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Background and Aims: Progressive multifocal leukoencephalopathy [PML], a brain infection associated with anti-integrin drugs that inhibit lymphocyte translocation from bloodstream to tissue, can be fatal. Decreased central nervous system [CNS] immune surveillance leading to this infection has been reported in patients with multiple sclerosis or Crohn's disease treated with antiintegrin antibody natalizumab. PF-00547659 is an investigational human monoclonal antibody for inflammatory bowel disease, targeted against α4β7-mucosal addressin cell-adhesion molecule-1 [the integrin ligand selectively expressed in the gut]. We hypothesised that this selective agent would not affect central nervous system immune surveillance. Methods: Cerebrospinal fluid from five healthy volunteers, and from 10 patients with Crohn's disease previously treated with immunosuppressants, was evaluated to assess the feasibility of the study. Subsequently, 39 patients with active Crohn's disease and previous immunosuppression were evaluated over 12 weeks of PF-00547659-induction therapy. We measured total lymphocytes, T cell subsets in cerebrospinal fluid, and circulating β7+ memory cells. Disease activity was assessed using the Harvey-Bradshaw Index. Results: Patients treated with PF-00547659 had no reduction of cerebrospinal fluid lymphocytes, T-lymphocyte subsets, or CD4:CD8 ratio, whereas circulating β7+ memory cells increased significantly. A total of 28/35 [80%] patients had a clinical response and 27/34 [79%] had disease remission. Treatment-related adverse events, none serious, were reported in 23/49 [47%] patients. Conclusions: In patients with active Crohn's disease, natalizumab therapy increases the risk for PML, and the increased risk is thought to be associated with iatrogenic leukopenia within the CNS. PML under PF-00547659 may be a lesser concern, as this agent did not reduce lymphocytes or T cell subsets in the cerebrospinal fluid., SCOPUS: ar.j, info:eu-repo/semantics/published

ObjectiveThis phase II, randomised, double-blind, placebo-controlled clinical trial was designed to evaluate the efficacy and safety of PF-00547659, a fully human monoclonal antibody that binds to human mucosal addressin cell adhesion molecule (MAdCAM) to selectively reduce lymphocyte homing to the intestinal tract, in patients with moderate-to-severe Crohn’s disease (CD).DesignEligible adults were aged 18–75 years, with active moderate-to-severe CD (Crohn’s Disease Activity Index (CDAI) 220–450), a history of failure or intolerance to antitumour necrosis factor and/or immunosuppressive agents, high-sensitivity C reactive protein >3.0 mg/L and ulcers on colonoscopy. Patients were randomised to PF-00547659 22.5 mg, 75 mg or 225 mg or placebo. The primary endpoint was CDAI 70-point decrease from baseline (CDAI-70) at week 8 or 12.ResultsIn all, 265 patients were eligible for study entry. Although CDAI-70 response was not significantly different with placebo versus PF-00547659 treatment at weeks 8 or 12, remission rate was greater in patients with higher baseline C reactive protein (>5 mg/L vs >18.8 mg/L, respectively). Soluble MAdCAM decreased significantly from baseline to week 2 in a dose-related manner and remained low during the study in PF-00547659-treated patients. Circulating β7+ CD4+ central memory T-lymphocytes increased at weeks 8 and 12 with PF-00547659 treatment. No safety signal was seen.ConclusionsClinical endpoint differences between PF-00547659 and placebo did not reach statistical significance in patients with moderate-to-severe CD. PF-00547659 was pharmacologically active, as shown by a sustained dose-related decrease in soluble MAdCAM and a dose-related increase in circulating β7+ central memory T cells.Trial registration numberNCT01276509; Results.

Objective To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn’s disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody. Methods In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, β7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing. Results A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [–87% to –98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for β7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for β7+ effector memory T cells [placebo, 8%; PF-00547659, 84–138%] and β7+ naïve T cells [8%; 13–50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in β7+ T cells. Conclusions Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes.

This study evaluated the short-term effects of tofacitinib treatment on peripheral blood leukocyte phenotype and function, and the reversibility of any such effects following treatment withdrawal in healthy volunteers. Cytomegalovirus (CMV)-seropositive subjects received oral tofacitinib 10 mg twice daily for 4 weeks and were followed for 4 weeks after drug withdrawal. There were slight increases in total lymphocyte and total T-cell counts during tofacitinib treatment, and B-cell counts increased by up to 26%. There were no significant changes in granulocyte or monocyte counts, or granulocyte function. Naive and central memory T-cell counts increased during treatment, while all subsets of activated T cells were decreased by up to 69%. T-cell subsets other than effector memory cluster of differentiation (CD)4+, activated naive CD4+ and effector CD8+ T-cell counts and B-cell counts, normalized 4 weeks after withdrawal. Following ex vivo activation, measures of CMV-specific T-cell responses, and antigen non-specific T-cell-mediated cytotoxicity and interferon (IFN)-γ production, decreased slightly. These T-cell functional changes were most pronounced at Day 15, partially normalized while still on tofacitinib and returned to baseline after drug withdrawal. Total natural killer (NK)-cell counts decreased by 33%, returning towards baseline after drug withdrawal. NK-cell function decreased during tofacitinib treatment, but without a consistent time course across measured parameters. However, markers of NK-cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity and IFN-γ production were decreased up to 42% 1 month after drug withdrawal. CMV DNA was not detectable in whole blood, and there were no cases of herpes zoster reactivation. No new safety concerns arose. In conclusion, the effect of short-term tofacitinib treatment on leukocyte composition and function in healthy CMV+ volunteers is modest and largely reversible 4 weeks after withdrawal.

Safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ATR-107, a fully human monoclonal anti-IL-21 receptor (IL-21R) antibody, administered as ascending single doses, subcutaneously or intravenously, was evaluated in a placebo-controlled, double-blind trial in healthy subjects. The dose levels were 3-300 mg by SC and 30-120 mg by IV. The most important adverse events were hypersensitivity reactions occurring in three out of six subjects in 300 mg SC cohort and considered as dose limiting toxicity. More than 75% of the subjects who received ATR-107 developed anti-drug antibodies (ADAs), which had no discernible impact on PK or safety. The PK of ATR-107 appeared to be dose -proportional. T1/2 was shorter than typical therapeutic antibodies. Bioavailability of ATR-107 was about 30%. IL-21R occupancy was measured in circulating B cells in the 60 and 120 mg IV cohort. The data indicated that single dose of ATR-107 was able to maximally occupy IL-21Rs through at least Day 42. Further escalation in the FIH study was halted partially due to the high rates of ADA formation. In conclusion, ATR-107 had a prolonged PD effect measured by IL-21R occupancy; was highly immunogenic after single dose administration and had PK properties with rapid clearance and low bioavailability.

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. Results: Sorted DNA/Syto16+CD45−CD31+CD146+ CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 ± 171/mL in healthy subjects (n = 37) and 951 ± 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 ± 14% in healthy subjects and 43 ± 23% in cancer patients (P < 0.0001). CEPs were 181 ± 167/mL in healthy donors and 429 ± 507/mL in patients (P = 0.00019). Coefficients of variation were 4 ± 4% (intrareader), 17 ± 4% (interreader), and 17 ± 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 ± 8% (intrareader), 16 ± 10% (interreader), and 26 ± 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

SummaryThis study’s objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14+ CD16+ monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14+ CD16+ cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points.

The potency and physical properties of a previously reported 7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine series of human eosinophil phosphodiesterase inhibitors were improved by tying the lactam moiety into a triazolo ring. The resulting 5,6-dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-triazolo[4,3-alpha]pyridine series provided nonionizable analogs with melting point properties suitable for micronization. Substitution at the 3-position of the 5,6-dihydro-(9H)-pyrazolo[3,4-c]-1,2,4-triazolo[4,3-alpha]pyridine tricycle led to a 2-thienyl analog, 19 (tofimilast), a potent PDE4 inhibitor with low oral bioavailability and no emesis-associated behaviors in ferrets at plasma concentrations up to 152 ng/mL.

Nicotinamide (2) is a potent and selective inhibitor of the PDE4D isozyme and as a chemical tool selectively blocks eosinophil mediator release and chemotaxis thus linking the role of PDE4D to eosinophil function.

The ability of various C-C chemokines to elicit tissue eosinophil infiltration following intradermal injection or peripheral blood eosinophilia following intravenous injection were compared in the Brown-Norway rat. Eotaxin (0.1–3 μg site−1) of human and murine origin produced equivalent, dose-dependent increases in eosinophil peroxidase activity in rat dermis 4 h post-injection. Human eotaxin-2 was equipotent with human eotaxin in terms of dermal eosinophil recruitment. Other human CCR3 agonists, such as MCP-3, RANTES and MCP-4 failed to increase dermal eosinophil peroxidase activity at doses up to 1 μg site−1 whereas the latter did produce a small effect at 3 μg site−1. Consistent with observations in vivo, human eotaxin displaced [125I]-eotaxin from rat spleen membranes more potently (IC50=2 nM) than did MCP-4 (IC50=500 nM). RANTES did not compete with the radiolabelled chemokine at concentrations up to 1 μM. Human eotaxin (5 μg) administered intravenously increased circulating eosinophils ∼3 fold whereas MCP-4 (5 μg, i.v.) increased circulating monocytes ∼3 fold without affecting eosinophil numbers. Dexamethasone pretreatment inhibited eotaxin-induced dermal eosinophil influx only at a steroid dose (0.1 mg kg−1, s.c.) which significantly reduced circulating eosinophil numbers. The steroid also reduced eosinophilia in peripheral blood resulting from systemic eotaxin administration (5 μg, i.v.). These data suggest differences in rat CCR3 relative to other species as surmised from a distinctive rank order of chemokine potency. In addition to its chemotactic effects eotaxin, but not MCP-4, promotes eosinophil recruitment into the circulation. One of the mechanisms by which glucocorticoids, such as dexamethasone, acutely inhibits eotaxin-induced dermal eosinophil influx is to diminish the circulating numbers of these cells available for tissue recruitment. British Journal of Pharmacology (1999) 128, 788–794; doi:10.1038/sj.bjp.0702835

Exploration of the indole nitrogen region of Zafirlukast (1) has uncovered a potent series of cysteinyl leukotriene D4 (LTD4) antagonists. These studies showed that a variety of functionality could be incorporated in this region of the molecule without sacrificing potency. Efforts to exploit this site in order to improve oral efficacy are discussed.

We have cloned and characterized the first human isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE9A. By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity). We report the cloning of human cDNA encoding a full-length protein of 593 amino acids, including a 261-amino acid region located near the C terminus that is homologous to the approximately 270-amino acid catalytic domain of other PDEs. PDE9A is expressed in all eight tissues examined as a approximately 2. 0-kilobase mRNA, with highest levels in spleen, small intestine, and brain. The full-length PDE9A was expressed in baculovirus fused to an N-terminal 9-amino acid FLAG tag. Kinetic analysis of the baculovirus-expressed enzyme shows it to be a very high affinity cGMP-specific PDE with a Km of 170 nM for cGMP and 230 microM for cAMP. The Km for cGMP makes PDE9A one of the highest affinity PDEs known. The Vmax for cGMP (4.9 nmol/min/microg recombinant enzyme) is about twice as fast as that of PDE4 for cAMP. The enzyme is about twice as active in vitro in 1-10 mM Mn2+ than in the same concentration of Mg2+ or Ca2+. PDE9A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, vinpocetine, SKF-94120, dipyridamole, and 3-isobutyl-1-methyl-xanthine but is inhibited (IC50 = 35 microM) by zaprinast, a PDE5 inhibitor. PDE9A lacks a region homologous to the allosteric cGMP-binding regulatory regions found in the cGMP-binding PDEs: PDE2, PDE5, and PDE6.

The SAR of a series of 7-chromanylacetic acids has been investigated with the aim of identifying potent and selective LTB4 receptor antagonists. We found optimal activity in derivatives with c~,c~-disubstitution on the acetic acid and a C-4 hydroxy group and a C-3 lipophilic group on the chromane ring. CP-105696 (43), which contains a 4-phenylbenzyl C-3 substituent, was selected for development. ~ 1997 Elsevier Science lad. LTB4 is a potent chemoattractant for granulocytes (e.g., neutrophils and eosinophils) and stimulates functional responses such as secretion and cytokine synthesis in these cells as well as in mononuclear cells and lymphocytes. Its presence in relevant tissues has implicated it to be a likely mediator in a number of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma. Receptor antagonists of LTB4 are thus expected to be useful therapeutics tbr these diseases. To date, compounds from several structural classes have been discovered to display LTB4 antagonismJ During the course of our investigations on leukotriene D4 antagonists, we observed that certain chromanol derivatives displayed modest LTB4 antagonism in addition to LTD4 antagonism. We also noted that of all the NSAIDs examined, only the propionic acid class showed any significant parallel between their ability to block (3H) LTB4 binding to high affinity receptors on guinea pig spleen membranes and their ability to inhibit LTB4-induced human neutrophil chemotaxis} These observations led us to prepare hybrid molecules with the aim of identifying potent and selective LTB4 antagonists. Accordingly, the propionic acid derivatives 1

CP-80,633, un inhibiteur selectif de la phosphodiesterase (PDE) 4, neutralise fortement la bronchoconstriction induite par l'histamine chez des cobayes anesthesies (ED 5 0 10 μg/kg), mais ne decontracte que legerement la trachee de cobaye contractee par l'histamine (EC 5 0 137 μM). Utilisant CP-80,633 comme un inhibiteur prototypique de PDE4, nous avons verifie l'hypothese que la bronchodilatation induite par les inhibiteurs de PDE4 n'est pas vehiculee par une relaxation directe du muscle lisse des voies aeriennes. Chez les cobayes anesthesies, une dose bronchodilatatrice de CP-80,633 n'a pas augmente les catecholamines plasmatiques; de plus, la capacite de CP-80,633 de neutraliser la bronchoconstriction induite par l'histamine n'a pas ete modifiee de maniere significative par un pretraitement au propranolol. Dans une preparation d'organes isoles, l'activite des bronchodilatateurs pourrait etre attenuee par l'absence d'activateurs endogenes de l'adenylyl cyclase ou par la diminution des taux de nucleotides cycliques intracellulaires. Un pretraitement avec l'agoniste des adrenorecepteurs β, salbutamol, ou avec l'inhibiteur de PDE3, imazodan, n'a pas potentialise la capacite de bronchodilatation de CP-80,633. Un pretraitement au milrinone a augmente la puissance de CP-80,633 a decontracter les anneaux tracheaux contractes par le carbachol, mais seulement aux concentrations ou aucun effet specifique n'a ete signale. En comparant les capacites bronchodilatatrices des inhibiteurs de PDE dans les anneaux de trachee pourvus ou non d'endothelium, nous avons determine que l'epithelium n'a pas constitue une barriere a la penetration medicamenteuse. Ainsi, CP-80,633 est un puissant bronchodilatateur in vivo dont l'activite n'est ni vehiculee par la relaxation directe du muscle lisse des voies aeriennes ni dependante des catecholamines endogenes.

Increased cyclic AMP-phosphodiesterase activity in peripheral blood leukocytes is associated with the immune and inflammatory hyperreactivity that characterizes atopic dermatitis. Atopic phosphodiesterase has high sensitivity to a variety of enzyme inhibitors, suggesting an increased therapeutic advantage. The objective of this study was to use in vitro assays to identify a potent phosphodiesterase inhibitor and then to investigate its effectiveness in treating atopic dermatitis. Leukocyte enzyme activity was measured by radioenzyme assay, whereas prostaglandin E 2 and interleukins 10 (IL-10) and 4 (IL-4) were measured in 24-h culture supernatants of mononuclear leukocytes by immunoassays. The effect of a topical phosphodiesterase Inhibitor on atopic dermatitis lesional skin was assessed by double-blind, paired comparisons of active drug and placebo ointments applied to symmetrically involved sites over a 28-d period. Using in vitro assays, we demonstrated the ability of selective high-potency phosphodiesterase inhibitors to reduce prostaglandin E 2 , IL-10, and IL-4 production in atopic mononuclear leukocyte cultures. We selected the Type 4 phosphodiesterase inhibitor, CP80,633, based on its inhibitory potency, for clinical testing by topical, bilateral paired comparisons in 20 patients with atopic dermatitis and demonstrated significant reductions of all inflammatory parameters. Phosphodiesterase inhibitors modulate several pathways contributing to the exaggerated immune and inflammatory responses, which characterize atopic dermatitis. This in vivo demonstration of anti-inflammatory efficacy may provide a useful alternative to the over-reliance on corticosteroid therapy in atopic disease.

1. The role of adrenal hormones in the regulation of the systemic and local production of tumour necrosis factor (TNF alpha) was examined in male Balb/c mice. 2. Intraperitoneal injection of 0.3 mg E. coli lipopolysaccharide (LPS, 0111:B4) led to high levels of circulating TNF alpha without stimulating TNF alpha production in the peritoneal cavity. Systemic production of TNF alpha in response to LPS was increased in adrenalectomized animals and in normal animals treated with the beta-adrenoceptor antagonist, propranolol. The glucocorticoid antagonist, RU 486, did not modify systemic TNF alpha production. These results indicate that systemic TNF alpha production is regulated by adrenaline but not by corticosterone. 3. When mice were primed with thioglycollate, TNF alpha was produced in the peritoneal cavity in response to low dose LPS (1 micrograms). The levels of TNF alpha in the peritoneal cavity were not enhanced by adrenalectomy or by treatment with either propranolol or RU 486, indicating local production of TNF alpha in the peritoneal cavity is not regulated by adrenaline or corticosterone. 4. The phosphodiesterase type IV (PDE-IV) inhibitor, rolipram, inhibited both the systemic production of TNF alpha in response to high dose endotoxin (ED50 = 1.3 mg kg-1) and the local production of TNF alpha in the peritoneal cavity in response to low dose endotoxin (ED50 = 9.1 mg kg-1). In adrenalectomized mice there was a slight reduction in the ability of rolipram to inhibit the systemic production of TNF alpha (ED50 = 3.3 mg kg-1) while the ability of rolipram to inhibit the local production of TNF alpha in the peritoneal cavity was virtually abolished (24% inhibition at 30 mg kg-1). The glucocorticoid antagonist, RU 486, also reduced the ability of rolipram to inhibit local TNF alpha production while propranolol was without effect. 5. Systemic treatment with rolipram increased the plasma concentrations of corticosterone in normal mice but not in adrenalectomized mice indicating that rolipram can cause adrenal stimulation in vivo. 6. In summary, these data indicate that systemic production of TNF alpha in response to high dose endotoxin is controlled differently from the local production of TNF alpha in response to low dose endotoxin. The systemic production of TNF alpha is regulated by catecholamines, but not by corticosterone, while the local production of TNF alpha in the peritoneal cavity is not regulated by basal levels of either catecholamines or corticosterone. 7. These data also show that the ability of rolipram to inhibit the local production of TNF alpha is dependent on the release of corticosterone from the adrenal glands.

The synthesis and the in vitro properties of a novel series of potent and selective phosphodiesterase type IV (PDE IV) inhibitors is described. Despite bearing structural similarity to rolipram, several of these compounds have much reduced affinity for the [3H]rolipram binding site.

The synthesis and biological properties of a novel series of potent and selective phosphodiesterase type IV (PDE IV) inhibitors are described. These catechol benzimidazoles were designed from rolipram and initial compounds reflected a similarly high affinity for the [ 3 H]rolipram b binding site (500 to 1000X greater affinity for the [ 3 H]rolipram binding site over the PDE IV inhibitory site). However, SAR studies on the 3-alkoxy position revealed that this [ 3 H]rolipram binding site affinity could be attenuated, while potentiating the PDE IV inhibitory activity. This resulted in the 2-indanyl analog 13 which is a potent, selective PDE IV inhibitor with a 15X differential in favor of PDE IV binding.

A series of pyridine chromanols were synthesized and evaluated as LTD 4 -antagonists (LTD 4 -A). The quinoline sidechain of this class of such agents, as exemplified by REV-5901, has until now been deemed as essential for potent activity. However, by manipulating substituents on a pyridine ring, quinoline-like potency can be achieved. The results indicate that this is a function of pKa.

The combination of key structural pharmacophores found in known leukotriene D4 (LTD4) receptor antagonists with those of potent platelet activating factor (PAF) receptor antagonist UK-74,505 has led to the synthesis of hybrid compounds CP-96,021 and CP-96,486. These compounds represent the first known balanced, combined and orally active LTD4/PAF receptor antagonists.

The development of two novel LTD 4 receptor antagonists as clinical candidates for the treatment of asthma is described. The first generation compound, CP-80,798, was found to be a balanced 5-lipoxygenase inhibitor (5-LOI)/LTD 4 antagonist (LTD 4 -A), while the second generation compound, CP-85,958, is a selective LTD 4 antagonist.

The development of novel LTB 4 antagonists from a class of quinolylmethyl LTD 4 antagonists is described. These α-methyl quinolylmethyl chromanols were found to have good vitro activity.

The 125I analog of MK-0591, 1, has been prepared for use as a radioligand for developing a 5-lipoxygenase activating protein (FLAP) binding assay. The radiosynthesis involves a two step oxidative iododestannylation-saponification procedure. A FLAP binding assay has been developed in human neutrophil membranes. The binding of 1 to human neutrophil FLAP is rapid, reversible, of high affinity, saturable and selective for FLAP inhibitors.

Tumor necrosis factor-α (TNF-α) has been implicated as a key mediator in the progression of rheumatoid arthritis. Inhibitors of phosphodiesterase IV (PDE IV) have been shown to inhibit the production of TNF-α by elevating intracellular levels of cyclic adenosine monophosphate (cAMP). Our efforts in a series of biarylcarboxamides have led to the identification of 8j (CP-353,164) as a potent inhibitor of PDE IV and TNF-α production.

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).

A new series of cysLT1 receptor antagonists represented by CP-288,886 (7) and CP-265,298 (8) were developed which are equipotent to clinical cysLT1 receptor antagonists Zafirlukast (1) and Pranlukast (2).

Peptidoleukotrienes (LTC4 , LTD, and LTE4) are implicated in the pathogenesis of asthma 1-3 although proof of their role is contingent upon the outcome of clinical trials with LT antagonists/inhibitors in asthmatics. Asthma is a complex, heterogeneous and chronic inflammatory disease of the lung . Therefore, a single mediator antagonist/inhibitor, such as a LTD, antagonist, may not be very useful for the treatment of asthma ; a speculation supported by clinical results with some of the first LTD, antagonists .', ' However, while still not conclusive, results of several recent small, double-blinded, placebo-controlled studies using a new generation of LTD, antagonists suggest that LTD 4 may be an important mediator in asthma . This conclusion is based on the finding that when given either orally, intravenously (i .v .) or by aerosol, these new compounds significantly block antigeninduced early and late phase bronchoconstriction in mild and moderate allergic asthmatics . Before these results can be said to have definitely established the role of LTD4 in asthma, more clinical trials of these compounds, e .g. with an increased patient population and different subsets of patients, as well as with end points other than bronchoconstriction (such as mucus secretion, cell infiltration, bronchial hyperresponsiveness), are undoubtedly needed . Since the discovery of FPL-55,712 as an LT (SRSA) antagonist in 1973, synthesis of acetophenone-like compounds proceeds with the intent of enhancing oral bioavailability/biological half-life and potency of LTD4 antagonism . Clinical results with some analogs from this chemical series, LY-171,883 4 and L649,923, 5 were unrewarding because of the modest ability of these compounds to suppress either cold airinduced bronchoconstriction or antigen-induced early

Naive guinea-pigs were passively sensitized with varying amounts of affinity column purified, homologous, anti-ovalbumin IgG1 (anti-OA IgG1) and then examined for a) the capacity of lung tissue to release mediators (histamine and LTB4/LTD4) in response to antigen-challenge ex vivo and b) the attendant circulating levels of anti-OA IgG1. Intraperitoneal administration of anti-OA IgG1 (0.125-0.75 mg/kg) to guinea-pigs facilitated the synthesis of LTB4 (8-25 ng/g lung) and LTD4 (18-80 ng/g) and the release of histamine (1-7 ug/g) from lung tissue after exposure to 10 micrograms/ml of ovalbumin for 20 min ex vivo. Peak levels of mediators were found using 0.5 mg/kg anti-OA IgG1 with an ED50 = 0.35 mg/kg. LTD4/LTB4 synthesis and histamine release were both antigen concentration- and time-dependent, and LT synthesis was observable in non-perfused lungs and in lungs perfused free of blood. Maximum sensitization occurred at 1-2 days post i.p. administration of anti-OA IgG1 and was maintained up to 7 days. Measurement of anti-OA IgG1 using an enzyme-linked immunosorbent assay demonstrated that circulating antibody levels were 2-6 micrograms/ml at the doses which caused sensitization. The level of anti-OA IgG1 found in passively sensitized animals was at least 100-fold less than that found in actively-sensitized guinea-pigs despite the similar magnitude in LTD4/LTB4 synthesized and the amount of histamine released. Using purified antibody, the results demonstrate that in guinea-pigs, IgG1 can play a prominent role in regulating lung LT synthesis and histamine release, and that microgram per ml circulating levels of this antibody are sufficient to sensitize naive lungs.

Anti-MAdCAM-1 Antibody (PF-00547659) for Active Refractory Crohn's Disease: Results of the OPERA study G. D'Haens*1, S. Lee2, D. Tarabar3, E. LOUIS4, M. Klopocka5, D.I. Park6, J. Claus7, X. Hebuterne8, W. Reinisch9, S. Schreiber10, K.J. Gorelick11, J. Cheng11, M. Hassan-Zahraee11, L. Brown12, A. Ahmad13, A. Banerjee13, F. Cataldi11, W. Sandborn14 1University of Amsterdam, Academic Medical Center, Amsterdam, Netherlands, 2University of Washington, Gastroenterology, Seattle, WA, United States, 3MMA, Gastroenterology, Belgrade, Serbia, 4University Hospital CHU of Liege, Gastroenterology, Liege, Belgium, 5Nicolaus Copernicus University in Torun, Gastroenterology, Bydgoszcz, Poland, 6Kangbuk Samsung HospitalSungkyunkwan University, Gastroenterology, Seoul, South Korea, 7Universitatsklinikum Ulm, Gastroenterology, Ulm, Germany, 8CHU de Nice et Universite de Nice Sophia-Antipolis, Gastroenterology, Nice, France, 9Medical University of Vienna, Internal Medicine/ Gastroenterology, Vienna, Austria, 10University of Kiel, Gastroenterology, Kiel, Germany, 11Pfizer Inc., Gastroenterology, Cambridge, United States, 12Pfizer Inc., Gastroenterology, Cambridge, MA, United Kingdom, 13Pfizer Inc., Gastroenterology, Cambridge, MA, United States, 14Univeristy of California San Diego, Division of Gastroenterolgy, San Diego, United States

CP-199,330 (3) and CP-199,331 (4) are cysLT1 receptor antagonists that are equipotent to marketed cysLT1 receptor antagonists zafirlukast and pranlukast, show good pharmacokinetics in rats and monkeys, and are devoid of liver toxicity in monkeys as seen in CP-85,958 (1).

A new series of cysLT1 receptor antagonists represented by CP-288,886 (7) and CP-265,298 (8) were developed which are equipotent to clinical cysLT1 receptor antagonists Zafirlukast (1) and Pranlukast (2).

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).

As part of our efforts to understand the regulation of intracellular cAMP and to generate new targets for pharmacological intervention, we have cloned and characterized the first isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE8A. PDE8A is most similar to PDE4 (38.5% amino acid identity in the catalytic domain), but is clearly not a member of any of the seven known PDE families. We report the cloning of human cDNA encoding the C-terminal 713 amino acids of the protein, including a 283 amino acid region located near the C-terminus that is homologous to the approximately 270 amino acid catalytic domain of other PDEs. In addition, we found cDNA sequences consistent with alternative 5' mRNA splicing analogous to that seen in other PDE genes. PDE8A is expressed in a wide variety of tissues as a approximately 4.5 kb mRNA, with highest levels in testis, ovary, small intestine, and colon. The C-terminal 545 amino acids of PDE8A (the region shared among all splice variants) were expressed in baculovirus. Kinetic analysis of the baculovirus expressed enzyme shows it to be a very high affinity cAMP specific PDE with a Km of 55 nM for cAMP and 124 microM for cGMP. The Vmax (150 pmol/min/microgram recombinant enzyme) is about 10 times slower than that of PDE4. The cAMP hydrolytic activity of PDE8A is not modulated by cGMP at concentrations up to 100 microM. The enzyme requires the presence of at least 1 mM Mn2+ or Mg2+ for maximal activity in vitro, while 100 mM Ca2+ restores only about 20% activity. PDE8A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, zaprinast, vinpocetine, SKF-94120, and IBMX, but is inhibited (IC50 = 9 microM) by the PDE inhibitor dipyridimole. To give PDE8A a descriptive name that distinguishes it from the other two known high affinity cAMP-specific phosphodiesterases (PDE4 and PDE7), we denote PDE8A as the high affinity cAMP-specific and IBMX-insensitive PDE.

In addition to having desirable inhibitory effects on inflammation, anaphylaxis, and smooth muscle contraction, PDE-IV inhibitors also produce undesirable side effects including nausea and vomiting. In general, compounds that inhibit PDE-IV also potently displace [3H]rolipram from a high-affinity binding site in rat cortex. While this binding site has not been identified, it has been proposed to be an allosteric binding site on the PDE-IV enzyme. Preliminary studies have suggested that the emetic potency of PDE-IV inhibitors is correlated with affinity for the brain rolipram binding site rather than potency at inhibiting PDE-IV enzyme activity. Efforts to eliminate the emetic potential of PDE-IV inhibitors were directed toward developing compounds with decreased [3H]rolipram binding affinity while retaining PDE-IV potency. Thus, a novel series of 4-(3-alkoxy-4-methoxyphenyl)benzoic acids and their corresponding carboxamides were prepared and evaluated for their PDE-IV inhibitory and rolipram binding site properties. Modification of the catechol ether moiety led to phenylbutoxy and phenylpentoxy analogues that provided the desired activity profile. Specifically, 4-[3-(5-phenylpentoxy)-4-methoxyphenyl]-2-methylbenzoic acid, 18, was found to exhibit potent PDE-IV inhibitory activity (IC50 0.41 microM) and possessed 400 times weaker activity than rolipram for the [3H]rolipram binding site. In vivo, compound 18 was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED50 8.8 mg/kg, po) and demonstrated a significant reduction in emetic side effects (ferret, 20% emesis at 30 mg/kg, po).

Publisher Summary This chapter discusses the two molecular targets, such as modulators of intracellular cyclic adenosine monophosphate (cAMP) and inhibitors of lymphocyte-derived cytokines. Current asthma therapies, such as theophylline and steroids, are used to stimulate the cAMP pathways. Cyclic nucleotide PDEs are viewed as attractive molecular targets for the treatment of asthma and inflammation. PDE isozymes are classified into five families based on their specificity, for either cAMP or cGMP, inhibitor sensitivity and calciumcalmodulin dependence. PDE IV and PDE III/lV inhibitors possess both bronchodilatory and anti-inflammatory activity in animal models. A major obstacle in the development of PDE IV inhibitors is the elimination of emetic side effects. PDE IV inhibitor 1 reduces the abnormal levels of IL-4 production, for controlling the defective immune function in atopic dermatitis. Eosinophil counts in conjunctival epithelium in LT-challenged guinea pigs that receive are reduced compared to the LT-challenged controls. The pulmonary anti-allergic activity of theophylline results in part from the inhibition of PDE IV and the bronchodilator activity of theophylline results in part from the inhibition of PDE 111. Several isoforms of the PDE IV isozyme have been cloned, from rat and from man, and access to these isoforms lead to more selective PDE inhibitors. Elevated cAMP binds the two regulatory subunits of the protein kinase A (PKA) holoenzyme, promoting dissociation of two monomeric catalytic subunits.

We have characterized [3H]leukotriene D4 binding to guinea pig lung homogenates. Both biphasic dissociation kinetics and curvilinear Scatchard plots indicated the presence of [3H]leukotriene high and low affinity states of the binding sites. The rank order of potency for the competition study was leukotriene C4 = leukotriene D4 greater than leukotriene E4 much greater than arachidonic acid, and for their contractile effect on lung strips was leukotriene C4 = leukotriene D4 = leukotriene E4 much greater than arachidonic acid. FPL-55712 was the only other agent tested that inhibited binding. These results suggest that binding of [3H]leukotriene D4 to the homogenate is consistent with its binding to specific leukotriene D4 receptor sites.

To establish a simple and sensitive quantitation of leukotriene B/sub 4/ (LTB/sub 4/), they developed a radioreceptor assay (RRA) using a highly specific (/sup 3/H)leukotriene B/sub 4/((/sup 3/H)LTB/sub 4/) binding to a guinea pig spleen homogenate. The assay detected LTB/sub 4/ levels as low as 0.12 pmol per tube. 50% inhibition of bound (/sup 3/H)LTB/sub 4/ was obtained by 2.5 nM of unlabeled LTB/sub 4/. (/sup 3/H)LTB/sub 4/ competition studies indicated that 20-hydroxy-LTB/sub 4/ was 8 times, 6 trans-LTB/sub 4/ was 640 times and 20-carboxy-LTB/sub 4/ was 1000 times less effective than LTB/sub 4/. The peptide leukotrienes C/sub 4/, D/sub 4/ and E/sub 4/ showed no effect on (/sup 3/H)LTB/sub 4/ binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB/sub 4/. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB/sub 4/ released from human granulocytes stimulated with calcium ionophore A23187. The LTB/sub 4/ level was maximal at 10 min and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B/sub 4/ is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive thanmore » other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B/sub 4/.« less

1. 1. The H 1 receptor blocking agents (H 1 blockers) (promethazine, diphenhydramine, chlorpheniramine and tripelennamine) but not H 2 blockers (burimamide and metiamide) were able to relax the rabbit cerebral, coronary and mesenteric arteries which were contracted with potassium (30 mM). However, these agents failed to decrease the active tension of rabbit aortic strips. 2. 2. Promethazine was the most potent vascular relaxants among the agents tested. In addition, the potencies of the H 1 blockers were higher in cerebral arteries than in the coronary and mesenteric arteries. 3. 3. H 2 blockers also did not alter either the relaxing response to H 1 blockers or the contractile effect of histamine on the cerebral arteries. Propranolol did not inhibit the relaxing effect of H 1 blockers. 4. 4. Although the mode of relaxing action of H 1 blockers is not clear it may be due to the direct action on the vascular smooth muscle.

Guinea pigs were sensitized by daily intraperitoneal injections of 100 mg of egg albumin for 3 consecutive days and sacrificed at 30–35 days after the last injection. The in vitro sensitivity and response of the sensitized tracheal muscle to methacholine and histamine were significantly less than those of littermate controls. However, there was no difference between the dissociation constants and concentrations of pulmonary muscarinic receptor binding sites of the control and sensitized guinea pigs. We conclude that subsensitivity and hyporesponsiveness to methacholine and histamine occur in the airway muscle of this guinea pig model of experimental asthma and suggest that the decreased reactivity to methacholine might be due to defects beyond the receptor.

The relaxation of thoracic and abdominal aortae of the spontaneously hypertensive rat (SHR) during prehypertensive (3--5 week old) and hypertensive (12--16 and 22--25 week old) stages was compared to that of matched normotensive Wistar rats (NWRs). In the thoracic aorta, the relaxation response to isoproterenol, acetylcholine, Mg2+, Mn2+, Co2+, and La3+ was less in both prehypertensive and hypertensive SHRs than in the matched NWRs; however, such difference was not evident in the abdominal aorta. Similarly, the relaxing effect of nitroglycerin and papaverine was not different in the aorta preparations of the SHR and NWR. After chronic reserpine treatment of the prehypertensive and hypertensive SHRs, the thoracic aorta still showed less relaxation in response to the aforementioned agents. Isoproterenol, but not other agents tested, produced less relaxation in the thoracic aorta from the renal hypertensive rat than from the control. Our results suggest that the decreased relaxation of the SHR thoracic aorta is not a consequence of hypertension and that the defect in the SHR thoracic aorta cannot be generalized to other vascular beds.