Phase 2a, randomized, double-blind, placebo-controlled, multicentre, parallel-group study of an H4 R-antagonist (JNJ-39758979) in adults with uncontrolled asthma.

BACKGROUND: The effects of H4 R antagonists in preclinical asthma models support the study of antagonists of the H4 R in the treatment of asthma in humans. JNJ-39758979 is a potent and highly selective oral H4 R antagonist. OBJECTIVE: We sought to evaluate the safety and efficacy of the H4 R-antagonist JNJ-39758979 in adult patients with uncontrolled asthma. METHODS: One hundred and fifteen eligible patients were randomly assigned to JNJ-39758979 300 mg or placebo once daily for 12 weeks in this phase 2a, double-blind, multicenter, placebo-controlled study. Primary efficacy was assessed via week-12 change from baseline in pre-bronchodilator forced expiratory volume in 1 second (FEV1 ). Secondary efficacy assessments included patient-reported outcome (PRO) asthma assessments (Asthma Daily Diary data [AM and PM peak expiratory flow rate, number of puffs of albuterol/salbutamol, the presence of nocturnal awakenings and asthma symptom score]). RESULTS: The study did not meet the primary end-point. However, nominally significant improvements in pre-bronchodilator FEV1 were observed with JNJ-39758979 versus placebo at week 12 in pre-specified subgroups with elevated exhaled nitric oxide, sputum eosinophils or blood eosinophils at baseline. Nominally significant improvements across PRO assessments were consistently observed in the overall population, as well as in eosinophilic subgroups. Safety, such as adverse event rates, was comparable between JNJ-39758979 and placebo. No serious adverse events were reported. No clinically relevant changes in laboratory values were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The findings suggest potential benefit of H4 R antagonists on lung function and asthma control in eosinophilic asthma patients and warrant further evaluation of this mechanism in asthma with eosinophilic inflammation. NCT00946569.

The histamine H4 receptor antagonist JNJ7777120 induces increases in the histamine content of the rat conjunctiva.

OBJECTIVES: Although the H(4) receptor localisation in the eye is unresolved, this study aimed to investigate the effects of the H(4) receptor antagonist JNJ7777120 in a model of experimental conjunctivitis. METHODS: JNJ7777120, at 0.005-1 mmol/l, was instilled into the lower conjunctival fornix of normal and compound 48/80 (C48/80)-challenged eyes of male Wistar rats, in the absence or presence of 40 mg/ml disodium cromoglycate (DSCG). Conjunctival histamine content was quantified 20 min post-challenge. Statistical analyses were performed by ANOVA. RESULTS: JNJ7777120 increased dose-dependently (r = 0.784, p < 0.001) the conjunctival histamine content. In the C48/80-challenged eye no effect of the antagonist was observed. Co-administration of JNJ7777120 with DSCG resulted in a biphasic action of JNJ7777120, implying a competitive action of the two agents. CONCLUSIONS: These data suggest a functionality of the H(4) receptor in the rat eye and address questions on the localization and the role of the receptor in ocular inflammation.

Kinetics of small molecule inhibitor binding to p38 kinase.

p38 mitogen-activated protein kinase (MAPK) (p38/p38-alpha/CSBP2/RK) has been implicated in the regulation of many proinflammatory pathways. Because of this, it has received much attention as a potential drug target for controlling diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, osteoporosis, and many others. A number of small molecule inhibitors of this kinase have been described, and in this paper we have used surface plasmon resonance to directly measure and quantitate their binding to p38. Despite the relatively low molecular mass (approximately 400 Da) of these inhibitors, specific binding can be observed. For the two most potent inhibitors studied, SB 203580 and RWJ 67657, dissociation constants, K(d)'s, of 22 and 10 nm, respectively, were obtained. These values closely match the IC(5)0 values observed in a cell-based TNF alpha release assay implying that p38 plays a major role in TNF alpha release. The association and dissociation rates for the binding of these inhibitors to p38 have also been quantitated. SB 203580 and RWJ 67657 have very similar association rates of around 8 x 10(5) m(-1) x s(-1), and the differences in affinity are determined by different dissociation rates. The weaker binding compounds have dissociation rates similar to SB 203580, but the association rates vary by an order of magnitude or more. The direct measurement of compounds binding to p38 may help in understanding the difference between potency and efficacy for these inhibitors. This in turn may yield clues on how to develop better inhibitors.

Composite membrane deformation on the mesoscopic length scale.

The physics of soft materials can be investigated using nuclear spin-lattice relaxation, which depends on the spectral densities of motion in the MHz range. For the first time, NMR relaxation has been used to study influences of the acyl length, polar head groups, a cosurfactant, and cholesterol on the viscoelastic properties of membrane lipids. The results imply the concept of elastic deformation is relevant on lengths approximately equal to the bilayer thickness and less, involving a broad spectrum of collective modes which contribute to the forces between lipid bilayers.

Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide.

We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.

Structure and function in rhodopsin: peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase.

Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in Vmax for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R/R135E showed a 50% reduction in Vmax relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the Km by 5-fold for E239Q and E247Q/K248L/E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.

Structure and function in rhodopsin: rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state.

The Glu-134-Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as "molecular sensors" of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.

Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.

Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.

Membrane thickness and molecular ordering in Acholeplasma laidlawii strain A studied by 2H NMR spectroscopy.

Since Acholeplasma laidlawii can be restricted to incorporating fatty acids from the growth medium into its membrane lipids, it is possible to study the effects of the length of the acyl chains on the properties of the membrane of the organism. A. laidlawii strain A-EF22 was grown with mixtures of one perdeuterated saturated fatty acid and one monounsaturated fatty acid. The average length () of the acyl chains in the membrane lipids varied from 14.6 to 19.9, and the degree of unsaturation ranged from 21 to 79 mol %. 2H nuclear magnetic resonance (NMR) spectra were recorded on whole cells, on intact membranes, and on lipids extracted from these membranes. It was found that the NMR spectra for all three cases were very similar, yielding deuterium quadrupolar splittings typical for the lamellar liquid-crystalline phase (L alpha) found in model membrane systems. The use of a perdeuterated acyl chain as a reporter molecule allowed for the calculation of order parameters averaged over the entire system. These measurements yielded a wide range of average order parameters varying from 0.136 to 0.186 for the membranes and from 0.137 to 0.181 for the extracted lipids. From the order parameters the average acyl chain length can be calculated, which is related to the average membrane thickness. This value ranged from 23.2 to 30.6 A. When either the order or the membrane thickness of the intact membranes was compared to that of the extracted lipids, only slight or even undetectable differences were found. This implies that the proteins associated with the membranes do not have any large effect on the overall packing of the membrane lipids, even though the membrane thickness varied by approximately 8 A over the series studied. A decrease in the ordering of the acyl chains was observed when the length of the acyl chains incorporated from the growth medium was increased in either the membranes or the extracted lipids. This decrease correlated with the decrease in the fraction of monoglucosyldiacylglycerol (MGlcDAG) found in the membrane. Since both the average order and the membrane thickness varied, it is proposed that by changing the mole fraction of MGlcDAG the organism regulates either the membrane curvature energy or the permeability, both of which are related to lipid packing in the bilayer.

Curvature, order, and dynamics of lipid hexagonal phases studied by deuterium NMR spectroscopy.

Solid-state deuterium (2H) NMR spectroscopy enables one to study both equilibrium and dynamical properties of membrane constituents at the molecular level and can yield significant insights regarding the organization of non-bilayer lipid aggregates. We have investigated a representative unsaturated phosphatidylethanolamine, viz., 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero- 3-phosphoethanolamine, PLPE-d31, in the lamellar, or L alpha, phase and the reversed hexagonal, or HII, phase. Phosphorus-31 (31P) NMR studies of PLPE-d31 in the HII phase revealed that the chemical shift anisotropy of the phosphoethanolamine head groups, delta sigma, was scaled by the expected geometrical factor of -1/2 relative to the lamellar state. However, we found the occurrence of a further reduction in the 2H NMR quadrupolar splittings, delta vQ, of the 2H-labeled palmitoyl acyl chain segments. These observations point toward the role of interfacial curvature with regard to properties of reverse hexagonal phase lipids, and indicate that the pivotal position or neutral surface of approximately constant area may lie near the glycerol or polar head group region. Variations in the acyl chain packing due to curvature of the aqueous interface yield significant differences in the segmental order profiles as determined by 2H NMR spectroscopy. The latter reflect the local orientational order of the acyl chains and can be used together with simple statistical theories to extract positional or structural information. Average projected acyl chain lengths and mean interfacial or cross-sectional areas for PLPE-d31 in the different phases have been calculated. In addition, we describe a new means of estimating the radius of curvature of HII phase lipid aggregates utilizing 2H NMR spectroscopy, which is based on the difference between the lamellar and hexagonal phase order profiles. Here the radius of curvature, Rc, is defined as the distance from the center of the water core to the lipid/water interface, near the carbonyl segments of the acyl chains, giving Rc = 25.4-28.1 A for PLPE-d31 in the HII phase at 60 degrees C. This value is in good agreement with previous X-ray diffraction studies of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Alternatively, the data yield for the radius of the central water core that Rw = 17.8-20.5 A at 60 degrees C. The differences in geometry also lead to higher quadrupolar echo relaxation rates (R2e) for the lipid acyl segments closest to the aqueous interface in the HII versus the L alpha phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of bile salts on monolayer curvature of a phosphatidylethanolamine/water model membrane system.

A partial phase diagram of the ternary system dioleoylphosphatidylethanolamine (DOPE)/sodium cholate/water has been determined using 31P Nuclear Magnetic Resonance (NMR) spectroscopy. In the absence of cholate, it is well known that the DOPE/water system forms a reversed hexagonal (HII) phase. We have found that addition of even small amounts of cholate to the DOPE/water system leads to a transition to a lamellar (L alpha) phase. At higher cholate concentrations, a cubic (I) phase (low water content) or a micellar solution (L1) phase (high water content) is present. Thus, cholate molecules have a strong tendency to alter the lipid monolayer curvature. Increasing the concentration of cholate changes the curvature of DOPE from negative (HII phase), through zero (L alpha phase), and finally to a phase of positive curvature (micellar solution). This observation can be rationalized in terms of the molecular structure of cholate, which is amphipathic and has one hydrophobic and one hydrophilic side of the steroid ring system. The cholate molecules have a tendency to lie flat on the lipid aggregate surface, thereby increasing the effective interfacial area of the polar head groups, and altering the curvature free energy of the system.

Molecular areas of phospholipids as determined by 2H NMR spectroscopy. Comparison of phosphatidylethanolamines and phosphatidylcholines.

The role of lipid diversity in biomembranes is one of the major unsolved problems in biochemistry. One parameter of possible importance is the mean cross-sectional area occupied per lipid molecule, which may be related to formation of nonbilayer structures and membrane protein function. We have used 2H NMR spectroscopy to compare the properties of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the L alpha phase. We find that DPPE has greater segmental order than DPPC, and that this increase in order is related to the smaller area per acyl chain found for DPPE. Values of the mean cross-sectional chain area are calculated using a simple diamond lattice model for the acyl chain configurational statistics, together with dilatometry data. The results obtained for the mean area per molecule are comparable with those from low angle x-ray diffraction studies.

Influences of membrane curvature in lipid hexagonal phases studied by deuterium NMR spectroscopy.

The presence of reversed hexagonal phase, HII, favoring lipids in membranes has been proposed to be significant in various biological processes. Therefore an understanding of the HII phase and the transition from the lamellar to hexagonal phase is of importance. We have applied deuterium NMR spectroscopy to study the bilayer and reversed hexagonal phases of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamin e. The difference in packing between the HII and L alpha phases leads to smaller segmental order parameters in the former case. Since the order profiles are sensitive to the geometry of the aggregates, they can be used to extract structural information about the phases. We present a new means of calculating the radius of curvature, R1, for the HII phase from 2H NMR data. This method gives a value of R1 = 18.1 A, which is in agreement with current understanding of the structure of the HII phase and with x-ray diffraction data.

Magnetic alignment and orientational order of dipalmitoylphosphatidylcholine bilayers containing palmitoyllysophosphatidylcholine.

Mixed bilayers of 1-palmitoyl-sn-glycero-3-phosphocholine (palmitoyllysophosphatidylcholine; PaLPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoyl phosphatidylcholine; DPPC) have been investigated by 2H-NMR and 31P-NMR spectroscopy. Binary phospholipid mixtures were studied in which the acyl chains of one or the other component were perdeuterated. At temperatures below the main order-disorder phase transition, the mixed PaLPC/DPPC bilayers appear to coexist with PaLPC micelles. The micelles disappear at temperatures above the phase transition, where mixed bilayers in the liquid-crystalline state are formed. The orientational order of the alkyl chains of the PaLPC component is essentially identical to that of the DPPC component in the mixed bilayers, both in the low temperature and liquid-crystalline phases. However, the presence of PaLPC perturbs the segmental ordering of DPPC as compared to the pure system. The order is increased in the low-temperature phase, where effective diffusion of the chains about their long axes occurs, but is decreased in the liquid-crystalline phase compared to pure DPPC bilayers. The mixed liquid-crystalline bilayers orient preferentially with their director axes perpendicular to the magnetic field. This alignment is easily observed in 31P- and 2H-NMR spectra, where the intensity of the perpendicular edges of the lineshapes is pronounced. One possible explanation of the magnetic alignment involves alteration of the curvature free energy of the DPPC bilayer due to incorporation of PaLPC in the mixed membranes.