是人参在体内发挥活性作用的主要物质。近年来,在体内转化IH901的理论指导下,国内外学者通过体外酶转化和微生物转化等生物工程技术在大规模提取制备IH901等研究方面均取得突破性的进展。以下综述了稀有人参皂苷IH901在体内外的生物转化及其生物活性等研究进展。     

In vitro and in vivo evaluation of the subtype-selective muscarinic agonist PD 151832

PD 151832 is a potent partial muscarinic agonist that displays a high level of functional selectivity for the muscarinic m1 receptor subtype, as evidenced by its selective stimulation of PI turnover and cellular metabolic activity in transfected Hm1-CHO cells at concentrations that produce minimal stimulation of other cloned human muscarinic receptors. PD 151832 enhanced the amplification of Hm1-transfected NIH-3T3 cells at concentrations lower than those required to produce similar effects in Hm2 or Hm3-transfected cells. The functional m1 selectivity of PD 151832 is consistent with its improvement of mouse water maze performance at doses far lower than those required to produce peripheral parasympathetic side effects.