Assessing the emetic potential of PDE4 inhibitors in rats.

1. Type 4 phosphodiesterase (PDE4) inhibitors mimic the pharmacological actions of alpha(2)-adrenoceptor antagonists. This has been postulated as the mechanism by which PDE4 inhibitors induce emesis and was also demonstrated by their ability to reverse xylazine/ketamine-induced anaesthesia. We further characterized this latter effect since it appears to reflect the emetic potential of PDE4 inhibitors. 2. Selective inhibitors of PDE 1, 2, 3, 4 and 5 were studied in rats, on the duration of anaesthesia induced by the combination of xylazine (10 mg kg(-1), i.m.) and ketamine (10 mg kg(-1), i.m.). PMNPQ (i.e. 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline) - PDE4 inhibitor: 0.01 - 3 mg kg(-1)), like MK-912 (alpha(2)-adrenoceptor antagonist: 0.01 - 3 mg kg(-1)), dose-dependently reduced the duration of anaesthesia. In contrast, vinpocetine (PDE1 inhibitor), EHNA (PDE2 inhibitor), milrinone (PDE3 inhibitor) and zaprinast (PDE5 inhibitor) had no significant effect at the doses tested (1 - 10 mg kg(-1)). Analysis of plasma and cerebrospinal fluid (CSF) of treated animals confirmed the absorption and distribution to the brain of the inactive inhibitors. 3. Neither MK-912 (3 mg kg(-1)) nor PMNPQ (0.1 - 1 mg kg(-1)) altered the duration of anaesthesia induced via a non-alpha(2)-adrenoceptor pathway (sodium pentobarbitone 50 mg kg(-1), i.p.). 4. Central NK(1) receptors are involved in PDE4 inhibitor-induced emesis. Consistently, [sar(9), Met(O(2))(11)]-substance P (NK(1) receptor agonist, 6 microg i.c.v.) reduced the duration of anaesthesia induced by xylazine/ketamine. 5. In summary, this model is functionally coupled to PDE4, specific to alpha(2)-adrenoceptors and relevant to PDE4 inhibitor-induced emesis. It therefore provides a novel way of evaluating the emetic potential of PDE4 inhibitors in rats.

PDE4 inhibitors induce emesis in ferrets via a noradrenergic pathway.

The objective of this work was to assess the role of alpha(2)-adrenoceptors in emesis induced by inhibitors of type 4 phosphodiesterase (PDE4) in ferrets. Pre-treatment with yohimbine, MK-912 or MK-467 (alpha(2)-adrenoceptor antagonists) caused sudden and unexpected vomiting. In contrast, clonidine (alpha(2)-adrenoceptor agonist) did not induce emesis at doses ranging from 62.5-250 microg/kg s.c. At the dose of 250 microg/kg, clonidine also provided protection against emesis induced by the PDE4 inhibitors, PMNPQ (i.e. 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline, CT-2450 and R-rolipram. It was postulated that PDE4 inhibitors trigger emesis by mimicking the pharmacological actions of alpha(2)-adrenoceptor antagonists. This hypothesis was strengthened by the demonstration that PDE4 inhibitors can reverse the hypnotic effect of an alpha(2)-adrenoceptor mediated anaesthetic regimen in rats and ferrets. Similar to alpha(2)-adrenoceptor antagonists, PMNPQ, R-rolipram and S-rolipram dose-dependently decreased the duration of anaesthesia in rats injected with the combination xylazine/ketamine. While subcutaneous injections of CT-2450 (3-30 mg/kg) were without effect, a central infusion (6 microg i.c.v.) decreased the duration of anaesthesia. These studies suggest that the ferret is an appropriate model to study emesis induced by PDE4 inhibitors and that these compounds trigger the emetic reflex via a noradrenergic pathway, mimicking the pharmacological actions of a pre-synaptic alpha(2)-adrenoceptor inhibition.

Selective potentiating effect of RS14203 on a serotoninergic pathway in anesthetized rats.

The usefulness of selective inhibitors of type 4 phosphodiesterase (PDE4) in the treatment of inflammation and pulmonary diseases is limited by their side effects: nausea and vomiting. We studied the effect of three structurally diverse PDE4 inhibitors on the vagal nerve afferent and efferent fibers in anesthetized rats. The effects of RS14203, (R)-rolipram, and CT-2450 were evaluated on the von Bezold-Jarisch reflex (vagal afferent fibers) and in a model of vagal electrical stimulation (vagal efferent fibers). All three PDE4 inhibitors were administered at 1, 10, or 100 microg/kg (iv) 15 min prior to the induction of bradycardia by an iv injection of 2-methyl-5-HT (von Bezold-Jarisch reflex) or by vagal electrical stimulation. At 100 microg/kg, RS14203 significantly potentiated the 2-methyl-5-HT response. No statistically significant effects were observed with (R)-rolipram or CT-2450 at the doses studied. RS14203, (R)-rolipram, or CT-2450 (1-100 microg/kg iv) did not affect the bradycardia induced by vagal electrical stimulation. Consequently, our results show that RS14203 selectively facilitates serotoninergic neurotransmission in vagal afferent fibers. The emetic action of RS14203 may be mediated by this mechanism.

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217.

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.

2-heterosubstituted-3-(4-methylsulfonyl)phenyl-5-trifluoromethyl pyridines as selective and orally active cyclooxygenase-2 inhibitors.

A series of novel 2-alkoxy, 2-thioalkoxy and 2-amino-3-(4-methylsulfonyl)phenylpyridines has been synthesized and shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. Structure-activity relationship studies have demonstrated that central pyridine ring substituents play an important role in the COX-2 potency, selectivity vs the COX-1 enzyme, and oral activity.

Use of BONSAI decision trees for the identification of potential MHC class I peptide epitope motifs.

Recognition of short peptides of 8 to 10 mer bound to MHC class I molecules by cytotoxic T lymphocytes forms the basis of cellular immunity. While the sequence motifs necessary for binding of intracellular peptides to MHC have been well studied, little is known about sequence motifs that may cause preferential affinity to the T cell receptor and/or preferential recognition and response by T cells. Here we demonstrate that computational learning systems can be useful to elucidate sequence motifs that affect T cell activation. Knowledge of T cell activation motifs could be useful for targeted vaccine design or immunotherapy. With the BONSAI computational learning algorithm, using a database of previously reported MHC bound peptides that had positive or negative T cell responses, we were able to identify sequence motif rules that explain 70% of positive T cell responses and 84% of negative T cell responses.

An HLA-binding-motif-aided peptide epitope library: a novel library design for the screening of HLA-DR4-restricted antigenic peptides recognized by CD4+T cells.

Susceptibility to a series of autoimmune diseases is strongly associated with particular HLA class II alleles. Identification of T cell clones and antigenic epitopes bound by HLA class II molecules involved in autoimmune diseases is critical to understanding the etiology of these HLA class II-associated diseases. However, establishment of T cell clones in autoimmune diseases is difficult because the antigenic peptides are unknown. Peptide library methods which include all possible peptide sequences offer a potentially powerful tool for the detection of cross-reactive antigenic peptides recognized by T cells. Here, we reduced the number of peptides per mixture by utilizing the known binding motifs of peptides for the HLA-DRB1*0405 molecule and evaluated the effectiveness of this library design. Each library mixture evoked a strong proliferative response in the unprimed peripheral blood lymphocytes (PBL) from HLA-DRB1*0405-positive donors but little or no response in the PBL from HLA-DRB1*0405-negative donors. The library also detected antigenic peptides that activated three antigen-specific T cell lines restricted by HLA-DRB1*0405, with different specificities. The motif-based approach thus presents a powerful method for monitoring T cells in large, heterogeneous T cell populations and is useful for the identification of the mimic peptide epitopes of T cell lines and clones.

MHC class I bound peptides of a colon carcinoma cell line, a Ki-ras gene-targeted progeny cell line and a B cell line.

MHC class I associated peptides on cancer cells represent potential targets for CD8+ cytotoxic T cell activity against tumor cells. We eluted the naturally bound MHC class I peptides of a colon carcinoma cell line and compared them to peptides isolated from a B cell line and a slow-growing activated Ki-ras-disrupted colon cancer cell line. While we failed to detect any significant differences in class I associated peptides due to the presence or absence of activated Ki-ras in colon cancer cell lines, the colon cancer cell lines and B cell line presented vastly different peptide repertoires in the context of HLA-A*0201 molecules.

2-Pyridinyl-3-(4-methylsulfonyl)phenylpyridines: selective and orally active cyclooxygenase-2 inhibitors.

A series of novel 2-pyridinyl-3-(4-methylsulfonyl)phenylpyridines has been synthesized and evaluated with respect to their ability to inhibit the isozymes of cyclooxygenase, COX-1, and COX-2. Optimum COX-2 activity is observed by introduction of a substituent at C5 of the central pyridine. 5- Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine 33 was identified as the optimum compound in this series.

Differences in MHC class I self peptide repertoires among HLA-A2 subtypes.

To investigate how single amino acid substitutions in MHC class I molecules affect differences in peptide repertoires, we eluted and sequenced the naturally processed peptides from three HLA-A2 subtypes (HLA-A*0204, -A*0206, and -A*0207) that differ by a single amino acid residue substitution each with HLA-A*0201 at the floor of the binding groove. Allele-specific peptide motifs for each HLA-A2 subtype substantially differed from that of HLA-A*0201 in the dominant anchor residues. The relative signal intensities for 18 self peptides, determined by mass spectrometry, precisely reflected these peptide motifs. Some overlapping peptides were isolated from both HLA-A*0201 and a single HLA-A2 variant, but no peptide was ubiquitously found across all variants. To rationalize the differences in peptide motifs, possible conformations of each allele were computer modeled by energy minimization calculations based on the reported crystal structure of HLA-A*0201. According to our models, the differences in peptide motifs could be explained by substituted-residue-driven conformational changes for each MHC-peptide complex. These results demonstrate the fine differences between HLA-A2 subtype self peptide repertoires and contribute to the prediction of antigenic peptides.

Innocuity of agents for radiocontrast enhancement of urinary tract stones.

OBJECTIVES: Mineral kidney stones are frequently difficult to detect due to their radiotranslucency. We have recently developed a method that enhances the visibility of such stones by retrograde infusions of certain heavy metal salt solutions such as cesium or lanthanide gadolinium. This report describes toxicologic studies carried out on the use of those contrast agents to introduce this technique eventually into clinical trials. METHODS: Systemic absorption was assessed in dogs through infusion of radioactive contrast agent into the renal pelvis with or without ureteral obstruction. Radioactivity in urine and blood was monitored. Local toxicity was studied in animals infused with the contrast agent at intervals up to 4 weeks. RESULTS: Reabsorption studies under high intrapelvic pressures (70 cm H2O or higher), demonstrated reabsorption of cesium. However, at normal intrapelvic pressures, only a moderate reabsorption of cesium was observed. No gadolinium reabsorption was detected even at high intrapelvic pressures. Histopathologic studies showed no major urothelial lesions but only a transient inflammatory reaction that was undetectable 4 weeks following exposure to gadolinium salts. CONCLUSIONS: Gadolinium salt solutions are good positive radiocontrast agents for mineral kidney stones without having serious toxic effects or systemic reabsorption.

Involvement of NK1 and NK2 receptors in pulmonary responses elicited by non-adrenergic, non-cholinergic vagal stimulation in guinea-pigs.

Previous studies from our laboratory using exogenously administered neurokinin (NK) agonists have shown that both NK1- and NK2-receptor subtypes are involved in plasma extravasation in the guinea-pig airways. In the present study, we have extended these observations using antidromic vagal stimulation to stimulate sensory c-fibres as a means of eliciting the release of endogenous tachykinins in propranolol- and atropine-treated guinea-pigs. Antidromic vagal stimulation (5 ms, 30 s) induced frequency-dependent (1-10 Hz) bronchoconstriction that was completely abolished by co-administration of the NK1-selective antagonist CP-99,994 ((2s-methoxy-benzyl)-(2-phenyl-piperidin-3s-yl)-amine), and the NK2-selective antagonist SR-48,968 ((S)-N-methyl-N-[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide), each at a dose sufficient to block NK1 and NK2 receptors, respectively (each at 0.3 mg kg-1, i.v.). In contrast, SR-48,968 when given alone only partially blocked the vagal stimulation-induced bronchospasm, whereas CP-99,994 had no effect. Significant increases (2-3-fold) in plasma extravasation of [125I]fibrinogen in the trachea, main bronchi, distal airways and oesophagus following vagal stimulation (5 Hz, 5 min, 10 V, 5 ms) were observed. Pretreatment with the neutral endopeptidase inhibitor, thiorphan (1 mg kg-1, i.v.), and the angiotensin-converting enzyme inhibitor, enalapril (1 mg kg-1, i.v.), potentiated both vagal stimulation-induced bronchoconstriction and plasma leakage in all tissues examined. This potentiation was due to reduced metabolism of endogenously released tachykinins since enhanced plasma overflow of immunoreactive substance P was observed following vagal stimulation in thiorphan- and enalapril-treated guinea-pigs. CP-99,994 substantially blocked plasma leakage in all parts of the airways and in the oesophagus. In comparison, SR-48,968 had no significant effect in the trachea and the oesophagus but partially inhibited plasma leakage in the main bronchi and distal airways. Co-administration of both CP-99,994 and SR-48,968 abolished the residual plasma leakage in these two regions. These results support the hypothesis that both NK1 and NK2 receptors are involved in tachykinin-induced pulmonary responses in the airways.

Effect of dexamethasone on antigen-induced high molecular weight glycoconjugate secretion in allergic guinea pigs.

The ovalbumin-sensitized guinea pig is commonly used as a small animal model of allergic asthma. This animal model exhibits many of the hallmark characteristics observed in patients afflicted with asthma including nonspecific airway hyperreactivity, airway eosinophilia, early and late phase bronchoconstriction, and plasma extravasation into the airways. In addition, mucous hypersecretion in the airways of asthmatic patients is thought to be responsible for the plugging of distal airways and to contribute to the morbidity and mortality associated with the disease process. In this study we examined whether the allergic guinea pig model exhibits an increase in airway high molecular weight glycoconjugate (HMWG) secretion in response to an antigen challenge and whether dexamethasone exerts any modulatory effects upon the response. Ovalbumin (OVA) -sensitized guinea pigs were challenged with OVA 2 wk following the initial exposure. Trachobronchoalveolar lavages (TBAL) were performed, and the samples were assayed for total eosinophil cell number, eosinophil peroxidase activity (EPO), and both acidic and neutral HMWG content. Morphometric analysis of mucous-containing cells was also performed on tissue sections prepared from the trachea, mainstem bronchus, and three lobes of the left lung. Within 24 h of an antigen challenge, TBAL samples obtained from the allergic guinea pigs exhibited increases in eosinophil cell number, measured EPO enzyme activity, and acidic HMWG content compared to TBAL samples prepared from vehicle-exposed animals. These antigen-induced changes were dependent on the concentration of aerosolized OVA administered. Exposing the animals to 0.3% OVA provoked a 6.23-fold increase in airway eosinophils, 15-fold elevation in TBAL EPO enzyme activity, and 175% increase in TBAL acidic HMWG. No significant changes in TBAL neutral HMWG were measured. The changes in measured EPO activity correlated with the levels of acidic HMWG found in the TBAL samples (r = 0.73, P < or = 0.001). The measured increase in TBAL acidic HMWG was time dependent and was found to be maximal at 2 h post-antigen challenge. Morphometric analysis of Alcian blue (pH 2.5) -stained airway sections showed a decline in stored mucosubstances following the antigen exposure, supporting the notion that the allergic guinea pig model exhibits a mucosecretory component. Pretreating the animals with dexamethasone attenuated the antigen-induced release of HMWG and changes in measured EPO activity. In conclusion, these data indicate that the allergic guinea pig may be a useful model for examining the neural and cellular mechanisms underlying mucus hypersecretion in individuals afflicted with bronchial asthma.

Neurokinin receptors subserving plasma extravasation in guinea pig airways.

In the respiratory system the tachykinins substance P and neurokinin A exhibit a variety of effects on airway function that include bronchoconstriction, vasodilatation, and plasma extravasation. Increased microvascular permeability with accompanying plasma extravasation is a principal cause of tissue edema observed in asthma. In guinea pig airways it has been suggested that neurogenic plasma extravasation is mediated by tachykinins, released from sensory nerve terminals, acting via neurokinin (NK) receptors. We have characterized NK receptor mediated plasma extravasation in guinea pig airways, using 125I-labelled human fibrinogen as a marker for leakage. Extravasation was induced using selective NK1 and NK2 receptor agonists, capsaicin, or nonadrenergic, noncholinergic nerve stimulation. The inhibitory effects of the selective nonpeptide NK receptor antagonists (CP 99,994 for NI1 and SR 48,968 for NK2) were also examined. Results from our studies demonstrate conclusively that only NK1 receptors subserve plasma extravasation in the trachea and large airways of the guinea pig. In start contrast, extravasation in the lower airways (secondary bronchi and intraparenchymal airways) of the guinea pig is mediated by both NK1 and NK2 receptors.

Evaluation of bronchoconstriction induced by neurokinins and its inhibition by selective nonpeptide antagonists in conscious guinea pigs, using a double-chamber plethysmograph technique.

Bronchoconstriction induced by inhaled neurokinins, leukotriene D4 (LTD4), and histamine was examined in conscious guinea pigs, using a double-chamber plethysmography. The reliability of the plethysmograph was established by obtaining stable baseline values of key pulmonary parameters, including specific airway resistance, over a 4-day period. As well, the usefulness of the setup was confirmed using LTD4 and the LTD4 antagonist MK-571. Aerosols of MK-571 inhibited the bronchoconstriction induced by LTD4 (0.3 microM, 3 min aerosol) with an IC50 value of 65 +/- 16 nM. Inhaled neurokinin A (NKA), substance P (SP), [beta Ala8]NKA(4-10), or [Sar9,Met(O2)11]SP at concentrations up to 10 microM had no bronchoconstrictive effect, unless the guinea pigs were pretreated with the neutral endopeptidase inhibitor thiorphan (0.2 mg/mL, 5 min aerosol). The rank order of bronchoconstriction potency was LTD4 > [beta Ala8]NKA(4-10) approximately NKA > [Sar9,Met(O2)11]SP approximately SP >> histamine. Hyperresponsiveness to NKA-induced bronchoconstriction was evident after 1 day and lasted for 4 days. The response to NKA was not inhibited by mepyramine, indomethacin, or MK-571 but was significantly reduced by atropine and hexamethonium, suggesting the involvement of a cholinergic mechanism. Aerosols of SR-48,968 a selective NK2 antagonist, had potent effects on the bronchoconstriction induced by NKA (1 microM, 3 min aerosol), with an IC50 value of 17 +/- 3 nM. SR-48,968 was also active when administered intraperitoneally. The NK1 antagonist CP-99,994 (0.1 microM, 10 min aerosol) inhibited the responses to SP by 70% but had no effect on NKA-induced responses at concentrations up to 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of hamster brain polyribosome-cytomatrix complexes.

We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins. This method is based on gentle dispersion of brain tissue and low speed centrifugation. This fraction is enriched in typical cytoskeletal proteins as glial fibrillary protein, neurofilament proteins and actin. Messenger RNA did not seem to be involved in the polyribosome association to the cytomatrix as shown by the effect of exposure to micrococcal nuclease. On the other hand, in vivo disruption of protein synthesis by acute experimental phenylketonuria, hypothermia or heat-shock did not cause the release of ribosomes from the cytomatrix.