The effect of the lipid A analog E5531 on phospholipid membrane properties

The orphan nuclear receptor steroidogenic factor 1 (SF-1) was initially isolated as a key regulator of the cytochrome P450 steroid hydroxylases in adrenocortical and gonadal cells. Subsequent analyses of SF-1 knockout mice have expanded considerably our understanding of the roles that SF-1 plays in endocrine development. These SF-1 knockout mice lacked adrenal glands and gonads, with consequent male-to-female sex reversal of their internal and external genitalia. Thus, SF-1 is essential for the embryonic survival of the primary steroidogenic organs. They further exhibited impaired gonadotrope function and agenesis of the ventromedial hypothalamic nucleus, establishing that SF-1 contributes to reproductive function at all three levels of the hypothalamic-pituitary-gonadal axis. This report reviews experiments that have defined these critical roles of SF-1 in endocrine development, and highlights areas of ongoing investigation.     

X-ray structural characterization of SR 142948, a novel potent synthetic neurotensin receptor antagonist

In ob/ob mice, leptin deficiency results in hypogonadotrophic hypogonadism, impaired sexual maturation and infertility, which are all corrected by leptin administration. In humans, pubertal development and menarche are related to the attainment of a critical amount of body fat. To examine whether changes in circulating concentrations of leptin could be a hormonal signal influencing gonadotrophin secretion, we studied 98 adolescents and young adults of both sexes, aged 13-19 years, whose weight varied from normal to massively obese and whose sexual maturation was between Tanner stages 3 and 5. We measured leptin, sex steroids and circulating gonadotrophin concentrations in the basal state and in response to GnRH. In perimenarchial and young adult girls, we found that the LH and FSH responses to GnRH were negatively correlated with body mass index (BMI: r = -0.45 and -0.47 respectively, P < 0.0025) and circulating leptin (r = -0.53 and -0.49 respectively, P < 0.002). Decreased LH and FSH responses to GnRH were associated with increased adiposity and hyperleptinaemia. Our data do not establish, but are consistent with a direct neuroendocrine negative effect of excess leptin on the central reproductive system of obese girls. In boys of comparable adiposity, we found no influence of BMI or leptin on gonadotrophin concentrations, which is another aspect of the sexual dimorphism characterizing human leptin physiology.     

Induction of early gene expression in murine macrophages by synthetic lipid A analogs with differing endotoxic potentials

Numerous lipid A analogs have been synthesized in an attempt to dissociate endotoxic activities from beneficial immunomodulatory activities. In the present study, we have evaluated select lipid A analogs in macrophages for their ability to induce a panel of lipopolysaccharide (LPS)-inducible genes to gain insights into the molecular mechanisms which underlie endotoxicity. We evaluated three monosaccharide lipid A analogs: SDZ MRL 953, an agonist with an improved therapeutic margin over endotoxin; SDZ 281.288, a more toxic analog; and SDZ 880.431, an analog with proven LPS-inhibitory activity. In addition, three disaccharide lipid A analogs (i.e., lipid IVA, SDZ 880.611, and SDZ 880.924) that differ in acylation and phosphorylation patterns were also examined and compared with synthetic lipid A. With the exception of SDZ 880.431, each of these structurally diverse analogs was able to induce the complete panel of LPS-inducible genes, specifically genes which encode tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, 75-kDa type 2 TNF receptor (D7), IP-10, D3, and D8. These results underscore that macrophage stimulation by lipid A analogs is permissive to considerable structural diversity. Structures with favorable therapeutic indices (SDZ MRL 953, SDZ 880.611, and SDZ 880.924) were not different from structures with poor therapeutic indices (lipid A, lipid IVA, and SDZ 281.288) with regard to gene induction. Nonetheless, the nontoxic SDZ MRL 953 was approximately 1,000-fold less potent than synthetic lipid A at inducing TNF-alpha secretion, and perhaps this contributes to the lack of toxicity exhibited by this compound. The ability of compound SDZ 880.431 to inhibit TNF-alpha secretion induced by both SDZ MRL 953 and smooth LPS suggests that the monosaccharide and smooth LPS share a receptor or a portion thereof. A pattern of protein tyrosine phosphorylation similar to that induced by LPS was stimulated by the monosaccharide SDZ MRL 953 and SDZ 281.288 and disaccharides lipid IVA, SDZ 880.924, and SDZ 880.611, providing evidence for a common signalling pathway.     

CVT-124, a novel adenosine A1 receptor antagonist with unique diuretic activity

Administration of the selective adenosine A1 receptor antagonist, CVT-124, to conscious chronically instrumented rats resulted in significant increases in urine flow rate and sodium excretion without affecting potassium excretion or renal hemodynamics. Its maximum effect was twice that of hydrochlorothiazide which was associated with a significant kaliuresis. The diuretic effect of CVT-124 was less than that observed with furosemide; however, furosemide administration was associated with a large increase in potassium excretion as well as a reduction in glomerular filtration rate. When given at equinatriuretic doses, CVT-124 enhanced the diuretic and natriuretic activity of furosemide without further increasing potassium excretion. In contrast, the combination of hydrochlorothiazide and furosemide resulted in a 3-fold increase in potassium excretion. These data suggest that CVT-124 possesses unique diuretic activity and, as such, it represents a potential new therapeutic in fluid retaining disorders. In addition, its unique mechanism of action suggests that CVT-124 would be effective in otherwise diuretic-resistant patients.     

Conformational studies of synthetic lipid A analogues and partial structures by infrared spectroscopy

Synthetic lipid A analogues and partial structures were analyzed and compared with natural hexaacyl lipid A from E. coli applying Fourier transform infrared spectroscopy. The investigations comprised (i) the measurement of the beta <=> alpha phase transition of the acyl chains via monitoring of the symmetric stretching vibration of the methylene groups, (ii) an estimation of the supramolecular aggregate structures evaluating vibrations from the interface like ester carbonyl and applying theoretical calculations (iii) a determination of the inter- and intramolecular conformations monitoring functional groups from the interface and the diglucosamine backbone (ester carbonyl, phosphate). The phase transition temperature Tc was found to be nearly a linear function of the number of acyl chains for most bisphosphoryl compounds indicating comparable packing density, whereas the deviating behaviour of some samples indicated a higher packing density. From the determination of the supramolecular aggregate structures (cubic, HII) of natural hexaacyl lipid A by X-ray small-angle diffraction, the existence of the same aggregate structures also for the synthetic hexaacyl lipid A was deduced from the nearly identical thermotropism of the ester carbonyl band. From this, a good approximation of the supramolecular structures of all synthetic samples was possible on the basis of the theory of Israelachvili. The analysis of the main phosphate band, together with that of the Tc data and former colorimetric results, allowed the establishment of a model of the intermolecular conformations of neighbouring lipid A/LPS molecules. The biological relevance of the findings is discussed in terms of the strongly varying biological activity (between high and no activity) of the samples.     

Martefragin A, a novel indole alkaloid isolated from red alga, inhibits lipid peroxidation

Martefragin A (1), a novel indole alkaloid, was isolated from a red alga, Martensia fragilis, by repeated column chromatography. The structure of 1 was elucidated on the basis of spectral analysis of its methyl ester (2), including 1H- and 13C-NMR, 1H-1H correlation spectroscopy (COSY), and 13C-1H COSY. A single crystal X-ray analysis of the hydrochloride of 1 confirmed the assignment. Martefragin A (1) showed inhibitory activity on NADPH-dependent lipid peroxidation in rat liver microsomes. The IC50 values of 1, alpha-tocopherol and ascorbic acid were 2.8, 87 and 200 microM, respectively.     

The pharmacological characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist, NAD-299

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.     

Stereoselective synthesis of a novel and bifunctional endothelin antagonist, IRL 3630

IRL 3630 (3), a single enantiomer of IRL 3461 with more potency was identified. Coupling reaction of the racemic fragment (1) with the chiral (L)-valinesulfonamide (2) under a biphasic solvent system (CH2Cl2-H2O) successfully led to the predominant formation of the desired isomer (3) with concomitant isomerization of 1. IRL 3630, N-butanesulfonyl-[N-(3,5-dimethylbenzoyl)-N-methyl-3-[4-(5-+ ++isoxazolyl) -phenyl]-(D)-alanyl]-(L)-valineamide, is a highly potent and bifunctional (ETA + ETB) antagonist [Ki(ETA) = 1.5 nM, Ki(ETB) = 1.2 nM].     

Suppression of murine experimental autoimmune hepatitis by T-cell vaccination or immunosuppression

Patients with autoimmune hepatitis (AIH) usually require immunosuppressive therapy for many years, if not for a lifetime. Experimental immunotherapy such as T-cell vaccination aims at manipulating the immune system in such a way that autoimmunity is specifically regulated to enable long-lasting correction of the disease process. We aimed to test the feasibility of T-cell vaccination as well as conventional immunosuppression in the murine model of experimental autoimmune hepatitis (EAH). EAH was induced in 5- to 7-week-old BALB/c mice by immunization with syngeneic liver homogenate in complete Freund's adjuvant. For T-cell vaccination, splenocytes were removed from animals 14 days after induction of EAH and from control animals, and activated in vitro by mitogen stimulation with Concanavalin A (Con A). Activated T cells were irradiated and injected at 5 x 10(7) cells per animal as T-cell vaccine. Immunosuppression in control animals was performed with prednisolone with or without azathioprine. T-cell vaccination with T cells from EAH animals, but not with irrelevant T cells, was able to protect animals from EAH, reducing the average disease severity from 2.2 (+/-0.3) to 0.5 (+/-0.3) (P < .01). T-cell vaccination was also able to treat EAH, because application of the vaccine 2 weeks after induction of the disease significantly reduced disease activity at week 4 from 2.4 (+/-0.4) to 1.1 (+/-0.2) (P < .05). Both passive transfer of disease and the capacity to protect by T-cell vaccination was mediated by CD4 T cells. Specific cellular recognition of activated disease-inducing T cells could be detected in vaccinated animals. Immunosuppressive drugs could also suppress EAH. Thus, T-cell vaccination in EAH is feasible and effective. Stimulation of a regulatory T-cell network is the likely mechanism of action by which T-cell vaccination can suppress EAH.     

E2011 a novel, selective and reversible inhibitor of monoamine oxidase type A

E2011, (5R)-3-[2-((1S)-3-cyano-1hydroxypropyl)benzothiazol- 6-yl]-5-methoxymethyl-2-oxazolidine, is a novel inhibitor of monoamine oxidase type A (MAO-A). We have characterized the neurochemical and pharmacological profiles of E2011 and compared them with those of known inhibitors of MAO-A. E2011 potently inhibited MAO-A with more than 30,000 times higher selectivity for MAO-A relative to MAO-B in rat brain homogenate. E2011 did not affect putative neural receptors or reuptake of biogenic amines into synaptosomes of rat brain, which suggests that it is specific to monoaminergic systems. In vivo, E2011 at a dose of 0.3 mg/kg p.o. exhibited potent MAO-A inhibitory activity, whereas MAO-B inhibition was not observed even at 100 mg/kg p.o. E2011 inhibited monoamine metabolism in the rat brain, but the effect disappeared 24 h after administration. Like other reversible MAO-A inhibitors, E2011 did not show a cumulative inhibitory effect during repeated administration for 7 days. However, inhibition of MAO-A by E2011 in ex vivo experiments appeared to be less potent than that by moclobemide. The MAO-A inhibition by E2011 was partially but significantly reversed by dialysis at 4 degrees C for 24 h, which indicates that E2011 could be dissociated from the enzyme. These findings suggest that E2011 is a reversible and highly selective inhibitor of MAO-A. The potency of inhibition by highly reversible MAO-A inhibitors such as E2011 is likely to be underestimated in ex vivo studies because of dilution of the homogenate in the assay system.     

16-Epiestriol, a novel anti-inflammatory nonglycogenic steroid, does not inhibit IFN-gamma production by murine splenocytes

All the steroidal anti-inflammatory drugs currently available are glucocorticoids. The desired anti-inflammatory activities of glucocorticoids frequently are accompanied by adverse side effects, notably glycogenic activities and profound immunosuppression, that can limit clinical use. We recently identified 16-epiestriol, a naturally occurring steroid, as exhibiting significant anti-inflammatory activity without glycogenic activity. In the present study, we compared the effects of 16-epiestriol and hydrocortisone on the capacity of murine splenocytes to produce interferon-y (IFN-y). We injected young adult male BDF1 mice once with 20 mg/kg hydrocortisone or 20, 5, or 1 mg/kg 16-epiestriol and 4 h later harvested the splenocytes. Flow cytometric analysis confirmed that 16-epiestriol did not alter the number of CD3+ T cells in the spleen. In contrast to the suppressive effects of hydrocortisone, none of the 16-epiestriol concentrations inhibited concanavalin A-stimulated IFN-gamma production by spleen cells, as determined by ELISA. Incubating spleen cells from untreated mice in concentrations of 16-epiestriol ranging from 1 mg/ml to 100 pg/ml did not alter profiles of IFN-gamma production, in contrast to the suppressive dose-response effects of hydrocortisone. Collectively, these results support the contention that 16-epiestriol may be a clinically useful safe anti-inflammatory steroid without profound immunosuppressive activities.     

Conformational rigidity versus flexibility in a novel peptidic neurokinin A receptor antagonist

Neurokinin A receptor antagonists have been proposed as a new class of drugs for several applications in humans (asthma, intestinal motility, etc.). The rational design, synthesis, structural characterization and biological activity evaluation of a new potent, highly selective, long-lasting, peptide-based receptor antagonist are reported. The structure-activity relationship indicates that the conformational rigidity determines potency, specificity and especially the long life of the molecule in the living body. MEN10627 is the prototype of a new class of cyclic, peptide-based, neurokinin A receptor antagonists and it is a suitable candidate for clinical testing in humans.     

Implantation of a synthetic cornea: design, development and biological response

Our goal was to evaluate 3 different designs of synthetic corneas in vivo. All devices had a transparent hydrogel center molded to a porous peripheral skirt. Over 30 devices were implanted into rabbits and followed for up to 6 months. The devices were preseeded with rabbit stromal fibroblasts, which enhanced the rate of fibroplasia. The anterior surface of the hydrogel was modified using argon rf plasma treatments. Clinical examinations were performed, and histological analyses were conducted on tissue throughout the time course. Our optimal model ranged from 4.5 to 6 mm and had an extended porous skirt increasing the surface area for fibroplasia and ultimate anchorage of the device. Fibroplasia occurred in this model, and collagen was detected by 28 days. The anterior chamber was normal with no detectable leakage of aqueous humor. Glycosaminoglycans were detected and followed the time course outlined previously when porous material itself was inserted into the stroma. We present the first demonstration that rabbit limbal epithelial cells can migrate onto the synthetic cornea in vivo.     

Anti-hepatitis A virus antibody response elicited in mice by different forms of a synthetic VP1 peptide

To explore factors associated with longevity, we studied geographic distribution of centenarians in Japan, based on 1990 the population census. We calculated the proportion of centenarians from ratio of number of centenarian to that of population aged 65 years or older. Centenarians in Japan consisted of number 4,152 persons. By prefecture, Tokyo had the most centenarians (383), followed by Okinawa (193) and Fukuoka (151) prefectures. Fukui had the least (24), followed by Akita (26) and Ishikawa (29) prefectures. The proportion of centenarians in Japan was 21.6 (per 100,000 populations) in 1990. By prefecture, the highest proportion lived in Okinawa (133.8), whereas the fewest were found in Akita (8.9). The relationship between geographic distribution of centenarians and environmental factors and nutritional factors were analyzed. Correlation coefficients between proportion of centenarians and mean temperature, high quality of welfare work and of medical services, and having much leisure time were positively significant. As for nutritional factor, correlation coefficients between proportion of centenarians and protein (% of energy) was positively significant, while intake of total energy was negatively significant.     

State-dependent NMDA receptor antagonism by Ro 8-4304, a novel NR2B selective, non-competitive, voltage-independent antagonist

1. Subunit-selective blockade of N-methyl-D-aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B-selective NMDA antagonist, 4-?3-[4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxy-propoxy ?-benzamide (Ro 8-4304), which exhibits >100 fold higher affinity for recombinant NR1(001)/NR2B than NR1(001)/NR2A receptors. 2. Ro 8-4304 is a voltage-independent, non-competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state-dependent mode of action similar to that described for ifenprodil. 3. The apparent affinity of Ro 8-4304 for the NMDA receptor increased in an NMDA concentration-dependent manner so that Ro 8-4304 inhibited 10 and 100 microM NMDA responses with IC50s of 2.3 and 0.36 microM, respectively. Currents elicited by 1 microM NMDA were slightly potentiated in the presence of 10 microM Ro 8-4304, and Ro 8-4304 binding slowed the rate of glutamate dissociation from NMDA receptors. 4. These results were predicted by a reaction scheme in which Ro 8-4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist-unbound resting state. Additionally, Ro 8-4304 binding resulted in a 3 4 fold increase in receptor affinity for glutamate site agonists. 5. Surprisingly, whilst exhibiting a similar affinity for NR2B-containing NMDA receptors as ifenprodil, Ro 8-4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B-selective compounds.     

Crosslinking of apolipoprotein E by products of lipid peroxidation

Apolipoprotein E (APOE) genotype and advancing aging are interacting ri sk factors in the expression of late onset and sporadic Alzheimer's Disease (AD). We tested the hypothesis that 2 products of lipid peroxidation, malondialdehyde (MDA) and 4 hydroxy-2-nonenal (HNE), covalently modify APOE and alter its metabolism. In vitro, both HNE and MDA crosslinked purified APOE3 and APOE4. HNE was a more potent crosslinker than MDA, and purified APO3 was more susceptible to crosslinking by HNE than was purified APOE4. In P19 neuroglial cultures, oxidative stress with lipid peroxidation led to increased intracellular accumulation of anti-HNE and anti-APOE immunoreactive proteins of approximately 50 kDa. Intercellular accumulation of the 50 kDa APOE-immunoreactive protein (APOE-50) was not prevented by cyclohexamide, suggesting formation by post-translational mechanisms. In CSF, a 50 kDa APOE-immunoreactive protein co-migrated with proteins most immunoreactive for HNE and MDA adducts, containing NaB3H4-reducible bonds. These proteins were in CSF from adult subjects (with or without dementia), and in AD patients homozygous for APOE3 or APOE4 alleles. These data suggest that HNE covalently crosslinks APOE in P19 neuroglial cultures to form a 50 kDa protein, and that similar modifications of APOE appear to occur in vivo.     

Animal studies on the endocrinological profile of dienogest, a novel synthetic steroid

Dienogest is an orally active synthetic steroid that is used for contraception and is currently being studied for the possible treatment of endometriosis. Earlier we demonstrated that dienogest had therapeutic effects on experimental endometriosis in rats and that its mechanisms of action were different from those of drugs currently on the market for the treatment of endometriosis. We also reported preclinically that dienogest showed a potential anticancer action against hormone-dependent cancers that was different from that of progestins. Accordingly, we obtained preclinical background data for the above-described clinical applications and extension of the clinical use of the drug in the near future by investigating the endocrinological profile of dienogest in rabbits and rats. Dienogest was characterized by having a moderate binding affinity for progesterone receptors and by progestational activities: it stimulated endometrial proliferation (> or = 0.01 mg/kg) that was only partially inhibited by RU-486, and induced carbonic anhydrase activity in endometrium (> or = 0.01 mg/kg). Also, it was slightly uterotrophic (> or = 1 mg/kg) with very low binding affinity for oestrogen receptors but without biological androgenic and anabolic activities (100 mg/kg), with neither glucocorticoid activity nor mineralocorticoid activity (100 mg/kg), and with very slight binding affinity for human sex hormone-binding globulin. These findings suggest that dienogest is not a pure progestin and appears to induce fewer side effects than drugs currently on the market for the treatment of endometriosis.