WO2007053194A2 - Modulation des dysfonctionnements de la barriere cellulaire - Google Patents

Modulation des dysfonctionnements de la barriere cellulaire Download PDF

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Publication number
WO2007053194A2
WO2007053194A2 PCT/US2006/021604 US2006021604W WO2007053194A2 WO 2007053194 A2 WO2007053194 A2 WO 2007053194A2 US 2006021604 W US2006021604 W US 2006021604W WO 2007053194 A2 WO2007053194 A2 WO 2007053194A2
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WO
WIPO (PCT)
Prior art keywords
opioid receptor
aeruginosa
opioid
receptor antagonist
cell
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PCT/US2006/021604
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English (en)
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WO2007053194A3 (fr
Inventor
John C. Alverdy
Jonathan Moss
Mark W. Lingen
Patrick A. Singleton
Joe G.N. Garcia
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The University Of Chicago
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Publication date
Priority claimed from PCT/US2006/007892 external-priority patent/WO2006096626A2/fr
Priority to US11/914,984 priority Critical patent/US20080194611A1/en
Application filed by The University Of Chicago filed Critical The University Of Chicago
Priority to AU2006309292A priority patent/AU2006309292A1/en
Priority to JP2008514938A priority patent/JP2008542395A/ja
Priority to EP06844131A priority patent/EP1901742A2/fr
Priority to CA002609985A priority patent/CA2609985A1/fr
Publication of WO2007053194A2 publication Critical patent/WO2007053194A2/fr
Publication of WO2007053194A3 publication Critical patent/WO2007053194A3/fr
Priority to US13/483,932 priority patent/US20120316190A1/en
Priority to US14/061,331 priority patent/US20140142133A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/485—Morphinan derivatives, e.g. morphine, codeine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention generally relates to the field of prophylactic and therapeutic use of opioid receptor antagonists in modulating cell barrier dysfunction characteristic of a disorder or disease afflicting vertebrates (e.g., mammals) such as humans.
  • vertebrates e.g., mammals
  • Vertebrate (e.g., mammalian) cell barrier dysfunction results in a change in permeability of a cell barrier contributing to the internal compartmentalization of a multicellular organism and/or to the segregation of internal and external environments of such an organism.
  • cell barrier dysfunctions are revealed as an increase in the permeability of a particular cell layer, such as the layer of endothelial cells found in the vasculature of higher eukaryotes or the layer of epithelial cells found in tissues exposed to the external environment, including the skin, lung and gut.
  • a variety of disorders and diseases afflicting vertebrates such as humans can involve cell barrier dysfunction.
  • endothelial cells provide a semi-selective barrier between the blood and underlying vasculature. Disruption of this barrier results in increased vascular permeability and organ dysfunction.
  • the inflammatory process increases macromolecular transport by decreasing cell-cell and cell-matrix adhesion and by increasing centripetally directed tension, resulting in the formation of intercellular gaps.
  • Agents that enhance endothelial cell barrier function provide a desirable therapeutic strategy for a variety of inflammatory diseases, atherosclerosis and acute lung injury.
  • Microbial pathogens such as P. aeruginosa can express various peptides and virulence factors that can disrupt barrier function.
  • Microbiologists have long recognized that many bacteria activate their virulence genes in response to ambient environmental cues. In general such physico-chemical cues signal environmental stress or adversity, such as changes in redox status, pH, osmolality, and the like.
  • P. aeruginosa and other bacteria can express a lectin/adhesin PA-I.
  • PA-I The distribution of PA-I in bacteria can be either primarily intracytoplasmic or extracellular, depending on its environment.
  • CyaB two sensor proteins located within the cell membrane of P. aeruginosa
  • GacS two sensor proteins located within the cell membrane of P. aeruginosa
  • CyaB via cAMP
  • GacS 4 via phosphorylation
  • Vfr the transcriptional regulators
  • GacA the transcriptional regulators
  • PcrA the transcriptional regulators
  • Mutant strains defective in CyaB and GacS have attenuated lethality in mice following lung instillation.
  • Opioids comprise a large group of compounds that are distributed in virtually every tissue of the body and are abundantly released in response to various stress conditions; for example dynorphin and ⁇ -endorphin appear to be the predominantly released endogenous opioids following stress (S. Yoshida, et al., Surg Endosc 14, 137 (2000), C. Sternini, S. Patierno, I. S. Selmer and A. Kirchgessner, Neurogastroenterol Motil 16 Suppl 2, 3 (2004)).
  • Morphine and morphine derivatives are among the most widely used analgesic drugs in the world and are often administered at high doses even at continuous dosing intervals in post-operative care, chronic pain management, and in critically ill patients such as patients with advanced cancer or AIDS.
  • Intravenously applied morphine has been demonstrated to accumulate at tissues sites of bacterial infection such as the intestinal mucosa, at concentrations as high as 100 ⁇ M (P. Dechelotte, A. Sabouraud, P. Sandouk, I. hackbarth and M. Schwenk, Drug Metab Dispos 21, 13 (1993)) and has been shown to readily cross the intestinal wall into the lumen (M. M. Doherty and K. S.
  • opioids have been shown to suppress a variety of immune cells resulting in impaired clearance of bacteria and enhanced mortality in animals (Wang, et al., J Leukoc Biol 71, 782 (2002)), it has not been previously considered that opioid compounds might also directly activate the virulence of bacteria.
  • Opioids and opioid antagonists such as morphine and DAMGO (([D-AIa 2 , N- MePhe 4 , Glycol], a mu opioid enkephalin) bind to the mOP-R present in the central nervous system (CNS) and peripheral tissue.
  • the mOP-R is expressed in a variety of cell types including endothelial cells and epithelial cells.
  • the mOP-R is a G protein-coupled receptor with multiple isoforms resulting from alternative splicing of mRNA encoded from a single gene.
  • Most mu opioid receptor antagonists, including naloxone exist in an uncharged state and readily pass through the blood-brain barrier (BBB) to reverse CNS-dependent analgesic effects.
  • BBB blood-brain barrier
  • MNTX is a charged molecule that is known to be unable to penetrate the BBB.
  • QDNM quaternary derivatives of noroxymorphone
  • SlP sphingosine-1 -phosphate
  • Edg receptors endothelial differentiation gene
  • Src (pp ⁇ OSrc, c-Src tyrosine kinase) is a non-receptor tyrosine kinase that contains an amino-terminal myristoylation site, Src Homology (SH) sites (i.e., SH2 and SH3), a tyrosine kinase catalytic domain and regulatory tyrosine phosphorylation sites.
  • SH Src Homology
  • Protein tyrosine phosphatases are a diverse superfamily encoded by over 100 genes that regulate a myriad of cellular events.
  • PTP protein tyrosine phosphatases
  • RPTP ⁇ receptor-like protein tyrosine phosphatase mu
  • RPTP ⁇ is composed of extracellular MAM (Meprin-A5-protein-M-type-RPTP (RPTP ⁇ ), Immunoglobulin (Ig)-like and Fibronectin type 3 (FN3)-like domains and intracellular PTP catalytic domains.
  • RPTP ⁇ is localized at endothelial cell junctions and regulates vascular integrity.
  • the invention satisfies at least one of the foregoing needs in the art in providing compositions and methods for preventing or treating cell barrier dysfunction by administering an effective amount of an opioid receptor antagonist (OP-RA).
  • OP-RA opioid receptor antagonist
  • the invention is directed in important embodiments to preventing or treating an endothelial or epithelial cell barrier dysfunction.
  • the invention relates to the cell barrier dysfunction inhibitory effect of opioid receptor antagonists, including peripherally restricted antagonists (e.g., polar or charged antagonists typified by methylnaltrexone) as well as centrally acting antagonists.
  • the methods are effective in preventing or treating the barrier dysfunction and attendant conditions and symptoms arising therefrom, associated with a variety of diseases and disorders, such as inflammation, atherosclerosis, and microbial pathogenesis.
  • the conditions with which the cell barrier dysfunction occurs may be gut-derived sepsis, a burn injury, a chemical contact injury, acute lung injury, neonatal necrotizing enterocolitis, severe neutropenia, toxic colitis, inflammatory bowel disease, Crohn's disease, enteropathy, transplant rejection, pouchitis, pig-bel, uremic pericardial effusion, leakage in the vitreous of the eye, macular degeneration, retinal dysfunction, and infection (e.g., viral infection, bacterial infection, opportunistic bacterial infection, Clostridium pere infection, Pseudomonas aeruginosa infection, Pseudomonas- mediated ophthalmologic infection, Pseudomonas-mQdiated otologic infection and Pseudomon ⁇ s-mediated cutaneous infection).
  • infection e.g., viral infection, bacterial infection, opportunistic bacterial infection, Clostridium pere infection, Pseudomona
  • opioid receptor antagonists useful in the inventions described herein are set forth more comprehensively in the detailed description below, which description is incorporated into this summary by reference.
  • suitable opioid receptor antagonists include heterocyclic amine compounds that belong to several classes of compounds.
  • One class is the tertiary derivatives of morphinan and, in particular, the tertiary derivatives of noroxymorphone.
  • the tertiary derivative of noroxymorphone e.g., naloxone or naltrexone
  • Another class is the quaternary derivatives of morphinan and, in particular, the quaternary derivatives of noroxymorphone.
  • Another class is the N-substituted piperidines.
  • the opioid receptor antagonist is a peripheral ⁇ -opioid receptor antagonist, such as N-methylnaltrexone, alvimopan, ADL 08-0011, a piperidine-N-alkylcarboxylate, a quaternary morphinan, an opium alkaloid derivative or a quaternary benzomorphan compound.
  • the quaternary morphinan compound may be a quaternary salt of N-methylnaltrexone, N-methylnaloxone, N-methylnalorphine, N-diallylnormorphine, N-allyllevallorphan or N-methylnalmefene.
  • the quaternary benzomorphan compound is 2'-hydroxy-5,9-dimethyl-2,2- diallyl-6,7-benzomorphanium-bromide; 2'-hydroxy-5,9-dimethyl-2-n-propyl-6,7- benzomorphan; 2'-hydroxy-5,9-dimethyl-2-allyl-6,7-benzomorphan; 2'-hydroxy-5,9- dimethyl-2-n-propyl-2-allyl-6,7-benzomorphanium bromide; 2'-hydroxy-5,9-dirnethyl-2-n- propyl-2-propargyl-6,7-benzomorphanium bromide ; or 2'-acetoxy-5,9-dimethyl-2-n-propyl- 2-allyl-6,7-benzomo ⁇ hanium bromide.
  • the method further comprises administration of a high molecular weight polyethylene glycol-like compound having an average molecular weight of at least 15 kilodaltons
  • the antagonist is a mu opioid receptor antagonist.
  • the antagonist is a peripheral opioid receptor antagonist, e.g., MNTX, which may also inhibit VEGF, platelet-derived growth factor (PDGF), sphingosine-1- phosphate (SlP) and/or hepatocyte growth factor (HGF)-stimulated or induced cell barrier dysfunction.
  • PDGF platelet-derived growth factor
  • SlP sphingosine-1- phosphate
  • HGF hepatocyte growth factor
  • the opioid receptor antagonist is a mu opioid receptor antagonist.
  • the opioid receptor antagonist is a kappa opioid receptor antagonist.
  • the invention also encompasses administration of more than one opioid receptor antagonist, including combinations of mu opioid receptor antagonists, combinations of kappa opioid receptor antagonists and combinations of mu and kappa opioid receptor antagonists, for example, a combination of methylnaltrexone and alvimopan (or ADL 08-0011), or a combination of naltrexone and methylnaltrexone.
  • the invention described herein involves the prevention and/or treatment of cell barrier dysfunction in vertebrates, and more preferably mammals.
  • Important subjects or "patients" to be treated are farm animals (e.g., horses, goats, cows, sheep, pigs, fish and chickens), domestic animals (dogs and cats) and humans.
  • the invention described herein involves prevention or treatment of cell barrier dysfunction.
  • Prevention as used herein means administration of an opioid receptor antagonist to a patient at risk of a cell barrier dysfunction in an amount effective to inhibit the appearance of, to lessen the development of or to prevent altogether the appearance of a symptom or adverse medical condition arising from the cell barrier dysfunction.
  • Treatment as used herein means administration of an opioid receptor antagonist to a patient having or believed to have a condition or symptom associated with a cell barrier dysfunction in an amount effective to inhibit, to halt the further development of, to lessen or to eliminate altogether a symptom or adverse medical condition arising from the cell barrier dysfunction.
  • An opioid receptor antagonist such as a mu opioid receptor antagonist (mOP- RA) like rnethylnaltrexone (MNTX), inhibits cell barrier dysfunction.
  • mOP-RA mu opioid receptor antagonist
  • MNTX rnethylnaltrexone
  • mu opioid receptor antagonists including MNTX, inhibit opiate-, thrombin- and LPS-induced endothelial cell barrier dysfunction by mu opioid receptor (mOP-R)-dependent, and - independent, mechanisms.
  • mOP-R-independent mechanisms of mOP-RA include activation of receptor-like protein tyrosine phosphatase mu (RPTP ⁇ ) and inhibition of thrombin- and LPS-induced, Src-dependent, S1P3 receptor transactivation (tyrosine phosphorylation).
  • RPTP ⁇ receptor-like protein tyrosine phosphatase mu
  • tyrosine phosphorylation tyrosine phosphorylation
  • the invention described herein provides methods for enhancing cell barrier function (e.g., endothelial and/or epithelial cell barrier function), comprising administering to a patient in need of such treatment a composition comprising an effective amount of one or more opioid receptor antagonists.
  • cell barrier function can be disrupted in certain inflammatory syndromes.
  • the invention provides a method of preventing or treating inflammatory syndromes, e.g., acute lung injury, as well as atherosclerosis and microbial pathogenesis (e.g., infection), which are characterized by a cell barrier dysfunction, typically an epithelial or endothelial cell barrier dysfunction.
  • the methods described herein also involve treating or preventing a symptom arising from cell barrier dysfunction associated with any of these diseases.
  • the patient preferably is a human.
  • the human patient is free of cancer, and/or is not in a methadone maintenance program, and/or is not immunosuppressed.
  • the patient is not experiencing post operative bowel dysfunction.
  • the patient may be, or may not be, on concurrent opioid therapy, depending on the particular disorder the patient has, the severity of the disorder, and the need the patient has for pain management.
  • the patient is taking concurrent opioid therapy.
  • the patient is not taking concurrent opioid therapy.
  • the patient is taking concurrent chronic opioid therapy.
  • the patient is not taking concurrent chronic opioid therapy.
  • the patient is receiving a dose of an opioid antagonist that is independent of any dose of opioid therapy concurrently administered.
  • the effective amount is such that the patient has effective circulating blood plasma levels of the opioid antagonist continuously for at least 1 week, at least 2 weeks, at least three weeks and, even at least 4 weeks.
  • the opioid antagonists are used peri-operatively. By peri-operatively, it is meant before ( e.g., in preparation for), during, and/or immediately after a surgical procedure (i.e., up to three or even up to five days). The opioid antagonists.
  • the invention also includes the co-administration of the opioid antagonists with agents that are not opioid antagonists, but which are nonetheless useful in treating a disorder, condition or symptom associated with a cell barrier dysfunction.
  • agents include anti-cancer agents , anti-neovascularization agents (for example, anti-VEGF monoclonal antibody), anti-infective agents (e.g., antibacterial agents and anti-viral agents), anti-inflammatory agents, anti-atherosclerotic agents, anti-thrombotic agents, and the like.
  • An aspect of the invention provides a method of treating a disorder characterized by a cell barrier dysfunction comprising administering to a subject free of an opioid-induced side effect an effective amount of a ⁇ -opioid receptor antagonist.
  • the opioid- induced side effects include opioid-induced constipation, irritable bowel syndrome, postoperative ileus or bowel dysfunction, opioid-induced nausea, opioid-induced vomiting, urinary retention, delayed gastrointestinal tract emptying, reduced gastrointestinal tract motility and opioid-induced suppression of the immune system.
  • the cell barrier dysfunction may be attributable to endothelial cells, epithelial cells, or both types of cells.
  • Another aspect of the invention provides a method of reducing the risk of developing a disorder characterized by a cell barrier dysfunction comprising administering to a subject at risk of developing the disorder a prophylactically effective amount of an opioid receptor antagonist.
  • Another aspect of the invention provides a method of reducing a symptom associated with a cell barrier disorder, comprising administering to a subject in need thereof an opioid receptor antagonist, wherein the compound is administered in an amount effective to reduce at least one symptom of the disorder.
  • Another aspect of the invention is a method of preventing tumor cell metastasis comprising peri-operatively administering an effective amount of an opioid receptor antagonist to a patient having a tumor amenable to surgical intervention.
  • the tumor cell is not an endothelial cell tumor.
  • Another aspect of the invention provides a method for preventing an infection or for lowering the risk of an infection by administering to a patient in need of such treatment an effective amount of an opioid receptor antagonist.
  • the patient has a traumatic injury, such as an internal injury, a surgery, an acute lung injury, or a burn.
  • the patient is subjected to high levels of stress.
  • the infection is from an opportunistic infectious agent.
  • the infection is a bacterial infection.
  • the infectious agent is Clostridium pere, or another bacterium capable of developing a virulent phenotype, such as Pseudomonas aeruginosa.
  • Another aspect of the invention provides a method of inhibiting the expression of a bacterial PA-I lectin/adhesin by a bacterium in a patient comprising administering an effective amount of an opioid receptor antagonist to a subject at risk of developing or suffering from bacterial pathogenesis.
  • Any known bacterial pathogen such as Clostridium pere, or bacterium capable of developing a virulent phenotype, such as Pseudomonas aeruginosa, that is further capable of expressing a PA-I lectin/adhesin ortholog is contemplated.
  • Another aspect of the invention provides a method for modulating the activity of a bacterial MvfR protein comprising administering an effective amount of an opioid receptor antagonist to a subject at risk of developing or suffering from bacterial pathogenesis.
  • Another aspect of the invention provides a method of decreasing the permeability of, or preventing the increase in permeability of, an epithelium to a bacterial toxin comprising administering to a subject an amount of an opioid receptor antagonist effective in reducing, or inhibiting an increase in, transepithelial cell electrical resistance.
  • Another aspect of the invention provides a method for preventing or treating sepsis by administering to a patient in need of such treatment an effective amount of an opioid receptor antagonist.
  • Another aspect of the invention provides a method for preventing or treating inflammation by administering to a patient in need of such treatment an effective amount of an opioid receptor antagonist.
  • the patient has inflammation from a traumatic injury, such as an internal injury, a surgery, an acute lung injury, or a bum.
  • the patient has inflammation from an infection.
  • the infection is a bacterial infection.
  • the infectious agent is Clostridium perme, or another bacterium capable of developing a virulent phenotype, such as Pseudomonas aeruginosa.
  • Another aspect of the invention provides a method of mitigating a cell barrier dysfunction free of ⁇ -opioid receptor-dependent effects, comprising administering to a subject free of an opioid-induced side effect an effective amount of a peripheral ⁇ -opioid receptor antagonist.
  • the peripheral ⁇ -opioid receptor antagonist is N- methylnaltrexone.
  • the cell barrier dysfunction is induced by an inducing agent selected from the group consisting of thrombin and bacterial lipopolysaccharide.
  • This aspect of the invention also extends to methods wherein a protein phosphatase is activated in the cell, such as methods in which an S1P3 receptor phosphorylation is reduced.
  • a protein tyrosine phosphatase such as a receptor protein tyrosine phosphatase ⁇ , is activated.
  • Yet another aspect of the invention is a method of mitigating a cell barrier dysfunction induced by transactivation of a S1P3 receptor, comprising administering to a subject free of an opioid-induced side effect an effective amount of a peripheral ⁇ -opioid receptor antagonist.
  • the peripheral ⁇ -opioid receptor antagonist is N- methylnaltrexone.
  • Still another aspect according to the invention is a method of using an opioid receptor antagonist in the preparation of a medicament for treating, ameliorating, or preventing a disorder or a symptom of a disorder selected from the group consisting of inflammation, atherosclerosis, acute lung injury, gut-derived sepsis, a burn injury, a chemical contact injury, neonatal necrotizing enterocolitis, severe neutropenia, toxic colitis, inflammatory bowel disease, Crohn's disease, enteropathy, transplant rejection, pouchitis, pig-bel, uremic pericardial effusion, leakage in the vitreous of the eye, macular degeneration, retinal dysfunction, infection (e.g., viral infection, bacterial infection, opportunistic bacterial infection, Clostridium pere infection, Pseudomonas aeruginosa infection, Pseudomonas- mediated ophthalmologic infection, Pseudomonas-mediated otologic infection and Pseudomon ⁇ s-mediated cutaneous
  • host stress-derived BSCs host cell-derived Bacterial Signaling Compounds
  • IFN- ⁇ Interferon gamma
  • PA-I PA-I lectin/adhesin
  • MvfR a transcriptional regulator of virulence gene expression
  • the data provide evidence for a model in which opportunistic pathogens sense host stress and vulnerability by perceiving key mediators released by the host into the intestinal tract during stress, such as the stress resulting from surgery. These host stress-derived compounds directly activate critical genes in P. aeruginosa leading to enhanced virulence.
  • Opioids released in increased amount during physiological.stress, directly induce the expression of several quorum sensing-dependent virulence factors in P. aeruginosa, such as pyocyanin, biofilm, and the lectin/adhesin PA-I.
  • pyocyanin a quorum sensing-dependent virulence factors in P. aeruginosa
  • U-50,488 (bremazocine, i.e., trans-3,4-dichloro-N-methyl-N[2-(l- pyrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate, an exemplary ⁇ -opioid receptor agonist, induces pyocyanin production in P. aeruginosa via the global virulence transcriptional regulator MvfR.
  • U-50,488 also induces pyocyanin at cell densities below those that would normally produce pyocyanin.
  • An opioid-induced side effect includes an opioid-induced constipation, irritable bowel syndrome, post-operative ileus or bowel dysfunction, opioid-induced nausea, opioid-induced vomiting, urinary retention, delayed gastrointestinal tract emptying, reduced gastrointestinal tract motility and opioid-induced suppression of the immune system, hi some embodiments, the patient will not be undergoing treatment for cancer or methadone treatment for drug addiction, hi some embodiments, the subject will not be receiving or experiencing an exogenous or an endogenous opioid.
  • the invention thus provides a method of reducing the risk of developing a disorder characterized by a cell barrier dysfunction (e.g., an epithelial cell or an endothelial cell) comprising administering to a subject at risk of developing the disorder a prophylactically effective amount of a ⁇ -opioid receptor antagonist.
  • a cell barrier dysfunction e.g., an epithelial cell or an endothelial cell
  • Another aspect of the invention is drawn to a method of reducing a symptom associated with a cell barrier disorder (e.g., an epithelial or endothelial cell barrier disorder), comprising administering to a subject in need thereof a ⁇ -opioid receptor antagonist, wherein the compound is administered in an amount effective to reduce at least one symptom of the disorder.
  • Another aspect of the invention is a method of inhibiting the expression of a bacterial PA-I lectin/adhesin comprising administering an effective amount of a ⁇ -opioid receptor antagonist to a subject at risk of developing or suffering from bacterial pathogenesis, hi some embodiments of this method, the bacterial PA-I lectin/adhesin is found in a bacterium residing in a mammalian intestine, hi some embodiments of this aspect, the bacterial PA-I lectin/adhesin is a Pseudomonad PA-I lectin/adhesin. An important Pseudomonad is Pseudomonas aeruginosa.
  • Another aspect of the invention is directed to a method of modulating the activity of a bacterial MvfR protein comprising administering an effective amount of a ⁇ -opioid receptor antagonist to a subject at risk of developing or suffering from bacterial pathogenesis.
  • the bacterial MvfR protein is found in a bacterium residing in a mammalian intestine.
  • the bacterial MvfR protein is a Pseudomonad MvfR protein, preferably a Pseudomonas aeruginosa MvfR protein.
  • the invention provides a method of decreasing the permeability of, or preventing the increase in permeability of, an epithelium to a bacterial toxin comprising administering to a subject an amount of a ⁇ -opioid receptor antagonist effective in reducing, or inhibiting an increase in, transepithelial cell electrical resistance (i.e., transcellular electrical resistance of an epithelium).
  • An epithelium in the context of this aspect of the invention comprises at least two epithelial cells.
  • the epithelial cells are intestinal epithelial cells.
  • any mode of administering the opioid receptor antagonist that is known in the art is contemplated, and in particular, delivery by parenteral, oral, subcutaneous, transcutaneous, subcutaneous implantation, intramuscular, intravenous, intrathecal, intraocular, intravitreous, ophthalmologic, intraspinal, intrasynovial, topical, rectal, transepithelial including transdermal, buccal, sublingual, intramuscular, intracavity, and aural routes, as well as by nasal inhalation including via insufflation and aerosol.
  • Microbial pathogens such as P.
  • the invention comprehends administering the opioid receptor antagonist by direct routes, e.g., as by topical delivery, cutaneous delivery, intravitreous delivery, and intracerebroventricular delivery, to achieve localized, therapeutically useful concentrations of the antagonist, hi addition, the invention comprehends treatment of any disorder caused, at least in part, by a microbial pathogen such as P.
  • a microbial pathogen such as P.
  • aeruginosa which includes Pseudomonas-mediated ophthalmologic, Pseudomonas-medmted otologic or Pseudomonas-mediated cutaneous disorders, by administering an opioid receptor antagonist through conventional systemic routes, including intravitreously, intracerebroventricularly, and topically (e.g., ophthalmologically, otologically, cutaneously), at levels sufficient to achieve therapeutically useful systemic levels of the antagonist.
  • FIG. 1 Brief Description of the Drawing Figure 1 is a panel of graphs, bar graphs and immunoblots showing that IFN- ⁇ induces the expression of the PA-I lectin in P. aeruginosa.
  • Figure 2 is a panel of bar graphs showing that the presence of rhlland rhlR, core quorum sensing signaling elements in P. aeruginosa, are required for the PA-I expression and pyocyanin production in response to IFN- ⁇ .
  • Figure 3 is a panel of graphs, an epimicrograph, immunoblots and MS/MS spectra showing the identification of the IFN- ⁇ binding site to solubilized membrane fractions of P. aeruginosa (PAOl).
  • Figure 4 is a panel of bar charts and graphs showing the binding characteristics of the IFN- ⁇ to membrane fractions of P. aeruginosa (PAOl).
  • Figure 5 is a panel of graphs, bar charts and immunoblots showing that IFN- ⁇ binds to OprF and induces PA-I expression.
  • Figure 6 is a panel of bar graphs and graphs showing that MvfR plays a key role in the effect of U-50,488 and C4-HSL on PCN production.
  • Figure 7 is a bar graph showing the inhibition of mo ⁇ hine-induced PA-I lectin/adhesin expression in the separate presences of ⁇ -opioid receptor antagonists methylnaltrexone (MNTX) and naloxone (NAL).
  • MNTX methylnaltrexone
  • NAL naloxone
  • Figure 8 is a panel of graphs and bar graphs showing the effects of adenosine and inosine on PA-I expression.
  • Figure 9 is a panel of graphs and bar graphs showing the effects of methylnaltrexone (MNTX) and DAMGO on human endothelial cell barrier regulation.
  • Figure 10 is a panel of graphs showing the effects of MNTX effects on non- opioid agonist-induced human endothelial cell barrier regulation.
  • Figure 11 is a bar graph showing the differential effects of MNTX and naloxone on agonist-induced human endothelial cell barrier disruption.
  • Figure 12 is a panel of bar graphs and immunoblots showing the effects of silencing Mu opioid receptor, SlPi receptor or SlP 3 receptor on MNTX-induced protection from human endothelial cell barrier disruption.
  • Figure 13 is a panel of immunoblots and bar graphs showing the effects of MNTX, naloxone and Src on SlP 3 receptor transactivation (tyrosine phosphorylation).
  • Figure 14 is a panel of bar graphs showing the analysis of agonist-induced total cellular tyrosine phosphatase activity in human endothelial cells.
  • Figure 15 is a panel of graphs and immunoblots showing the effects of SlP 3 receptor transactivation and endothelial cell barrier function by receptor tyrosine phosphatase mu (RPTP ⁇ ).
  • Figure 16 is a panel of bar graphs showing the regulation of agonist-induced total cellular tyrosine phosphatase activity and MNTX-induced protection from human endothelial cell barrier disruption by RPTP ⁇ .
  • Figure 17 is a panel of immunhistochemical stains and bar graphs showing the effect of MNTX on LPS-induced pulmonary vascular hyper-permeability in vivo.
  • Figure 18 is a panel of bar graphs showing the effects of silencing mu opioid receptor expression using siRNA on agonist-induced barrier function.
  • Figure 19 is a schematic illustration of pathways relevant to cell barrier function, and dysfunction.
  • a wide variety of inflammatory disorders, tumor metastasis, and a variety of other diseases and disorders are characterized by a cell barrier dysfunction manifested as an increased cell barrier permeability or loss of selective permeability and concomitant exudation of cells, cellular contents, fluid or protein across the barrier.
  • a cell barrier dysfunction manifested as an increased cell barrier permeability or loss of selective permeability and concomitant exudation of cells, cellular contents, fluid or protein across the barrier.
  • an endothelial cell barrier dysfunction can lead to increased vascular permeability and a resulting extravasation of protein and fluids, characteristic of inflammatory processes.
  • McVerry et al., Cell. Signal. 17:131-139 (2005) Analogously, a cell barrier dysfunction can become permissive for tumor cell metastasis.
  • Microbial pathogenesis can be the product of infection by a pathogen (e.g., Clostridium perflora) or by the phenotypic shift of a normally benign member of the normal flora associated with an organism (e.g., intestinal flora) to a pathogenic or virulent state (e.g., Pseudomonas aeruginosa).
  • a pathogen e.g., Clostridium peroxib
  • a normally benign member of the normal flora associated with an organism e.g., intestinal flora
  • a pathogenic or virulent state e.g., Pseudomonas aeruginosa
  • multi-cellular organisms such as vertebrates (e.g., mammals, including humans) generally exhibit supracellular compartmentalization resulting in discrete spacings for tissues, organs, and organ systems. Chief contributors to this necessary compartmentalization are the several kinds of cell barriers. Exemplified in terms of endothelial and epithelial cell barriers, there are cell barriers associated with most tissues, organs, and organ systems, e.g., brain (e.g., cerebral endothelial lining/blood brain barrier), spleen, liver, eye, lung, vasculature (blood and lymph), kidney, bladder, ureter, urethra, alimentary canal, including the small and large intestines, lung, and the like.
  • the invention provides methods for preventing, reducing or eliminating a cell barrier dysfunction associated with a disease or disorder that is capable of lowering the quality of life or that deleteriously impacts the health of a subject or patient that has the disease or disorder.
  • host stress signaling compounds and the membrane receptors to which they bind such as receptors on host cells (e.g., epithelial and endothelial cells) as well as receptors on pathogenic microbes such as infectious bacteria, will lead to the discovery of therapeutic targets that will allow for prevention or treatment in a variety of cell barrier diseases and disorders, including the infection, at its most proximate point.
  • conserved receptors e.g., bacterial receptors common to other microbial species
  • Such an approach of rendering recipient cells (e.g., colonizing pathogens) insensate to host stress activators has the potential to provide efficacious and cost-effective treatment for a wide variety of diseases and disorders characterized by cell barrier dysfunction.
  • abnormal condition is broadly defined to include mammalian diseases, mammalian disorders and any abnormal state of mammalian health that is characterized by a cell barrier dysfunction.
  • Exemplary cells that may exhibit a cell barrier dysfunction, or be at risk of developing such a dysfunction include endothelial cells and epithelial cells.
  • the abnormal conditions may be found in humans, non-human mammals, or any mammal.
  • “Burn injury” means (i) damage to mammalian tissue resulting from exposure of the tissue to heat, for example in the form of an open flame, steam, hot fluid, and a hot surface.
  • a “chemical contact injury” refers to an injury caused by direct contact with a chemical and can involve a chemical burn or other injury. "Severe neutropenia” is given its ordinary and accustomed meaning of a marked decrease in the number of circulating neutrophils.
  • administering is given its ordinary and accustomed meaning of delivery of a therapeutic to an organism in need by any suitable means recognized in the art.
  • exemplary forms of administering include delivery by parenteral, oral, subcutaneous, transcutaneous, subcutaneous implantation, intramuscular, intravenous, intrathecal, intraocular, intravitreous, ophthalmologic, intraspinal, topical, rectal, transdermal, sublingual, intramuscular, intracavity, and aural routes, as well as by nasal inhalation (e.g., nebulizing spray).
  • the mechanism of delivery may be direct puncture or injection, or gel or fluid application to an eye, ear, nose, mouth, anus or urethral opening, as well as cannulation.
  • an "effective dose” is that amount of a substance that provides a beneficial effect on the organism receiving the dose and may vary depending upon the purpose of administering the dose, the size and condition of the organism receiving the dose, and other variables recognized in the art as relevant to a determination of an effective dose.
  • the process of determining an effective dose involves routine optimization procedures that are within the skill in the art.
  • an "animal” is given its conventional meaning of a non-plant, non-protist living being.
  • a preferred animal is a mammal, such as a human.
  • a “need” is an organismal, organ, tissue, or cellular state that could benefit from administration of an effective dose to an organism characterized by that state.
  • a human at risk of developing gut-derived sepsis, or presenting a symptom thereof is an organism in need of an effective dose of a product, such as a pharmaceutical composition, according to the present invention.
  • Average molecular weight is given its ordinary and accustomed meaning of the arithmetic mean of the molecular weights of the components (e.g., molecules) of a composition, regardless of the accuracy of the determination of that mean.
  • polyethylene glycol, or PEG having an average molecular weight of 3.5 kilodaltons may contain PEG molecules of varying molecular weight, provided that the arithmetic mean of those molecular weights is determined to be 3.5 kilodaltons at some level of accuracy, which may reflect an estimate of the arithmetic mean, as would be understood in the art.
  • PEG 15-20 means PEG whose molecular weights yield an arithmetic mean between 15 and 20 kilodaltons, with that arithmetic mean subject to the caveats noted above.
  • PEG molecules include, but are not limited to, simple PEG polymers. For example, a plurality of relatively smaller PEG molecules (e.g., 7,000 to 10,000 daltons) may be joined, optionally with a linker molecule such as a phenol, into a single molecule having a higher , average molecular weight (e.g., 15,000 to 20,000 daltons).
  • PA-I PA-I lectin/adhesin
  • PA-IL PA-IL expression means the production or generation of an activity characteristic of PA-I lectin/adhesin.
  • PA-I lectin/adhesin expression involves translation of a PA-I lectm/adhesin-encoding mRNA to yield a PA-I lectin/adhesin polypeptide having at least one activity characteristic of PA-I lectin/adhesin.
  • PA-I lectin/adhesin further includes transcription of a PA-I lectin/adhesin-encoding DNA to yield the aforementioned mRNA.
  • Intestinal pathogen means a microbial pathogen capable of causing, in whole or part, gut-derived sepsis in an animal such as a human. Intestinal pathogens known in the art are embraced by this definition, including gram negative bacilli such as the Pseudomonads (e.g., Pseudomonas aeruginosa).
  • Pathogenic quorum means aggregation or association of a sufficient number of pathogenic organisms (e.g., P. aeruginosa) to initiate or maintain a quorum sensing signal or communication that a threshold concentration, or number, of organisms (e.g., intestinal pathogens) are present, as would be known in the art.
  • pathogenic organisms e.g., P. aeruginosa
  • a threshold concentration, or number, of organisms e.g., intestinal pathogens
  • Transcellular Electrical Resistance or TER, is given the meaning this phrase has acquired in the art, which refers to a measurement of electrical resistance across cells of a given type (e.g., epithelial or endothelial cells), which is non-exclusively useful in assessing the status of tight junctions between such cells.
  • TEER is used herein to refer to "transepithelial cell electrical resistance,” or “transendothelial cell electrical resistance,” and the particular usage will be apparent from context.
  • “Pharmaceutical composition” means a formulation of compounds suitable for therapeutic administration, to a living animal, such as a human patient. Preferred pharmaceutical compositions according to the invention are described in the copending U.S. Patent Publication No. 20040266806 the contents of which are herein incorporated herein by reference in their entireties.
  • the pharmaceutical compositions of the invention may comprise a solution balanced in viscosity, electrolyte profile and osmolality, comprising an electrolyte, dextran-coated L-glutamine, dextran-coated inulin, lactulase, D-galactose, N-acetyl D- galactosamine and 5-20% PEG (15,000-20,000).
  • the compounds are preferably combined with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice as described, for example, in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980), the disclosures of which are hereby incorporated herein by reference, in their entireties.
  • a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice as described, for example, in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980), the disclosures of which are hereby incorporated herein by reference, in their entireties.
  • adjuvants are each given the meanings those terms have acquired in the art.
  • An adjuvant is one or more substances that serve to prolong the immunogenicity of a co-administered immunogen.
  • a carrier is one or more substances that facilitate the manipulation, such as by translocation of a substance being carried.
  • a diluent is one or more substances that reduce the concentration of, or dilute, a given substance exposed to the diluent.
  • Alkyl refers to an aliphatic hydrocarbon group which is saturated and which may be straight, branched or cyclic and has from 1 to about 10 carbon atoms in the chain, as well as all combinations and subcombinations of chains therein.
  • exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.
  • “Lower alkyl” refers to an alkyl group having 1 to about 6 carbon atoms.
  • alkenyl refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and having from 2 to about 10 carbon atoms in the chain, as well as all combinations and sub- combinations of chains therein.
  • alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups.
  • Alkynyl refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and having from 2 to about 10 carbon atoms in the chain, as well as combinations and sub-combinations of chains therein.
  • exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl groups.
  • Alkylene refers to a bivalent aliphatic hydrocarbon group having from 1 to about 6 carbon atoms, and all combinations and subcombinations of chains therein.
  • the alkylene group may be straight, branched or cyclic.
  • alkenylene refers to an alkylene group containing at least one carbon- carbon double bond.
  • Cycloalkyl refers to any stable monocyclic or bicyclic ring having from about 3 to about 10 carbons, and all combinations and subcombinations of rings therein.
  • the cycloalkyl group may be substituted with one or more cycloalkyl-group substituents.
  • Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups.
  • Cycloalkyl-substituted alkyl refers to a linear alkyl group, preferably a lower alkyl group, substituted at a terminal carbon with a cycloalkyl group, preferably a C3-C8 cycloalkyl group.
  • exemplary cycloalkyl-substituted alkyl groups include cyclohexylmethyl, cyclohexylethyl, cyclopentylethyl, cyclopentylpropyl, cyclopropyhnethyl and the like.
  • Cycloalkenyl refers to an olefinically unsaturated cycloaliphatic group having from about 4 to about 10 carbons, and all combinations and subcombinations of rings therein.
  • Alkoxy refers to an alkyl substituted hydroxyl, or alkyl-O, group, where alkyl is as previously described.
  • exemplary alkoxy groups include, for example, methoxy, ethoxy, propoxy, butoxy and heptoxy.
  • Alkoxy-alkyl refers to a di-alkyl ether, or alkyl-O-alkyl, group, where alkyl is as previously described.
  • acyl means an alkyl-CO group wherein alkyl is as previously described.
  • exemplary acyl groups include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
  • Aryl refers to an aromatic carbocyclic group containing from about 6 to about 10 carbons, and all combinations and subcombinations of rings therein.
  • the aryl group may be substituted with one or two or more aryl group substituents.
  • Exemplary aryl groups include phenyl and naphthyl.
  • Aryl-substituted alkyl refers to a linear alkyl group, preferably a lower alkyl group, substituted at a terminal carbon with an optionally substituted aryl group, preferably an optionally substituted phenyl ring.
  • exemplary aryl-substituted alkyl groups include, for example, phenylmethyl, phenylethyl and 3-(4-methylphenyl)propyl.
  • Heterocyclic refers to a monocyclic or multicyclic ring system carbocyclic group or radical containing from about 4 to about 10 members, and all combinations and subcombinations of rings therein, wherein one or more of the members of the ring is an element other than carbon, for example, nitrogen, oxygen or sulfur.
  • the heterocyclic group may be aromatic or nonaromatic.
  • Exemplary heterocyclic groups include, for example, pyrrole and piperidine groups.
  • Halo refers to fluoro, chloro, bromo or iodo.
  • Opium alkaloid derivative refers to mu opioid receptor antagonists (e.g., peripheral antagonists) that are synthetic or semi-synthetic derivatives or analogs of opium alkaloids.
  • Substantially no agonist activity in connection with the opium alkaloid derivatives, means that, at a concentration of 1 ⁇ M, the maximal measured physiological response of a receptor, e.g., electrically stimulated guinea pig ileum, is about 60% or less relative to morphine.
  • HMW PEG-like compounds refer to relatively high molecular weight PEG compounds, defined as having an average molecular weight greater than 3.5 kilodaltons (kD).
  • HMW PEG has an average molecular weight greater than 5 kilodaltons and, in particular embodiments, HMW PEG has an average molecular weight at least 8 kilodaltons, more than 12 kilodaltons, at least 15 kilodaltons, and between 15 and 20 kilodaltons.
  • HMW PEG-like compounds includes HMW PEG derivatives wherein each such derivative is an HMW PEG containing at least one additional functional group. Preferred HMW PEG derivatives are cationic polymers.
  • Exemplary functional groups include any of the alkoxy series, preferably Cl-ClO, any of the aryloxy series, phenyl and substituted phenyl groups. Such functional groups may be attached at any point to an HMW PEG molecule, including at either terminus or in the middle; also included are functional groups, e.g., phenyl and its substituents, that serve to link to smaller PEG molecules or derivative thereof into a single HMW PEG-like compound.
  • the HMW PEG-like molecules having an additional functional group may have one such group or more than one such group; each molecule may also have a mixture of additional functional groups, provided such molecules are useful in stabilizing at least one therapeutic during delivery thereof or in treating, ameliorating or preventing a disease, disorder or condition of an epithelial cell.
  • peripheral opioid receptor antagonist designates an opioid receptor antagonist, including a ⁇ -opioid receptor antagonist, that acts primarily on physiological systems and components external to the central nervous system, i.e., the antagonist does not readily cross the blood-brain barrier.
  • the peripheral opioid receptor antagonists employed in the methods of the invention exhibit high levels of activity with respect to gastrointestinal tissue, while exhibiting reduced, and preferably substantially no, central nervous system (CNS) activity.
  • CNS central nervous system
  • substantially no CNS activity means that less than 20% of the pharmacological activity of the peripheral opioid receptor antagonists exhibited outside the CNS is exhibited inside the CNS.
  • the peripheral opioid receptor antagonists employed in the inventive methods exhibit less than 15% of their pharmacological activity in the CNS, with less than about 10% being more preferred. In even more preferred embodiments, the peripheral opioid receptor antagonists employed in the methods of the invention exhibit less than 5% of their pharmacological activity in the CNS, with about 0% (i.e., no CNS activity) also being more preferred.
  • Preferred peripheral opioid receptor antagonists of the invention are quaternary derivatives of noroxymorphone, such as R-methylnaltrexone.
  • mice In general terms, a model of lethal sepsis in mice has been developed which provides unique insight into the process by which microbial pathogens can cause lethal sepsis syndrome from within the intestinal tract of a physiologically stressed host. Three physiologic "hits" result in mortality, e.g., surgical stress (30% hepatectomy), starvation (48 hour of water only) and the introduction of P. aeruginosa into the distal intestinal tract (cecum). This model results in 100% mortality, whereas elimination of any one of the three factors results in complete survival.
  • PA-I Pseudomonas aeruginosa
  • PA-I lectin/adhesin plays a key role in the lethal effect of this organism by creating a permeability defect to potent and lethal cytotoxins of P. aeruginosa, such as exotoxin A and elastase.
  • the lethal effect of intestinal P. aeruginosa appears to occur completely independent of its extraintestinal dissemination (translocation).
  • systemic injection intravenous, intraperitoneal
  • P. aeruginosa in this model produces no mortality and no systemic inflammation.
  • BSC host stress-derived bacterial signaling compounds
  • aeruginosa causing sepsis following lung instillation are not those that display the most invasive (translocating/disseminating) phenotype, but rather are those strains that are most disruptive of cellular integrity and epithelial permselectivity to its locally released cytotoxins.
  • PA-I the lecA gene
  • the gene encoding PA-I is an ideal biological "read-out” and reporter gene in which to examine overall virulence gene expression in P. aeruginosa in response to host stress-derived BSCs.
  • Opioids are highly conserved compounds and various bacteria and fungi, including P. aeruginosa, synthesize and metabolize morphine.
  • elements of the immune system such as IFN- ⁇ , can also serve as potent host stress-derived BSCs.
  • P. aeruginosa is able to sense the presence of the IFN- ⁇ and respond by expressing two quorum sensing dependent virulence factors, PA-I and pyocyanin. From the perspective of P.
  • aeruginosa the ability to sense and respond to host immune activation, in particular to IFN- ⁇ whose function is directed at bacterial clearance, provides this organism with a counternieasure against host immune activation, hi particular, Interferon- ⁇ is shown below to bind to an outer membrane protein in P. aeruginosa, OprF, resulting in the expression of a quorum sensing-dependent virulence determinant, the PA-I lectin. IFN- ⁇ also bound E. coli membranes.
  • C4-HSL also requires intact MvfR to produce PCN, coupled with the finding of highly up-regulated PCN production in strains harboring multiple mvJR genes, is consistent with quorum sensing activation relying not only on the binding of QS signaling molecules to their core QS transcriptional regulators (i.e., RhIR, LasR), but also having QS signals activating proximal transcriptional regulators.
  • QS transcriptional regulators i.e., RhIR, LasR
  • opioid compounds may vary in their ability to induce a particular virulence phenotype in P. aeruginosa. It is contemplated that there are multiple host-stress-derived bacterial signaling compounds that are able to influence the state of virulence in P. aeruginosa.
  • Norepinephrine can also affect the QS-dependent virulence factor PA-IL in P. aeruginosa (J. Alverdy, et al., Ann Surg 232, 480 (2000)) and soluble compounds released into the media by hypoxic intestinal epithelial cells also induce PA-IL expression. Consistent with these disclosures is the disclosure that norepinephrine directly affects QS circuitry in E. coli (V. Sperandio, A. G. Torres, B. Jarvis, J. P. Nataro and J. B. Kaper, Proc Natl Acad Sci U S A 100, 8951 (2003)).
  • the invention provides methods of screening for modulators of the signaling induced by one or more BSCs, including such modulators as opioid receptor agonists, morphine, and interferon gamma.
  • modulators as opioid receptor agonists, morphine, and interferon gamma.
  • These therapeutics are delivered to an organism, such as a human patient, in need thereof. Dosage levels and delivery routes and schedules will vary depending upon circumstances readily identified and accommodated by those skilled in the art using routine procedures.
  • the therapeutics according to the invention may further comprise a HMW PEG-like compound, which may be administered by any means suitable for the condition or disorder to be treated.
  • the compound(s) may be delivered orally, such as in the form of tablets, capsules, granules, powders, or with liquid formulations including syrups; by sublingual; buccal; or transdermal delivery; by injection or infusion parenterally, subcutaneously, transcutaneously, subcutaneous implantation, intravenously, intramuscularly, intrathecally, intraocularly, ophthalmologically, intraspinally, topically, or intrasternally (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); orally, nasally, such as by inhalation spray; aurally, rectally such as in the form of suppositories; vaginally or urethrally via suppository or infusion, e.g., via cannulation, or liposomally, and intracavity delivery.
  • parenterally subcutaneously, transcutaneously, subcutaneous implantation, intravenously, intramuscularly, intrathecally, intraocularly,
  • Dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents may be administered.
  • the compounds may be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved with suitable pharmaceutical compositions known in the art.
  • compositions for oral administration include suspensions which may contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, sweeteners or flavoring agents such as those known in the art; and immediate release tablets which may contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants, such as those known in the art.
  • the inventive compounds may be orally delivered by sublingual and/or buccal administration, e.g., with molded, compressed, or freeze-dried tablets.
  • compositions may include fast-dissolving diluents such as mannitol, lactose, sucrose, and/or cyclodextrins.
  • excipients such as a relatively high molecular weight cellulose (AVICEL ® ) or a polyethylene glycol (PEG; GoLytely ® , 3.34 kD); an excipient to aid mucosal adhesion such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC), and/or maleic anhydride copolymer (e.g., GANTREZ ® ).
  • Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use.
  • compositions for nasal aerosol or inhalation administration include solutions which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance absorption and/or bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
  • compositions for intestinal administration include solutions or suspensions which may contain, for example, suitable non-toxic diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides and fatty acids, including oleic acid.
  • suitable non-toxic diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides and fatty acids, including oleic acid.
  • suitable non-irritating excipients such as cocoa butter, synthetic glyceride esters or polyethylene glycols (e.g., GoLytely ® ).
  • the effective amount of a compound of the present invention may be determined by one of ordinary skill in the art.
  • the specific dose level and frequency of dosage for any particular subject may vary and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.
  • Preferred subjects for treatment include animals, most preferably mammalian species such as humans, and domestic animals such as dogs, cats, horses, and the like, at risk of developing a microbe-mediated epithelial condition or disease, such as gut-derived sepsis, or at risk of developing an inflammatory disorder, e.g., acute lung injury, characterized by cell barrier dysfunction.
  • the peripheral opioid receptor antagonists of the invention are administered in an effective amount, e.g., from 10 ⁇ 6 M to 10 "9 M.
  • Patient drug plasma levels may be measured using routine HPLC methods known to those of skill in the art.
  • the invention provides methods of administering opioid receptor antagonists to treat, prevent, or alleviate a symptom associated with, a disease or disorder characteristically exhibiting a cell barrier dysfunction.
  • the opioid receptor antagonist may be a mu opioid antagonist, or the antagonist may be a kappa opioid antagonist.
  • the invention also encompasses administration of more than one opioid antagonist, including combinations of mu antagonists, combinations of kappa antagonists, and combinations of at least one mu antagonist and at least one kappa antagonist; the invention further comprehends administration of combinations of at least one centrally acting opioid receptor antagonist and at least one peripherally restricted opioid receptor antagonist. For example, a combination of methylnaltrexone and either alvimopan or its metabolite ADL 08-0011, or a combination of naltrexone and methylnaltrexone, may be administered.
  • both morphine and DAMGO induce cell barrier dysfunction, such as pulmonary microvascular endothelial cell barrier disruption.
  • Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells.
  • Certain inflammatory syndromes for example, acute lung injury and sepsis, reduce barrier function.
  • Such barrier disruption results in increased vascular permeability and organ dysfunction.
  • a peripheral opioid receptor antagonist in accordance with the invention enhanced endothelial cell barrier function. Specifically, the cell barrier disruption is blocked by pretreatment with a peripheral opioid receptor antagonist.
  • peripheral opioid receptor antagonist e.g., MNTX
  • a peripheral opioid receptor antagonist e.g., MNTX
  • the peripheral opioid receptor antagonist is also useful in protecting against cell barrier dysfunction arising from both ⁇ opioid receptor- dependent effects, e.g., effects of ⁇ opioid receptor agonist (e.g., morphine) binding, and ⁇ opioid receptor-independent effects, e.g., effects realized without a contribution from a ⁇ opioid receptor, such as thrombin- and/or lipopolysaccharide (LPS)-dependent cell barrier dysfunction or disruption, such as in endothelial cells.
  • ⁇ opioid receptor- dependent effects e.g., effects of ⁇ opioid receptor agonist (e.g., morphine) binding
  • ⁇ opioid receptor-independent effects e.g., effects realized without a contribution from a ⁇ opioid receptor, such as thrombin- and/or lipopolysaccharide (LPS)-dependent cell barrier dysfunction or disruption, such as in endot
  • ⁇ opioid receptor antagonists e.g., peripheral ⁇ opioid receptor antagonists
  • inflammatory syndromes e.g., acute lung injury, atherosclerosis, and other diseases characterized by a cell barrier dysfunction.
  • the methods of the invention have therapeutic value in the treatment of those syndromes characterized by barrier dysfunction or disruption, e.g., atherosclerosis, acute lung injury, microbial infection, and the like. It is, therefore, contemplated that the invention includes methods of reducing cell barrier disruption by administering to the cells an effective amount of a cell barrier enhancement protective agent, e.g., MNTX.
  • a cell barrier enhancement protective agent e.g., MNTX.
  • the methods of the invention also encompass treating patients who are undergoing treatment with opioid receptor agonists, although in some embodiments, the patients are not chronic recipients of any opioid receptor agonist.
  • the opioid receptor agonists may be exogenously or endogenously supplied, and the agonist may be a naturally occurring opioid or a non-naturally occurring synthetic compound.
  • cancer patients frequently receive morphine to manage pain associated with advanced stages of the disease and, while the ⁇ opioid receptor antagonists are useful in this context in providing beneficial effects on cell barrier dysfunction without undermining efforts to manage pain, these ⁇ opioid receptor antagonists also find use in treating cancer at a much earlier stage.
  • the ⁇ opioid receptor antagonists are beneficially administered to cancer patients having pre-metastatic stage tumors, e.g., peri-operatively, where pain management may not dictate the need for a ⁇ opioid receptor agonist such as morphine.
  • a ⁇ opioid receptor antagonist provides therapeutic support of normal cell barrier function, facilitating resistance to the metastatic processes (i.e., tumor cell seeding) that exploit cell barrier dysfunction. Consequently, ⁇ opioid receptor antagonists have a particular application in pre-metastatic cancer patient populations, which are populations typically free of chronic recipients of opioid receptor agonists like morphine.
  • a ⁇ opioid receptor antagonist e.g., a peripheral ⁇ opioid receptor antagonist such as MNTX
  • a ⁇ opioid receptor antagonist e.g., a peripheral ⁇ opioid receptor antagonist such as MNTX
  • the surgical intervention creates a host stress that may signal cells, such as endothelial and/or epithelial cells of a wide variety of tissues, organs and organ systems (e.g., lung, gut, vasculature, eye) in a manner that leads to a cell barrier dysfunction that facilitates cancer cell mobilization or metastasis.
  • Opioid receptor agonists include, but are not limited to, morphine, methadone, codeine, meperidine, fentidine, fentanil, sufentanil, alfentanil and the like. Opioid receptor agonists are classified by their ability to agonize one type of receptor an order of magnitude more effectively than another.
  • the relative affinity of morphine for the mu receptor is 200 times greater than for the kappa receptor, and it is therefore classified as a mu opioid receptor agonist.
  • Some opioid compounds may act as agonists towards one receptor type and as antagonists toward another receptor type; such and are classified as agonist/antagonists, (also known as mixed or partial agonists)., "Agonist/antagonist,” “partial agonist,” and “mixed agonist” are used interchangeably herein.
  • These opioids include, but are not limited to, pentazocine, butorphanol, nalorphine, nalbufine, buprenorphine, bremazocine, and bezocine.
  • opioids are agonists of the kappa receptors and antagonists of the mu receptors.
  • active metabolites of opioid receptor agonists will also be active in the methods of the invention.
  • the metabolites of morphine, morphine 3-glucuronide and morphine 6-glucuronide are expected to be active in preventing, reducing or eliminating cell barrier dysfunction.
  • peripheral opioid receptor antagonists form a class of compounds that can vary in structure while maintaining the restriction to peripheral receptor interaction. These compounds include tertiary and quaternary morphinans, in particular noroxymorphone derivatives, N-substituted piperidines, and in particular, piperidine-N-alkylcarboxylates, and tertiary and quaternary benzomorphans.
  • Peripherally restricted antagonists while varied in structure, are typically charged, polar and/or of high molecular weight, each of which impedes crossing of the blood-brain barrier.
  • the present methods involve the administration to a patient of a peripheral ⁇ -opioid receptor antagonist that is a piperidine-N-alkylcarboxylate compound.
  • Piperidine-N-alkylcarboxylate opioid antagonists include, for example, the compounds disclosed in U.S. Patent Nos. 5,250,542; 5,159,081; 5,270,328; and 5,434,171, the disclosures of which are hereby incorporated herein by reference, in their entireties.
  • a class of piperidine-N-alkylcarboxylate opioid antagonists include those having the following formula (I):
  • Rl is hydrogen or alkyl
  • R2 is hydrogen, alkyl or alkenyl
  • R3 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl or aryl-substituted alkyl;
  • R4 is hydrogen, alkyl or alkenyl
  • A is OR5 or NR6 R7; wherein:
  • R5 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl;
  • R6 is hydrogen or alkyl
  • R7 is hydrogen, alkyl, alkenyl, cycloalkyl, aryl, cycloalkyl-substituted alkyl, cycloalkenyl, cycloalkenyl-substituted alkyl, aryl-substituted alkyl, aryl-substituted alkyl, or alkylene substituted B or, together with the nitrogen atom to which they are attached, R6 and R7 form a heterocyclic ring;
  • B is
  • R8 is hydrogen or alkyl
  • R9 is hydrogen, alkyl, alkenyl, cycloalkyl-substituted alkyl, cycloalkyl, cycloalkenyl, cycloalkenyl-substituted alkyl, aryl or aryl-substituted alkyl or, together with the nitrogen atom to which they are attached, R8 and R9 form a heterocyclic ring;
  • W is ORlO, NRl 1 R12, or OE;
  • RlO is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl;
  • Rl 1 is hydrogen or alkyl
  • Rl 3 is alkyl substituted alkylene
  • R14 is alkyl
  • D is OR15 or NRl 6 Rl 7;
  • Rl 5 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl;
  • Rl 6 is hydrogen, alkyl, alkenyl, aryl, aryl-substituted alkyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl or cycloalkenyl-substituted alkyl;
  • Rl 7 is hydrogen or alkyl or, together with the nitrogen atom to which they are attached, Rl 6 and Rl 7 form a heterocyclic ring;
  • Y is OR18 orNR19 R20;
  • Rl 8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl;
  • Rl 9 is hydrogen or alkyl
  • R20 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl or, together with the nitrogen atom to which they are attached, Rl 9 and R20 form a heterocyclic ring;
  • R21 is hydrogen or alkyl;
  • n 0 to about 4;
  • Rl is hydrogen or alkyl.
  • Rl is hydrogen or Cl -C5 alkyl.
  • R2 is hydrogen, alkyl or alkenyl.
  • R2 is hydrogen, Cl -C5 alkyl or C2 -C6 alkenyl.
  • R2 is alkyl, with Cl -C3 alkyl being more preferred.
  • R3 is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl or aryl-substituted alkyl.
  • R3 is hydrogen, Cl -ClO alkyl, C3 -ClO alkenyl, phenyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C8 cycloalkyl- substituted Cl -C3 alkyl or phenyl-substituted Cl -C3 alkyl.
  • R3 is benzyl, phenyl, cyclohexyl, or cyclohexylmethyl.
  • R4 is hydrogen, alkyl or alkenyl.
  • R4 is hydrogen, Cl -C5 alkyl or C2 -C6 alkenyl.
  • R4 is Cl -C3 alkyl, with methyl being more preferred.
  • A is OR5 or NR6 R7.
  • R5 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl- substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl.
  • R5 is hydrogen, Cl -ClO alkyl, C2 -ClO alkenyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C8 cycloalkenyl-substituted Cl -C3 alkyl, or phenyl-substituted Cl -C3 alkyl.
  • R5 is hydrogen or alkyl, with Cl -C3 alkyl being more preferred.
  • R6 is hydrogen or alkyl.
  • R6 is hydrogen or Cl -C3 alkyl.
  • R6 is hydrogen.
  • R7 is hydrogen, alkyl, alkenyl, cycloalkyl, aryl, cycloalkyl- substituted alkyl, cycloalkenyl, cycloalkenyl-substituted alkyl, aryl-substituted alkyl, aryl- substituted alkyl or alkylene substituted B.
  • R7 is hydrogen, Cl -ClO alkyl, C3 -ClO alkenyl, phenyl, cycloalkyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C8 cycloalkenyl, C5 -C8 cycloalkenyl-substituted Cl -C3 alkyl, phenyl-substituted Cl -C3 alkyl or (CH2)q -B. In some embodiments, R7 is (CH2)q -B.
  • R6 and R7 form, together with the nitrogen atom to which they are attached, a heterocyclic ring.
  • R8 in the definition of B is hydrogen or alkyl. In some embodiments, R8 is hydrogen or Cl -C3 alkyl.
  • R9 in the definition of B is hydrogen, alkyl, alkenyl, cycloalkyl-substituted alkyl, cycloalkyl, cycloalkenyl, cycloalkenyl-substituted alkyl, aryl or aryl-substituted alkyl.
  • R9 is hydrogen, Cl -ClO alkyl, C3 -ClO alkenyl, cycloalkyl-substituted
  • Cl -C3 alkyl cycloalkyl, C5 -C8 cycloalkenyl, C5 -C8 cycloalkenyl-substituted Cl -C3 alkyl, phenyl or phenyl-substituted Cl -C3 alkyl.
  • R8 and R9 form, together with the nitrogen atom to which they are attached, a heterocyclic ring.
  • the group W in the definition of B is ORlO, NRl 1 R12 or OE.
  • the group RlO in the definition of W is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl.
  • RlO is hydrogen, Cl -ClO alkyl, C2 -ClO alkenyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C8 cycloalkenyl-substituted Cl -C3 alkyl, or phenyl-substituted Cl -C3 alkyl.
  • RlO is hydrogen, alkyl, Cl -C5 alkyl, phenyl-substituted alkyl, phenyl-substituted Cl -C2 alkyl, cycloalkyl or cycloalkyl-substituted alkyl, C5 -C6 cycloalkyl-substituted Cl -C3 alkyl.
  • Rl 1 in the definition of W is hydrogen or alkyl. hi some embodiments, Rl 1 is hydrogen or Cl -C3 alkyl.
  • Rl 2 is hydrogen, Cl -ClO alkyl, C3 -ClO alkenyl, phenyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -
  • Rl 2 is hydrogen, alkyl, some Cl -
  • Rl 2 The group Y in the definition of Rl 2 is ORIS or NRl 9 R20.
  • R12 and R13 form, together with the nitrogen atom to which they are attached, a heterocyclic ring.
  • E is:
  • the group Rl 3 in the definition of E is alkyl substituted alkylene. In some embodiments,
  • Rl 3 is Cl -C3 alkyl substituted methylene. In some embodiments, Rl 3 is — CH(CH3) ⁇ or —
  • Rl 4 in the definition of E is alkyl. In some embodiments, Rl 4 is Cl -ClO alkyl.
  • the group D in the definition of E is D is ORl 5 or NRl 6 Rl 7.
  • the group Rl 5 in the definition of D is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl.
  • Rl 5 is hydrogen, Cl -ClO alkyl, C2 -ClO alkenyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C8 cycloalkenyl-substituted Cl -C3 alkyl, or phenyl-substituted Cl -C3 alkyl. Also in some embodiments, Rl 5 is hydrogen or alkyl, with Cl -C3 alkyl being more preferred.
  • the group Rl 6 in the definition of D is hydrogen, alkyl, alkenyl, aryl, aryl-substituted alkyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl or cycloalkenyl-substituted alkyl.
  • R16 is hydrogen, Cl -ClO alkyl, C3 -ClO alkenyl, phenyl, phenyl- substituted Cl -C3 alkyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C8 cycloalkenyl-substituted Cl -C3 alkyl.
  • Rl 6 is methyl or benzyl.
  • Rl 7 in the definition of D is hydrogen or alkyl. In some embodiments, Rl 7 is hydrogen or Cl -C3 alkyl. In even more some embodiments, Rl 7 is hydrogen.
  • Rl 6 and Rl 7 form, together with the nitrogen atom to which they are attached, a heterocyclic ring.
  • the group Rl 8 in the definition of Y is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl.
  • Rl 8 is hydrogen, Cl -ClO alkyl, C2 -ClO alkenyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C8 cycloalkenyl-substituted Cl -C3 alkyl, or phenyl-substituted Cl -C3 alkyl. In some embodiments, Rl 8 is hydrogen or Cl -C3 alkyl.
  • Rl 9 in the definition of Y is hydrogen or alkyl. hi some embodiments, Rl 9 is hydrogen or Cl -C3 alkyl.
  • the group R20 in the definition of Y is hydrogen, alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, or aryl-substituted alkyl.
  • R20 is hydrogen, Cl -ClO alkyl, C3 -ClO alkenyl, phenyl, cycloalkyl, C5 -C8 cycloalkenyl, cycloalkyl-substituted Cl -C3 alkyl, C5 -C gcycloalkenyl- substituted Cl -C3 alkyl, or phenyl-substituted Cl -C3 alkyl. In some embodiments, R20 is hydrogen or Cl -C3 alkyl.
  • Rl 9 and R20 form, together with the nitrogen atom to which they are attached, a heterocyclic ring.
  • R21 in the definition of B is hydrogen or alkyl.
  • R21 is hydrogen or Cl -C3 alkyl.
  • R21 is hydrogen.
  • n is 0 to about 4. In some embodiments, n is about 1 or 2.
  • q is about 1 to about 4.
  • q is about 1 to about 3.
  • m is about 1 to about 4. hi some embodiments, m is about 1 to about 3.
  • the compounds of formula (I) can occur as the trans and cis stereochemical isomers by virtue of the substituents at the 3- and 4-positions of the piperidine ring, and such stereochemical isomers are within the scope of the claims.
  • the term "trans”, as used herein, refers to R2 in position 3 being on the opposite side from the methyl group in position 4, whereas in the "cis” isomer R2 and the 4-methyl are on the same side of the ring.
  • the compounds employed may be the individual stereoisomers, as well as mixtures of stereoisomers.
  • the methods of the present invention involve compounds of formula (I) wherein the group R2 at the 3-position is situated on the opposite side of the ring, i.e., trans to the methyl group in the 4-position and on the same side of the ring.
  • trans isomers can exist as the 3R,4R-isomer, or the 3S,4S-isomer.
  • R and S are used herein as commonly used in organic chemistry to denote specific configuration of a chiral center.
  • the term “R” refers to "right” and refers that configuration of a chiral center with a clockwise relationship of group priorities (highest to second lowest) when viewed along the bond toward the lowest priority group.
  • the term “S” or “left” refers to that configuration of a chiral center with a counterclockwise relationship of group priorities (highest to second lowest) when viewed along the bond toward the lowest priority group.
  • the priority of groups is based upon their atomic number (heaviest isotope first). A partial list of priorities and a discussion of stereochemistry is contained in the book: The Vocabulary of Organic Chemistry, Orchin, et al., John Wiley and Sons Inc., page 126 (1980), which is incorporated herein by reference in its entirety.
  • Piperidine-N-alkylcarboxylate compounds for use in the methods of the present invention are those of formula (I) in which the configuration of substituents on the piperidine ring is 3R and 4R.
  • R3 is not hydrogen
  • the carbon atom to which R3 is attached is asymmetric.
  • this class of compounds can further exist as the individual R or S stereoisomers at this chiral center, or as mixtures of stereoisomers, and all are contemplated within the scope of the present invention.
  • a substantially pure stereoisomer of the compounds of this invention can be used, i.e., an isomer in which the configuration at the chiral center to which R3 is attached is R or S, i.e., those compounds in which the configuration at the three chiral centers is 3R, 4R, S or 3R, 4R, R.
  • piperidine-N-alkylcarboxylate compounds for use in the methods of the present invention include the following:
  • Piperidine-N-alkylcarboxylate compounds for use in the methods of the present invention include the following:
  • the compound of formula (I) has the formula Q-CH2 CH(CH2 (C6 H5))C(O)OH, Q--CH2 CH2 CH(C6 H5)C(O)NHCH2 C(0)0CH2 CH2, Q--CH2 CH2 CH(C6 H5)C(O)NHCH2 C(O)OH, Q-CH2 CH2 CH(C6 H5)C(O)NHCH2 C(O)NHCH3, Q-CH2 CH2 CH(C6 H5)C(O)NHCH2 C(0)NHCH2 CH3, G ⁇ NH(CH2)2 C(0)NH2, G ⁇ NH(CH2)2 C(0)NHCH3, G-NHCH2 C(0)NH2, G-NHCH2 C(0)NHCH3, G-NHCH3 C(O)NHCH2 CH3, G-NH(CH2)3 C(0)0CH2 CH3, G-NH(CH2)3 C(0)NHCH3, G ⁇ NH(CH2)2 C(O)OH, G ⁇ NH(CH2)3 C(
  • the compound of formula (I) has the formula (3R,4R,S) ⁇ Z ⁇ NHCH2 C(O)OCH2 CH(CH3)2, (+)--Z ⁇ NHCH2 C(O)OH, (-)-Z-NHCH2 C(O)OH, (3R,4R,R)-Z-NHCH2 C(0) ⁇ 0CH2 CH(CH3)2, (3S,4S,S)--Z--NHCH2 C(0)0CH2 CH(CH3)2, (3 S,4S,R)-Z-NHCH2 C(0)0CH2 CH(CH3)2, (3R,4R) ⁇ Z ⁇ NHCH2 C(O)NHCH2 (C6 H5) or (3R,4R)-G-NH(CH2)3 C(O)OH, where Z and G are as defined above.
  • the compound of formula (I) has the formula (+) ⁇ Z— NHCH2 C(O)OH or (-) ⁇ Z-NHCH2 C(O)OH where Z is as defined above.
  • An embodiment of the present invention is the compound (+)- -Z-NHCH2 C(O)OH, i.e., the compound of the following formula (II).
  • the compound of formula (II) has low solubility in water except at low or high pH conditions.
  • Zwitterionic character may be inherent to the compound, and may impart desirable properties such as poor systemic absorption and sustained local affect on the gut following oral administration.
  • the methods of the present invention may involve administering to a patient a peripheral mu-opioid receptor antagonist that is a quaternary morphinan compound.
  • quaternary morphinan compounds that may be suitable for use in the methods of the present invention include, for example, quaternary salts of N- methylnaltrexone, N-methylnaloxone, N-methylnalorphine, N-diallylnormorphine, N- allyllevallorphan and N-methylnalmefene.
  • the methods of the present invention may involve administering to a patient a peripheral mu-opioid receptor antagonist in the form of an opium alkaloid derivative.
  • opioid alkaloid derivative refers to peripheral mu-opioid receptor antagonists that are synthetic or semi-synthetic derivatives or analogs of opium alkaloids.
  • the opium alkaloid derivatives employed in the methods of the present invention exhibit high levels of morphine antagonism, while exhibiting reduced, and preferably substantially no, agonist activity.
  • substantially no agonist activity as used herein in connection with the opium alkaloid derivatives, means that the maximal response with respect to electrically stimulated guinea pig ileum, at a concentration of 1 ⁇ M, is about 60% or less relative to morphine.
  • the opium alkaloid derivatives employed in the present methods have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 50% or less relative to morphine, with a maximal response of about 40% or less being more preferred. In some embodiments, the opium alkaloid derivatives employed in the present methods have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 30% or less relative to morphine, with a maximal response of about 20% or less.
  • the opium alkaloid derivatives employed in the present methods have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 10% or less relative to morphine. In certain embodiments, the opium alkaloid derivatives have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 0% (i.e., no response).
  • the opium alkaloid derivatives employed in the methods of the present invention have the following formulas (III) or (IV):
  • R is alkyl, cycloalkyl-substituted alkyl, aryl, aryl-substituted alkyl or alkenyl;
  • Z is hydrogen or OH
  • R' is X'-J(L)(T), wherein:
  • J is alkylene or alkenylene
  • L is hydrogen, amino, or alkyl optionally substituted with CO2 H, OH or phenyl
  • T is CO2 H, SO3 H, amino or guanidino
  • R" is NH-J(L)(T) or guanidino; or a stereoisomer, prodrug, or pharmaceutically acceptable salt, hydrate or N-oxide thereof.
  • R is alkyl, cycloalkyl-substituted alkyl, aryl, aryl-substituted alkyl or alkenyl.
  • R is Cl -C5 alkyl, C3 -C6 cycloakyl-substituted alkyl, aryl, arylalkyl or trans-C2 -C5 alkenyl.
  • R is Cl -C3 alkyl, allyl or cyclopropylmethyl, with cyclopropylmethyl being even more preferred.
  • Z is hydrogen or OH.
  • Z is OH.
  • R 1 is X-J(L)(T) and R" is NH-J(L)(T) or guanidino.
  • G is alkylene or alkenylene.
  • J is Cl -
  • C5 alkylene C2 -C6 alkylene interrupted by an oxygen atom, or C2 -C5 alkenylene.
  • L is hydrogen, amino, or alkyl optionally substituted with
  • L is hydrogen, amino, or Cl -C5 alkyl optionally substituted with CO2 H, OH or phenyl. In some embodiments, L is hydrogen or amino.
  • T is CO2 H, SO3 H, amino or guanidino. In some embodiments, T is CO2 H or guanidino.
  • Important opioid alkaloid derivatives that may be employed in the methods of the present invention include compounds of formula (III) wherein R is cyclopropylmethyl, Z is OH, and
  • the opioid alkaloid derivatives that may be employed in the methods of the present invention include compounds of formula (IV) wherein R is cyclopropylmethyl, Z is OH, and R" is NHCH2CO2H.
  • N-methylnaltrexone or methymaltrexone, MNTX
  • MNTX methymaltrexone
  • the methods of the present invention may involve administering to a patient a peripheral mu-opioid receptor antagonist compound in the form of a quaternary benzomorphan compound.
  • the quaternary benzomorphan compounds employed in the methods of the present invention exhibit high levels of morphine antagonism, while exhibiting reduced, and preferably substantially no, agonist activity.
  • substantially no agonist activity as used herein in connection with the quaternary benzomorphan compounds, means that the maximal response with respect to electrically stimulated guinea pig ileum, at a concentration of 1 ⁇ M, is about 60% or less relative to morphine.
  • the quaternary benzomorphan compounds employed in the present methods have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 50% or less relative to morphine, with a maximal response of about 40% or less being more preferred. In some embodiments, the quaternary benzomorphan compounds employed in the present methods have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 30% or less relative to morphine, with a maximal response of about 20% or less being.
  • the quaternary benzomorphan compounds employed in the present methods have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 10% or less relative to morphine. In certain embodiments, the quaternary benzomorphan compounds have a maximal response with respect to guinea pig ileum, at a concentration of 1 ⁇ M, of about 0% (i.e., no response).
  • the quaternary benzomorphan compounds employed in the methods of the present invention have the following formula (VI):
  • R24 is hydrogen or acyl
  • R25 is alkyl or alkenyl; or a stereoisomer, prodrug, or pharmaceutically acceptable salt, hydrate or N-oxide thereof.
  • R24 is hydrogen or acyl. In some embodiments, R24 is hydrogen or Cl -C6 acyl. m some embodiments, R24 is hydrogen or Cl -C2 acyl. In some embodiments, R24 is hydrogen or acetoxy, with hydrogen being still more preferred.
  • R25 is alkyl or alkenyl. In some embodiments, R25 is Cl -C6 alkyl or C2 -C6 alkenyl. hi some embodiments, R25 is Cl -C3 alkyl or C2 - C3 alkenyl. hi some embodiments, R25 is propyl or allyl.
  • Important quaternary benzomorphan compounds that may be employed in the methods of the present invention include the following compounds of formula (VI): 2'- hydroxy-5,9-dimethyl-2,2-diallyl-6,7-benzomorphanium-bromide; 2'-hydroxy-5,9-dimethyl- 2-n-propyl-6,7-benzomorphan; 2'-hydroxy-5,9-dimethyl-2-allyl-6,7-benzomorphan; 2'- hydroxy-5,9-dimethyl-2-n-propyl-2-allyl-6,7-benzomo ⁇ hanium-bromide; 2'-hydroxy-5,9- dimethyl-2-n-propyl-2-propargyl-6,7-benzomorphanium-bromide; and 2'-acetoxy-5,9- dimethyl-2-n-propyl-2-allyl-6,7-benzomo ⁇ hanium-bromide.
  • mu opioid receptor antagonists which may be employed in the methods and compositions of the present invention, in addition to those exemplified above, would be readily apparent to one of ordinary skill in the art, once armed with the teachings of the present disclosure.
  • prodrug is intended to include any covalently bonded carriers which release the active parent drug, for example, as according to formulas (I) or (II) or other formulas or compounds employed in the methods of the present invention in vivo when such prodrug is administered to a mammalian subject. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.) the compounds employed in the present methods may, if desired, be delivered in prodrug form. Thus, the present invention contemplates methods of delivering prodrugs.
  • Prodrugs of the compounds employed in the present invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the pharmacologically active moiety.
  • prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively.
  • Examples include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.
  • alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.
  • the compounds employed in the methods of the present invention maybe prepared in a number of ways well known to those skilled in the art.
  • the compounds can be synthesized, for example, by the methods described below, or variations thereon as appreciated by the skilled artisan. All processes disclosed in association with the present invention are contemplated to be practiced on any scale, including milligram, gram, multigram, kilogram, multikilogram or commercial industrial scale.
  • Compounds employed in the present methods may contain one or more asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms. Thus, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.
  • protecting groups present may contain protecting groups during the course of synthesis.
  • Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups may be employed with the present invention.
  • Protecting groups include the benzyloxycarbonyl group and the tert-butyloxycarbonyl group.
  • Other protecting groups that may be employed in accordance with the present invention may be described in Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis 2d. Ed., Wiley & Sons, 1991.
  • Piperidine-N-alkylcarboxylate compounds according to the present invention may be synthesized employing methods taught, for example, in U.S. Pat. Nos. 5,250,542, 5,434,171, 5,159,081, and 5,270,328, the disclosures of which are hereby incorporated herein by reference in their entireties.
  • the 3-substituted-4-methyl-4-(3-hydroxy- or alkanoyloxyphenyl)piperidine derivatives employed as starting materials in the synthesis of the present compounds may be prepared by the general procedure taught in U.S. Pat. No. 4,115,400 and U.S. Pat. No. 4,891,379, the disclosures of which are hereby incorporated herein by reference in their entireties.
  • the starting material for the synthesis of compounds described herein, (3R,4R)-4-(3-hydroxypheny)-3,4-dimethylpiperidine may be prepared by the procedures described in U.S. Pat. No. 4,581,456, the disclosures of which are hereby incorporated herein by reference, in their entirety, but adjusted as described such that the .beta. -stereochemistry is preferred.
  • the first step of the process may involves the formation of the 3- alkoxyphenyllithium reagent by reacting 3-alkoxybromobenzene with an alkyllithium reagent.
  • This reaction may be performed under inert conditions and in the presence of a suitable non-reactive solvent such as dry diethyl ether or preferably dry tetrahydrof ⁇ ran.
  • Preferred alkyllithium reagents used in this process are n-butyllithium, and especially sec- butyllithium. Generally, approximately an equimolar to slight excess of alkyllithium reagent may be added to the reaction mixture.
  • the reaction may be conducted at a temperature of from about -20°C and about -100 0 C, more preferably from about -50°C. to about -55°C.
  • a l-alkyl-4-piperidone may be added to the mixture while maintaining the temperature between -20°C and -100°C.
  • the reaction is typically complete after about 1 to 24 hours. At this point, the reaction mixture may be allowed to gradually warm to room temperature.
  • the product may be isolated by the addition to the reaction mixture of a saturated sodium chloride solution to quench any residual lithium reagent.
  • the organic layer maybe separated and further purified if desired to provide the appropriate l-alkyl-4-(3- alkoxyphenyl)piperidinol derivative.
  • the dehydration of the 4-phenylpiperidinol prepared above maybe accomplished with a strong acid according to well known procedures. While dehydration occurs in various amounts with any one of several strong acids such as hydrochloric acid, hydrobromic acid, and the like, dehydration is preferably conducted with phosphoric acid, or especially p-toluenesulfonic acid in toluene or benzene. This reaction may be typically conducted under reflux conditions, more generally from about 50°C and 150 0 C.
  • the product thus formed may be isolated by basifying an acidic aqueous solution of the salt form of the product and extracting the aqueous solution with a suitable water immiscible solvent. The resulting residue following evaporation can then be further purified if desired.
  • the l-alkyl-4-methyl-4-(3-alkoxyphenyl)tetrahydropyridine derivatives may be prepared by a metalloenamine alkylation. This reaction is preferably conducted with n- butyllithium in tetrahydrofuran (THF) under an inert atmosphere, such as nitrogen or argon. Generally, a slight excess of n-butyllithium may be added to a stirring solution of the 1-alkyl- 4-(3-alkoxyphenyl)-tetrahydropyridine in THF cooled to a temperature in the range of from about is -50°C to about 0°C, more preferably from about -20°C to -10°C.
  • THF tetrahydrofuran
  • This mixture may be stirred for approximately 10 to 30 minutes followed by the addition of approximately from 1.0 to 1.5 equivalents of methyl halide to the solution while maintaining the temperature of the reaction mixture below O 0 C. After about 5 to 60 minutes, water may be added to the reaction mixture and the organic phase may be collected.
  • the product can be purified according to standard procedures, but the crude product is preferably purified by either distilling it under vacuum or slurrying it in a mixture of hexanerethyl acetate (65:35, v:v) and silica gel for about two hours. According to the latter procedure, the product may be then isolated by filtration followed by evaporating the filtrate under reduced pressure.
  • the next step in the process may involve the application of the Mannich reaction of aminomethylation to non-conjugated, endocyclic enamines.
  • This reaction is preferably carried out by combining from about 1.2 to 2.0 equivalents of aqueous formaldehyde and about 1.3 to 2.0 equivalents of a suitable secondary amine in a suitable solvent. While water may be the preferred solvent, other non-nucleophilic solvents, such as acetone and acetonitrile can also be employed in this reaction.
  • the pH of this solution may be adjusted to approximately 3.0 to 4.0 with an acid that provides a non-nucleophilic anion.
  • acids include sulfuric acid, the sulfonic acids such as methanesulfonic acid and p-toluenesulfonic acid, phosphoric acid, and tetrafluoroboric acid, with sulfuric acid being preferred.
  • sulfuric acid being preferred.
  • To this solution may be added one equivalent of a l-alkyl-4-methyl-4-(3- alkoxyphenyl)tetrahydropyridine, typically dissolved in aqueous sulfuric acid, and the pH of the solution may be readjusted with the non-nucleophilic acid or a suitable secondary amine.
  • the pH is preferably maintained in the range of from about 1.0 to 5.0, with a pH of about 3.0 to 3.5 being more preferred during the reaction.
  • the reaction is substantially complete after about 1 to 4 hours, more typically about 2 hours, when conducted at a temperature in the range of from about 50°C to about 80°C, more preferably about 70°C.
  • the reaction may then be cooled .to approximately 30° C, and added to a sodium hydroxide solution.
  • This solution may then be extracted with a water immiscible organic solvent, such as hexane or ethyl acetate, and the organic phase, following thorough washing with water to remove any residual formaldehyde, may be evaporated to dryness under reduced pressure.
  • the next step of the process may involve the catalytic hydrogenation of the prepared l-alkyl-4-methyl-4-(3-alkoxyphenyl)-3-tetrahydropyridinemethanamine to the corresponding trans- 1 -alkyl-3,4-dimethyl-4-(3-alkoxyphenyl)piperidine.
  • This reaction actually occurs in two steps.
  • the first step is the hydro genolysis reaction wherein the exo C-- N bond is reductively cleaved to generate the 3-methyltetrahydropyridine.
  • the 2,3-double bond in the tetrahydropyridine ring is reduced to afford the desired piperidine ring.
  • the catalysts employed in the process may be chosen from among the various palladium and preferably platinum catalysts.
  • the catalytic hydrogenation step of the process is preferably conducted in an acidic reaction medium.
  • Suitable solvents for use in the process include the alcohols, such as methanol or ethanol, as well as ethyl acetate, tetrahydrofuran, toluene, hexane, and the like.
  • Proper stereochemical outcome may be dependent on the quantity of catalyst employed.
  • the quantity of catalyst required to produce the desired stereochemical result may be dependent upon the purity of the starting materials in regard to the presence or absence of various catalyst poisons.
  • the hydrogen pressure in the reaction vessel may not be critical but can be in the range of from about 5 to 200 psi. Concentration of the starting material by volume is preferably around 20 niL of liquid per gram of starting material, although an increased or decreased concentration of the starting material can also be employed. Under the conditions specified herein, the length of time for the catalytic hydrogenation may not be critical because of the inability for over-reduction of the molecule. While the reaction can continue for up to 24 hours or longer, it may not be necessary to continue the reduction conditions after the uptake of the theoretical two moles of hydrogen.
  • the product may then be isolated by filtering the reaction mixture for example through infusorial earth, and evaporating the filtrate to dryness under reduced pressure. Further purification of the product thus isolated may not be necessary and preferably the diastereomeric mixture may be carried directly on to the following reaction.
  • the alkyl substituent may be removed from the 1 -position of the piperidine ring by standard dealkylation procedures.
  • a chloroformate derivative especially the vinyl or phenyl derivatives, may be employed and removed with acid.
  • the prepared alkoxy compound may be dealkylated to the corresponding phenol.
  • This reaction may be generally carried out by reacting the compound in a 48% aqueous hydrobromic acid solution. This reaction may be substantially complete after about 30 minutes to 24 hours when conducted at a temperature of from about 50°C to about 150°C, more preferably at the reflux temperature of the reaction mixture.
  • the mixture may then be worked up by cooling the solution, followed by neutralization with base to an approximate pH of 8.
  • This aqueous solution may be extracted with a water immiscible organic solvent. The residue following evaporation of the organic phase may then be used directly in the following step.
  • the compounds employed as starting materials to the compounds of the invention can also be prepared by brominating the l-alkyl-4-niethyl-4-(3-alkoxyphenyl)-3- tetrahydropyridinemethanamine at the 3-position, lithiating the bromo compound thus prepared, and reacting the lithiated intermediate with a methylhalide, such as methyl bromide to provide the corresponding l-alkyl-3,4-dimethyl-4-(3- alkoxyphenyl)tetrahydropyridinemethanamine.
  • This compound may then be reduced and converted to the starting material as indicated above.
  • the compounds of the present invention can exist as the individual stereoisomers. Preferably reaction conditions are adjusted as disclosed in U.S. Pat. No.
  • Example 1 of U.S. Pat. No. 5,250,542 to be substantially stereoselective and provide a racemic mixture of essentially two enantiomers.
  • These enantiomers may then be resolved.
  • a procedure which may be employed to prepare the resolved starting materials used in the synthesis of these compounds includes treating a racemic mixture of alkyl-3,4-dimethyl-4-(3-alkoxyphenyl)piperidine with either (+)- or (-)- ditoluoyl tartaric acid to provide the resolved intermediate.
  • This compound may then be dealkylated at the 1 -position with vinyl chloroformate and finally converted to the desired 4- (3-hydroxyphenyl)piperidine isomer.
  • the individual enantiomers of the invention can also be isolated with either (+) or (-) dibenzoyl tartaric acid, as desired, from the corresponding racemic mixture of the compounds of the invention.
  • the (+)-trans enantiomer is obtained.
  • (+)trans-3,4 stereoisomer is preferred, all of the possible stereoiosmers of the compounds described herein are within the contemplated scope of the present invention. Racemic mixtures of the stereoisomers as well as the substantially pure stereoisomers are within the scope of the invention.
  • Intermediates can be prepared by reacting a 3,4-alkyl-substituted-4-(3- hydroxyphenyl)piperidine with a compound of the formula LCH2 (CH2),C1 CHR3 C(O)E where L is a leaving group such as chlorine, bromine or iodine, E is a carboxylic acid, ester or amide, and R3 and n are as defined hereinabove.
  • L may be chlorine and the reaction is carried out in the presence of a base to alkylate the piperidine nitrogen.
  • 4-chloro-2-cyclohexylbutanoic acid, ethyl ester can be contacted with (3R,4R)-4-(3- hydroxyphenyl)-3,4-dimethylpiperidine to provide 4-[(3R,4R)-4-(3-hydroxyphenyl)-3,4- dimethyl-l-piperidine]butanoic acid, ethyl ester.
  • the ester of the carboxylic acid may be preferred, the free acid itself or an amide of the carboxylic acid may be used.
  • the substituted piperidine can be contacted with a methylene alkyl ester to alkylate the piperidine nitrogen.
  • 2-methylene-3- phenylpropanoic acid, ethyl ester can be contacted with a desired piperidine to provide 2- benzyl-3-piperidinepropanoic acid ethyl ester.
  • Another synthetic route can involve the reaction of a substituted piperidine with a haloalkylnitrile.
  • the nitrile group of the resulting piperidine alkylnitrile can be hydrolyzed to the corresponding carboxylic acid.
  • the resulting ester or carboxylic acid can be reacted with an amine or alcohol to provide modified chemical structures.
  • the piperidine-carboxylic acid or -carboxylic acid ester may be reacted with an amine in the presence of a coupling agent such as dicyclohexylcarbodiimide, boric acid, borane-trimethylamine, and the like.
  • Esters can be prepared by contacting the piperidine- carboxylic acid with the appropriate alcohol in the presence of a coupling agent such as p- toluenesulfonic acid, boron trifluoride etherate or N,N'-carbonyldiimidazole.
  • the piperidine-carboxylic acid chloride can be prepared using a reagent such as thionyl chloride, phosphorus trichloride, phosphorus pentachloride and the like. This acyl chloride can be reacted with the appropriate amine or alcohol to provide the corresponding amide or ester.
  • a reagent such as thionyl chloride, phosphorus trichloride, phosphorus pentachloride and the like.
  • This acyl chloride can be reacted with the appropriate amine or alcohol to provide the corresponding amide or ester.
  • Opium alkaloid derivatives according to the present invention may be synthesized employing methods taught, for example, in U.S. Pat. Nos. 4,730,048 and 4,806,556, the disclosures of which are hereby incorporated herein by reference in their entireties.
  • opium alkaloid derivatives of formula (III) may be prepared by attaching hydrophilic, ionizable moieties R 1 and R" to the 6-amino group of naltrexamine (formula (III) where R is (cyclopropyl)methyl, Z is OH and R! is H) or oxymorphamine (formula (III) where R is CH3, Z is OH and R! is H).
  • deoxy-opiates of formulae (III) and (IV) wherein Z is hydrogen may be prepared from readily available starting materials.
  • the compounds of formula (VII) may be synthesized employing methods taught, for example, in U.S. Pat. No. 3,723,440, the disclosures of which are hereby incorporated herein by reference in their entirety.
  • the antagonist may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the antagonist may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the amount of antagonist in such therapeutically useful compositions is adjusted to achieve suitable dosages using routine techniques within the skill in the art.
  • An exemplary dosage for an antagonist is an oral dosage unit form containing from about 0.1 to about 1000 mg of antagonist.
  • the tablets, troches, pills, capsules and the like may also contain one or more of the following: a binder, such as gum tragacanth, acacia, corn starch or gelatin; an excipient, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; a sweetening agent such as sucrose, lactose, saccharin, and/or a flavoring agent, such as peppermint, oil of wintergreen or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • an excipient such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose, saccharin, and/or a
  • any material used in preparing any unit dosage form is preferably pharmaceutically pure and substantially non-toxic in the amount employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • the antagonist may also be administered parenterally or intraperitoneally.
  • Solutions of the antagonists in unmodified form or as pharmacologically acceptable salts are contemplated and can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • a dispersion can also be prepared in glycerol, liquid polyethylene glycols, preferably a high molecular weight polyethylene glycol of average molecular weight at least 15 kDa, mixtures thereof and in oils.
  • any route of administration disclosed herein or known in the art may be used.
  • Pharmacologically and pharmaceutically acceptable salts for inclusion in administrable compositions include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p- toluenesulfonic, tartaric, citric, methanesulfonic, formic, succinic, naphthalene-2-sulfonic, palmoic, S-hydroxy ⁇ -naphthalenecarboxylic, and benzene sulfonic.
  • Suitable buffering agents include, but are not limited to, acetic acid and salts thereof (1-2% WN); citric acid and salts thereof (1-3% WN); boric acid and salts thereof (0.5-2.5% WN); and phosphoric acid and salts thereof (0.8-2% WN).
  • Suitable preservatives include, but are not limited to, benzalkonium chloride (0.003-0.03% WN); 5 chlorobutanol (0.3-0.9% WIN); parabens (0.01- 0.25% WN) and thimerosal (0.004-0.02% WN).
  • a pharmaceutical composition of the peripheral opioid antagonist may also contain one or more pharmaceutically acceptable excipients, such as lubricants, diluents, binders, carriers, and disintegrants.
  • auxiliary agents may include, e.g., stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, coloring, flavoring and/or aromatic active compounds.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • suitable pharmaceutically acceptable carriers, diluents, solvents or vehicles include, but are not limited to, water, salt (buffer) solutions, alcohols, gum arabic, mineral and vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, vegetable oils, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like.
  • Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganism may be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like.
  • the dosage form of the antagonist(s) may be tablets, capsules, powders, suppositories, or lozenges. If a liquid carrier is used, soft gelatin capsules, transdermal patches, aerosol sprays, topical cream, syrups or liquid suspensions, emulsions or solutions may be the dosage form.
  • injectable, sterile solutions preferably non-aqueous or aqueous solutions, as well as dispersions, suspensions, emulsions, or implants, including suppositories.
  • Ampoules are convenient forms in which to administer unit dosages.
  • the pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form is preferably sterile; for administration via injection, the form is preferably sufficiently non-viscous to provide acceptable syringeability according to norms established in the art.
  • the antagonist forms are preferably stable under the conditions of manufacture and storage and are preferably resistant to untoward contamination.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sugars or sodium chloride.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the active compounds in the required amounts, in the appropriate solvent, with various of the other ingredients disclosed above, as required, followed by filter sterilization or sterilization via irradiation.
  • dispersions may be prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those disclosed above.
  • the preferred methods of preparation may include vacuum drying and/or a freeze drying technique which yields a powder of the active ingredient, plus any additional desired ingredient from the previously sterilized solution thereof.
  • An injectable depot form may also be suitable and may be made by forming a microcapsule matrix of the drug in a biodegradable polymer such as polylactide- polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
  • Suitable enteral application particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules such as soft gelatin capsules.
  • a syrup, elixir, or the like can be used wherein a sweetened vehicle is employed.
  • Another delivery system may include a time-release, delayed-release or sustained-release (extended release) delivery system.
  • a time-release, delayed-release or sustained-release (extended release) delivery system can avoid repeated administrations of a compound of the invention, increasing convenience to the patient and the physician and maintaining sustained plasma levels of compounds where desired.
  • Many types of controlled-release delivery systems are available and known to those of ordinary skill in the art.
  • Sustained- or controlled-release compositions can be formulated, e.g., as liposomes or by protecting the active compound with differentially degradable coatings, such as by microencapsulation, multiple coatings, and the like.
  • sustained-release matrices such as biodegradable polymers
  • a sustained-release matrix is a matrix typically composed of one or more polymers that are degradable by enzymatic or acid-base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
  • a sustained-release matrix may be desirably chosen from biocompatible materials such as liposomes, polymer-based systems such as polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyarihydrides, poly(ortho)esters, polysaccharides, polyamino acids, hyaluronic acid, collagen, chondroitin sulfate, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone, and silicone; nonpolymer systems are composed of chemical components such as carboxylic acids, fatty acids, phospholipids, amino acids, lipids such as sterols, hydrogel release systems, silastic systems, peptide-based systems, implants, and the like.
  • biocompatible materials such as liposomes
  • polymer-based systems such as polylactides (polylactic acid), polyglycolide (polymer
  • long-term sustained-release implant may be particularly suitable for treatment of chronic conditions.
  • Long-term release as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and suitably 30 to 60 days.
  • Long-term sustained-release implants are well-known to those of ordinary skill in the art and include some of the release system described above.
  • one embodiment employs, as a nonsprayable form, a viscous to semi-solid or solid form comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water.
  • Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, cream, ointments, powders, liniments, salves, aerosols, and the like, which are optionally sterilized or mixed with auxiliary agents, e.g., preservatives, and the like.
  • Transdermal or iontophoretic delivery of pharmaceutical compositions of the peripheral opioid antagonists is also contemplated.
  • the therapeutic compounds of this invention may be administered to a patient alone or in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may be determined, for example, by the solubility and chemical nature of the compounds, chosen route of administration and standard pharmaceutical practice.
  • a daily dosage may range from about 0.001 to about 100 milligrams of the peripheral ⁇ -opioid receptor antagonist (and all combinations and subcombinations of ranges therein), per kilogram of patient body weight.
  • the a daily dosage may be about 0.01 to about 10 milligrams of the peripheral ⁇ -opioid receptor antagonist per kilogram of patient body weight.
  • a daily dosage of about 0.1 milligrams of the peripheral ⁇ -opioid receptor antagonist per kilogram of patient body weight is preferred.
  • the peripheral ⁇ -opioid receptor antagonist is present in an amount of about 0.1 to about 4 milligrams.
  • the product is orally administered wherein an antagonist is enteric coated.
  • enteric coating an antagonist it is possible to control its release into the gastrointestinal tract such that the antagonist is not released in the stomach, but rather is released in the intestine.
  • Another embodiment of this invention where oral administration is desired provides for a combination product wherein one of the products, e.g., a ⁇ -opioid receptor antagonist , is coated with a sustained-release material which effects a sustained-release throughout the gastrointestinal tract and also serves to minimize physical contact between the ⁇ -opioid receptor antagonist and any other compound in the product.
  • the sustained-released component can be additionally enteric coated such that the release of this component occurs only in the intestine.
  • Still another approach involves the formulation of a combination product in which the one component is coated with a sustained and/or enteric release polymer, and the other component is also coated with a polymer such as a low-viscosity grade of hydroxypropyl methylcellulose (HPMC) or other appropriate material as known in the art, in order to further separate the active components.
  • HPMC hydroxypropyl methylcellulose
  • the polymer coating serves to form an additional barrier to interaction with the other component.
  • compounds of the invention are administered in a dosing regimen that provides a continuous dose of the compound to a subject, i.e., a regimen that eliminates the variation in internal drug levels found with conventional regimens.
  • a continuous dose may be achieved by administering the compound to a subject on a daily basis using any of the delivery methods disclosed herein.
  • the continuous dose may be achieved using continuous infusion to the subject, or via a mechanism that facilitates the release of the compound over time, for example, a transdermal patch, or a sustained release formulation.
  • compounds of the invention are continuously released to the subject in amounts sufficient to maintain a concentration of the compound in the plasma of the subject effective to inhibit or reduce cell barrier dysfunction.
  • Compounds in accordance with the invention are provided in an effective amount to prevent, reduce or eliminate a cell barrier dysfunction. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the level of ordinary skill in the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single-dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • single-dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • oral doses of the opioid receptor antagonists will range from about 1 to about 80 mg/kg body weight per day. It is expected that oral doses in the range from 2 to 20 mg/kg body weight will yield beneficial results.
  • parenteral administration including intravenous and subcutaneous administration, will range from about 0.001 to 5 mg/kg body weight. It is expected that doses ranging from 0.05 to 0.5 mg/kg body weight will yield the desired results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending on the mode of administration. For example, it is expected that the dosage for oral administration of the opioid antagonists in an enterically-coated formulation would be from 10 to 30% of the non-coated oral dose.
  • the opioid receptor antagonists are coadministered with an opioid compound.
  • co-administration is meant to refer to a combination therapy by any administration route in which two or more agents are administered to a patient or subject. Co-administration of agents may also be referred to as combination therapy or combination treatment.
  • the agents may be in the same dosage formulations or separate formulations. For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separate times.
  • the agents may be administered simultaneously or sequentially (i.e., one agent may directly follow administration of the other or the agents maybe given episodically, i.e., one can be given at one time followed by the other at a later time, e.g., within a week), as long as they are given in a manner sufficient to allow both agents to achieve effective concentrations in the body.
  • the agents may also be administered by different routes, e.g., one agent may be administered intravenously while a second agent is administered intramuscularly, intravenously or orally.
  • the co-administration of the opioid receptor antagonist compound with an opioid compound is suitably considered a combined pharmaceutical preparation which contains an opioid receptor antagonist and an opioid compound or agent, the preparation being adapted for the administration of the opioid receptor antagonist on a daily or intermittent basis, and the administration of the opioid agent on a daily or intermittent basis.
  • the opioid receptor antagonists may be administered prior to, concomitant with, or after administration of the opioids.
  • Co-administrable agents also may be formulated as an admixture as, for example, in a single formulation or single tablet. These formulations may be parenteral or oral, such as the formulations described in, e.g., U.S. Pat. Nos. 6,277,384; 6,261,599; 5,958,452 and PCT Publication No. WO 98125613, each hereby incorporated by reference.
  • any mode of administration disclosed herein or known in the art to be compatible with the contemplated co-administration is a suitable mode of administration.
  • the peripheral opioid receptor antagonist may be co-administered with an opioid or opioid receptor agonist, and another therapeutic agent that is not an opioid or opioid receptor agonist.
  • opioids and peripheral opioid receptor agonists are described above.
  • Suitable therapeutic agents include anti-biotics and anti-inflammatory agents.
  • the formulations may be prepared using standard formulation methods known to those of skill in the art.
  • Antibiotics include: Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispyrithione Mags
  • Antiviral agents include: Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Ribavirin
  • Antifungal agents include: Acrisorcin; Ambruticin; Amphotericin B;
  • Anti-inflammatory agents include: Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium
  • a plasmid containing the GFP-PA-I fusion construct was constructed using conventional recombinant DNA techniques.
  • the EGFP gene encoding green fluorescent protein was amplified using the pBI-EGFP plasmid (Clontech) as a template.
  • Xb ⁇ l and Pstl restriction sites were introduced using primers TCTAGAACTAGTGGATCCCCGCGGATG (SEQ ID NO: 1) and GCAGACTAGGTCGACAAGCTTGATATC (SEQ ED NO: 2).
  • the PCR product was cloned directly into the pCR 2.1 vector using a TA-cloning kit (Invitrogen), followed by transformation of the pCR2.1/EGFP construct into E.coli DH5a.
  • the EGFP gene was excised from this construct by digestion with Xbal and Pstl and the fragment containing the excised gene was cloned into the E.coli-P. aeruginosa shuttle vector pUCP24, which had been digested with the same restriction enzymes.
  • the resulting construct i.e., pUCP24/EGFP
  • pUCP24/EGFP containing the EGFP gene in the shuttle vector
  • Cells containing pUCP24/EGFP were selected by gentamicin (Gm)challenge, typically at 100 ⁇ g/ml.
  • a derivative of pUCP24/EGFP was generated that placed the PA-I lectin/adhesin gene in close proximity to the EGFP gene, effectively linking the genes genetically.
  • the construct contained the QS lux box and RpoS consensus sequences in the 5' non-coding region of lee A, along with rRNA sequence.
  • the derivative construct was termed pUCP24/PLL-EGFP.
  • One of skill would understand how to make and use the above-described construct, as well as other suitable constructs for providing lecA, alone or in physical proximity to a marker gene such as EGFP, using any of a variety of techniques.
  • PA-I lectin/adhesin was localized to a previously undescribed structural appendage on the outer surface of P. aeruginosa, using conventional techniques.
  • C. elegans is suitable as an in vivo model system for BSC signaling and its role in the production of PA-I.
  • C. elegans is accepted as a highly accurate and predictable model in which to study the host response to P. aeruginosa.
  • C. elegans worms feed on lawns of P. aeruginosa growing on solid agar and, thus, provides an ideal system in which to study microbial pathogenesis, especially in regard to gut-derived sepsis, since the mode of infectivity is via the digestive tract.
  • These nematodes readily feed on bacteria such as E. coli growing on solid agar plates, yet when fed specific strains of P. aeruginosa, mortality rates exceed 50% within 72 hours.
  • PA-I was functionally expressed in epithelial cell assays in the presence of the PA-I-inducing compounds.
  • Exotoxin A was labeled with AlexaFluor 594, and its transepithelial flux was measured at varying levels of decrease of transepithelial resistance (TEER) of MDCK monolayers that was achieved by apical application of MDCK cells to different concentrations of pure PA-I protein. A five-fold increase in exotoxin A flux across MDCK cells was found when transepithelial resistance was decreased below 50% of control.
  • Purified PA-I decreased the TEER of epithelial cells to the same degree as P. aeruginosa.
  • PA-I null mutants of P. aeruginosa had a significantly attenuated effect on the transepithelial resistance of MDCK cells. Techniques used in conducting the experiments are described in Example 23, below, or are conventional in the art.
  • the degree of cell polarity i.e. degree of cell confluency and tight junctional apposition
  • degree of cell confluency and tight junctional apposition has been shown to dictate the degree of response to purified PA-I protein.
  • Cells that were loosely confluent had a more profound fall in TEER in response to PA-I compared to "tighter" and more differentiated cell monolayers.
  • wounded monolayers exposed dense areas of PA-I binding.
  • Cell culturmg was performed as described in Example 24, below; relative confluency was assessed using conventional techniques as would be known in the art.
  • GFP-reporter strains permit demonstrations that virulence gene expression in P. aeruginosa is expressed in vivo within the intestinal tract of a stressed (30% hepatectomy) host.
  • EGPF reporter constructs were specifically designed to contain known upstream regulatory regions involved in PA-I expression (e.g., lux box (QS promoter elements) and RpoS).
  • the EGFP-PA-I reporter strain termed PLL-EGFP, was then injected into the cecum of sham-operated (control) mice and mice undergoing surgical hepatectomy. Twenty-four hours later, feces and washed cecal mucosa were then assayed for the presence of fluorescent bacteria.
  • PA-I was expressed in vivo (three- to six- fold over control levels) in response to elements of the local intestinal microenvironment (cecum) of mice subjected to catabolic (surgical) stress.
  • These findings were verified in the non-reporter strain, PA27853, using an assay in which bacterial RNA is extracted from fresh feces using an RNA protection system.
  • Reiterative studies were performed in which PA27853 was introduced into the cecum of control and hepatectomized mice and then bacterial RNA recovered from fresh feces 24 hours later for quantitative RT-PCR (QRT-PCR) of both PA-I and exotoxin A (about 600% and 800% respectively).
  • QRT-PCR quantitative RT-PCR
  • This assay provides a precise molecular "snapshot" of the effect of the in situ cecal environment on P. aeruginosa virulence gene expression. Results demonstrated that the cecal microenvironment of a stressed host induced PA-I and exotoxin A virulence gene expression. Next, in order to determine whether these findings were due to soluble factors released into the intestinal lumen, particulate-free filtrates were prepared from cecal luminal contents from control and hepatectomized mice and added to fresh cultures of the reporter strain PLL-EGFP.
  • aeruginosa to express PA-I; 2) these factors may originate from the intestinal tract itself, since during ischemia the intestine is isolated from systemic factors; 3) blood components do not induce PA-I expression; and 4) the presence of the normal flora, virtually absent in flushed small bowel segments, appears to play no role in this response.
  • P. aeruginosa strain PA-27853 and reporter strains (PLL-EGFP) were exposed to ambient hypoxia (0.3% O 2 ), pH changes (6-8), and 80% CO 2 . None of these conditions induced PA-I expression.
  • BSCs Bacterial Signaling Compounds inducing PA-I lectin/ adhesin expression are found in epithelial cells
  • PA-I expression is influenced by both membrane-bound and soluble factors, and it is contemplated that modulators of the bacterial signaling process include, but are not limited to, effectors (i.e., enhancers, activators, and inhibitors) of a soluble factor, a membrane-bound factor, or both.
  • effectors i.e., enhancers, activators, and inhibitors
  • Example 9 Stressed Caco-2 cells release soluble factors that induce PA-I lectin/ adhesin expression
  • Media from hypoxic and heat shock stressed Caco-2 cells were next fractionated into 5 molecular weight fractions ( ⁇ 3, 3-10, 10-20, 20-30, >30 kDa) using centricones, to determine if a specific MW fraction could be identified that induces PA-I expression.
  • the bacterial signaling compound (s) was a protein
  • fractions were treated with heat inactivation and the protein inhibitor, proteinase K.
  • the identified fraction was 10-30 kD and for the heat shock fraction the identified fraction was 30-50 kD. Both fractions were inactivated, consistent with the BSC being proteins.
  • Stimulated immune cells release factors that induce PA-I lectin/ adhesin expression
  • Immune elements released at the mucosal epithelial surface, the primary site of colonization for P. aeruginosa were considered to be suitable candidates to serve as host stress-derived bacterial signaling compounds.
  • As a physiologically relevant in vitro system to determine whether immune factors can activate P. aeruginosa virulence supernatants from antigen-stimulated T cells were evaluated for their ability to increase PA-I expression in the P. aeruginosa strain PLL-EGFP/27853, which carries a PA-I-GFP reporter construct.
  • P. aeruginosa cells were incubated with supernatants from stimulated T-cells and PA-I expression was assessed by GFP expression levels (fluorescence).
  • the reporter strain was exposed to various cytokines (human IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12, Interferon gamma (IFN- ⁇ ) and tumor necrosis factor alpha (TNF- ⁇ ) with only IFN- ⁇ showing a significant increase in PA-I expression beginning at early stationary phase of growth (Fig. 1C). None of the cytokines tested had any significant effect on bacterial growth (Fig. IB).
  • IFN- ⁇ Interferon gamma
  • TNF- ⁇ tumor necrosis factor alpha
  • IFN- ⁇ IFN- ⁇
  • TNF- ⁇ or other cytokines induced lecA mRNA
  • P. aeruginosa strains were exposed to media containing adenosine (released by Caco-2 cells in response to hypoxia) TNF ⁇ , IL-2, IL-6 IL-8 (released by epithelia in response to bacterial invasion/ischemia), and IFN ⁇ (released by intraepithelial lymphocytes in response to bacterial invasion/ischemia).
  • adenosine released by Caco-2 cells in response to hypoxia
  • TNF ⁇ IL-2
  • IL-6 IL-8 released by epithelia in response to bacterial invasion/ischemia
  • IFN ⁇ released by intraepithelial lymphocytes in response to bacterial invasion/ischemia
  • cytokines such as IFN- ⁇ are embraced by the invention as effective modulators of bacterial signaling and, ultimately, of eukaryotic (e.g., epithelial) cell barrier function.
  • QS quorum sensing signaling system
  • virulence in P. aeruginosa is highly regulated by the quorum sensing signaling system (QS), a hierarchical system of virulence gene regulation that is dependent on bacterial cell density and hence growth phase (M. Whiteley, K. M. Lee, E. P. Greenberg, Proc Natl Acad Sd USA 96, 13904 (Nov 23, 1999)) (S. P. Diggle, K. Winzer, A. Lazdunski, P. Williams, M.
  • QS quorum sensing signaling system
  • RhII is the gene required for the synthesis of C 4 -HSL (C 4 -homoserine lactone), a core quorum sensing signaling molecule that plays a key role in the expression of PA-I (M. R. Parsek, E. P. Greenberg, Proc Natl Acad Sd (USA) 97, 8789 (Aug 1, 2000)).
  • P P.
  • Example 11 Interferon- ⁇ binds to the surface of P. aeruginosa
  • IFN- ⁇ direct binding to a protein on the surface of P. aeruginosa in the course of virulence activation, was also investigated.
  • ELISA binding assays were performed by first coating microtiter plates with P. aeruginosa (strain PAOl), then adding recombinant human IFN- ⁇ (rH IFN- ⁇ ), followed by biotin-labeled anti-IFN- ⁇ antibody. IFN- ⁇ avidly bound to whole fixed cells of P. aeruginosa in a dose-dependent manner (Fig. 3A).
  • the ELISA data were confirmed by the results of immunofluorescent imaging of bacterial cells exposed to IFN- ⁇ followed by biotin-labeled anti- IFN- ⁇ antibody and Alexa 594-labeled streptavidin.
  • the binding capacity of the IFN- ⁇ to the P. aeruginosa was affected by bacterial growth phase (Fig. 4A).
  • P. aeruginosa PAOl
  • equal protein concentrations of membrane and cytosol fractions of P. aeruginosa were prepared and coated onto ELISA microtiter plates.
  • ELISA binding assays showed that IFN- ⁇ preferentially bound to membrane fractions of P. aeruginosa (Fig. 4B).
  • membrane fractions were treated with proteinase K for 3 hours and IFN- ⁇ binding assessed. Binding by IFN- ⁇ to P. aeruginosa membranes after treatment with proteinase K was decreased (Fig. 4C) suggested that IFN- ⁇ binds to protein on the bacterial cell membrane.
  • cytokines similarly would bind to P. aeruginosa cell membranes by performing reiterative binding studies with human TNF- ⁇ , IL-2, IL-4, IL-IO, EGF, and TGF- ⁇ . No binding was observed with any of these cytokines (Fig. 4D). Taken together these data indicate IFN- ⁇ bound to membrane protein on P. aeruginosa.
  • membrane protein was extracted from 4L of freshly grown P. aeruginosa and fractionated by molecular weight between 10-100 kD. Solubilized protein was then immunoprecipitated using TFN- ⁇ and anti-IFN- ⁇ antibody. BSA was used as a control. Immunoprecipitation resulted in the appearance of a distinct protein with a molecular weight of about 35 kD. To further confirm that the protein isolated by immunoprecipitation was dependent on the presence of IFN- ⁇ , equally divided solubilized membrane protein fractions were mixed with and without IFN- ⁇ and then immunoprecipitated with anti-IFN- ⁇ antibody.
  • the IFN- ⁇ -dependent band was identified by ESI-TRAP-Electrospray LC-MSMS Ion Trap as the P. aeruginosa outer membrane porin OprF (Fig. 3F).
  • Endogenous morphine has been documented to be released in direct proportion to the magnitude of surgical stress/injury in both animals and humans. Initially, morphine was assessed for its effects. Interestingly, exposure of Pseudomonas strain PA27853 to physiologic concentrations of morphine (13 ⁇ M) resulted in a four-fold increase in PA-I expression (in comparison, in the same assay C4-HSL induced about a 16-fold increase in PA-I expression).
  • morphine is considered to be a non-selective opioid
  • specific endogenous opioid agonists with high selective affinity to ⁇ , K and ⁇ receptors were tested for their abilities to induce PA-I lectin/adhesin expression in strains PA27853 and PAOl.
  • mice were implanted with slow release morphine pellets that release a .daily dose of morphine that is similar to that used clinically (pellets obtained from the National Institute on Drug Abuse (NIDA). Control mice were implanted with a placebo pellet. Mice drank infant formula spiked with a daily inoculum of Ix 10 cfu/ml of PA27853. AU the morphine treated mice developed severe sepsis (4/4) and significant mortality while none of the control mice appeared septic and all survived. Finally, agonists were tested for their ability to induce biofilm in PA27853, a quorum sensing dependent phenotype.
  • Biofilm production by P. aeruginosa and other organisms has been established to be a major phenotype indicative of enhanced virulence.
  • the opioid K and ⁇ agonists significantly increased biofilm production in strains PA27853, about 150% and 180% of PA27853 induction respectively.
  • these studies demonstrate that opioid agonists can directly influence the virulence, and potential lethality, of P. aeruginosa.
  • opioid agonists and antagonists whether found endogenously or not, and whether purified from a natural source, chemically synthesized, or produced by a combination thereof, are contemplated by the invention as useful modulators of the bacterial signaling affecting microbial pathogenesis generally, and eukaryotic (e.g., epithelial or endothelial) cell barrier function more specifically.
  • eukaryotic e.g., epithelial or endothelial
  • Opioid compounds known to accumulate in tissues such as the lung and intestine following stress, directly activate the virulence of P. aeruginosa as judged by pyocyanin production, biofilm formation, and the expression of the PA-IL protein. Specifically, pyocyanin production was enhanced in the presence of the selective ⁇ -opioid receptor agonist, U-50,488, and the naturally occurring endogenous peptide dynorphin, also a selective ⁇ -opioid receptor agonist. To understand the regulatory pathway(s) involved in opioid-induced virulence gene expression in P. aeruginosa, the effect of U-50,488 on multiple mutant P.
  • aeruginosa strains defective in key elements involved in pyocyanin production was examined. Results demonstrated that the global transcriptional regulator, MvfR, plays a key role in pyocyanin production in response to U-50,488. Intact MvfR was also shown to be required for P. aeruginosa to respond to C4-HSL, a key quorum sensing signaling molecule known to activate hundreds of virulence genes. Taken together, these studies indicate that opioid compounds serve as host-derived signaling molecules that can be perceived by bacteria during host stress for the purposes of activating their virulence phenotype. Bacterial strains and culture conditions. P.
  • aeruginosa strains PAOl and 27853, and their derivative strains were routinely grown in tryptic soy broth (TSB) supplemented when necessary with tetracycline (Tc), 60 ⁇ g/ml, and/or gentamicin (Gm), 100 ⁇ g/ml.
  • TTB tryptic soy broth
  • Tc tetracycline
  • Gm gentamicin
  • Alkaloid opiates morphine a preferable ⁇ -opioid receptor agonist
  • A. Shahbazian, et al., Br J Pharmacol 135, 741 (2002) U-50,488, a specific ⁇ -o ⁇ ioid receptor agonist (J.
  • Morphine was purchased from Abbott Laboratories, U-50,488, BW373U86, dynorphin, nor- binaltorphimine, and methyl anthranilate from Sigma- Aldrich, and C4-HSL from Fluka.
  • GacA mutant with gacA gene.
  • the gacA gene a member of a two-component signaling method involved in the elaboration of virulence in many gram-negative bacteria, was amplified and directly cloned into pCR2.1 (Invitrogen). The gene was then excised with Xbal-HinDIII restriction endonucleases and subcloned into pUCP24 under the Plac promoter to create pUCP24/gacA.
  • the plasmids pUCP24 (blank control) and pUCP24/gacA were electroporated in P. aeruginosa strain PAO6281, defective in GacA production, to create the P. aeruginosa strain PAO6281/ GacA (Tables 1, 2).
  • Truncation of MvfR PCR products of truncated mvfR genes amplified from pUCP24/MvfR and their respective primers (Tables 1, 2) were purified using a Geneclean kit (Qbiogene), digested with Xbal-HinDIII restriction endonucleases, and ligated into pUCP24 followed by electroporation into P. aeruginosa strain 13375.
  • PA-IL assays Immunoblotting and fluorescence of the GFP-PA-IL reporter strain were used to determine the effect of opioids on PA-IL expression.
  • a bacterial culture of the GFP -PAIL reporter strain 27853/PLL-EGFP (L. Wu, et al., Gastroenterology 126, 488 (2004)) was plated at a final concentration of 108 CFU/ml in 96- well fluorometry plates (Costar) in conventional media, i.e., HDMEM media containing 10% FBS and HEPES buffer with or without 60 ⁇ M of U-50,488. Incubation was performed at 37°C, 100 rpm, and fluorescence reading was performed hourly with a 96-well fluorometry Plate Reader (Synergy HT, Biotec Inc.) at excitation/emission of 485/528 nm. Fluorescence intensity was normalized to cell density measured at 600 nm.
  • Biofilm formation assay Bacterial cells were plated in quadruplicate in 96- well U-bottom plates (Falcon) at a concentration of 107 CFU/ml in M63S media (13.6 g KH2PO4 1-1, 2.0 g (NH4)2SO4 1-1, 0.5 mg FeSO4x7H20 1-1), supplemented with 0.5% casamino acids, ImM MgSO4x7H20 and 0.2% glucose, and incubated overnight at 37 0 C under static conditions. U-50,488 was added at the inoculation point. After inoculation, the wells were rinsed thoroughly with water and the attached material was stained with 0.1% crystal violet, washed with water, and solubilized in ethanol.
  • Solubilized fractions were collected and absorbance measured at 590 nm as described (G. A. O'Toole and R. Kolter, MoI Microbiol 28, 449 (1998)) with a Plate Reader.
  • ⁇ -opioid receptor agonists U-50,488 and dynorphin stimulate pyocyanin production in P. aeruginosa.
  • P. aeruginosa harvested from the intestine of surgically stress mice appeared intensely green compared to P. aeruginosa from the intestines of sham-operated control mice.
  • P. aeruginosa might be responding to a signal to produce increased amounts of pyocyanin (PCN) in response to environmental cues unique to the intestinal tract of stressed mice.
  • PCN pyocyanin
  • Pyocyanin a redox active compound that increases intracellular oxidant stress, has been shown to play a key role in the virulence of P. aeruginosa in animal models mediating tissue damage and necrosis during lung infection (G. W. Lau, H. Ran, F. Kong, D. J. Hassett and D.
  • P. aeruginosa PAOl was exposed to peptide opioids and alkaloid opiates representing groups of ⁇ -, K-, and ⁇ - opioid receptor agonists. Results indicated that following overnight exposure, the alkaloid opiate U-50,488, a specific ⁇ -opioid receptor agonist, induced an intensely bright green color in P. aeruginosa PAOl , while no such effect was observed with any of the remaining compounds. To verify that the color change was due to PCN production, pyocyanin was measured at OD520 nm (D. W. Essar, L. Eberly, A. Hadero and I. P.
  • the ⁇ -opioid-receptor agonist U-50,488 shifts pyocyanin production at lower cell densities in P. aeruginosa.
  • the ⁇ -opioid-receptor agonist U-50,488 exerts its inducing effect on pyocyanin production via elements of the quorum sensing system in Pseudomonas aeruginosa.
  • the pathways of PCN regulation and biosynthesis have been described in ' detail (D. V. Mavrodi, et al., J Bacteriol 183, 6454 (2001), E. Deziel, et al., Proc Natl Acad Sci U S A 101, 1339 (2004), T. R. de Kievit, Y. Kakai, J. K. Register, E. C. Pesci and B. H. Iglewski, FEMS Microbiol Lett 212, 101 (2002), S. L.
  • MvfR is involved in the ability of U-50,488 and C4-HSL to enhance PCN production in PAOl.
  • Intact substrate-binding and DNA-binding domains of MvfR are required for U-50,488 to enhance PCN production in PAOl.
  • MvfR belongs to a family of prokaryotic LysR transcriptional regulators that possess a helix-turn-helix DNA-binding motif at the N terminus and a substrate binding domain at the C terminus.
  • a NCBI conserveed Domain Search revealed similar domains in MvfR: a LysR DNA-binding domain located at 6-64 aa, and a LysR substrate binding domain located at 156-293 amino acids. Therefore PAOl mutants were constructed producing N- and C-terminus-truncated MvfR to determine if specific domains could be identified that play a functional role in mediating the ⁇ -opioid receptor agonist effect on PCN production.
  • MvfR might play a critical role in PCN production via positive transcriptional regulation of the phnAB and PQS ABCDE operons that encode two 12 precursors of PQS, anthranilic acid (AA) and 4-hydroxy-2-heptylquinolone (HHQ) (E. Deziel, et al., Proc Natl Acad Sci U S A 101 , 1339 (2004)). Therefore the mutants ⁇ PhnA and ⁇ PqsA were examined for their ability to produce PCN in the presence of U-50,488. Neither mutant produced PCN. Exposure of each mutant to U-50,488 resulted in a slight increase in PCN production, although the increase was much less than that observed with the wild-type strain PAOl .
  • U-50,488 stimulates other QS-regulated virulence determinants in P. aeruginosa including biofilm formation and PA-IL production.
  • PA-IL expression was dynamically tracked in response to U-50,488 using the green fluorescent PA-IL reporter strain P. aeruginosa 27853/PLL-EGFP (L. Wu, et al., Gastroenterology 126, 488 (2004)). Marked fluorescence was observed in this strain following 9 hours of growth in HDMEM media. Results were confirmed in strain PAOl by immunoblotting using rabbit polyclonal antibody against PA-IL .
  • the effect of U-50,488 on PCN production in P. aeruginosa can be inhibited by the anti-infective high molecular weight polymer PEG 15-20.
  • the capacity of PEG 15-20 to interfere with the U-50, 488 effect on P. aeruginosa was assessed by measuring PCN production in the media of P. aeruginosa PAOl incubated in the presence of 5% PEG 15-20 and 0.5 mM U-50,488 or 0.2 mM C4-HSL. Results demonstrated that PEG 15-20 had a strong inhibitory effect on both U-50, 488- and C4-HSL-mediated up- regulation of PCN production.
  • P. aeruginosa PAOl expresses abundant PA-I and alters MDCK monolayer permeability in a PA-I-dependent manner
  • PA-I induced permeability defect in MDCK cells was of sufficient magnitude to permit the apical to basolateral flux of exotoxin A across the monolayers, with a PA-I-induced TEER decrease of over 50% resulting in a five-fold increase in exotoxin A flux.
  • PA-I protein has been shown to be abundantly expressed in PAOl when strains were exposed to the various opioid agonists.
  • the ⁇ agonist BW373U86
  • induced a response equal to C4-HSL The data establish that PA-I expression affects eukaryotic cell barrier function.
  • modulators of PA-I expression will be useful in affecting the virulence phenotype of microbial pathogens and will be useful in affecting the eukaryotic (e.g., epithelial) cell barrier dysfunction associated with that phenotype.
  • Host cell-derived bacterial signaling components enhance the barrier-dysregulating properties of P. aeruginosa against epithelial cells
  • PA-I is expressed in vivo within the digestive tube of Caenorhabditis elegans
  • the PA-I-GFP reporter plasmid was introduced into P. aeruginosa strain PAl 4, a strain highly lethal to C. elegans, by electroporation. Worms were then fed GFP- tagged PA14 and PA27853 and examined for fluorescent bacteria. Worms feeding on lawns of PA14 and PA27853 displayed fluorescent bacteria within the digestive tube, whereas no fluorescence was seen within the surrounding media, indicating that PA-I promoter activity is activated by local factors within the worm digestive tube. Finally the killing dynamics of strain PA- 14, a highly lethal strain in this model, was compared to the dynamics associated with the completely sequenced PAOl strain. The strain of E.
  • PAOl upon which worms normally feed, resulted in 100% survival, whereas, PA- 14 displayed fast killing dynamics, as predicted.
  • the PAOl strain displayed slow killing with only a 50% mortality rate at 80 hours.
  • PAOl exhibits killing dynamics that will allow assessments of whether host stress-derived BSCs shift the killing curve to that of a more virulent strain. It is expected that BSCs, whether soluble or membrane-bound, will shift the killing dynamics of relatively quiescent, or benign, microbes towards the dynamics exhibited by lethal microbial strains. Stated in the alternative, it is expected that a BSC will shift the phenotype of a microbe towards a virulent phenotype.
  • Modulators of such activities are expected to be useful in preventing and treating disorders associated with the display of a virulence phenotype by any such microbe and in particular by P. aeruginosa. Such modulators are also expected to be used in methods for ameliorating a symptom of such a disorder.
  • PA-I expression is dependent on multiple elements of the virulence gene regulatory circuitry in P. aeruginosa, including the quorum sensing signaling system (QS) and RpoS.
  • QS quorum sensing signaling system
  • RpoS quorum sensing signaling system
  • At least two techniques are contemplated for use in gene identification: 1) perform transcriptome analysis on P. aeruginosa strain PAOl exposed to morphine, K and ⁇ opioid receptor agonists, and IFN- ⁇ , and 2) establish a functional role for candidate genes identified in the transcriptome analysis by screening the corresponding transposon mutants for their ability to up-regulate PA-I protein expression in response to opioids and IFN- ⁇ .
  • Transcriptome analyses is performed using Affymetrix GeneChip genome arrays in strain PAOl to identify the genes that respond to the host cell elements such as morphine (non-selective opioid receptor agonist), U-50488 (K receptor agonist) , BW373U86 ( ⁇ opioid receptor agonist), and IFN- ⁇ .
  • morphine non-selective opioid receptor agonist
  • U-50488 K receptor agonist
  • BW373U86 ⁇ opioid receptor agonist
  • IFN- ⁇ IFN- ⁇
  • bacteria are grown in TSB overnight and diluted 1 : 100 in TSB containing either morphine (20 ⁇ M), K agonist (80 ⁇ M), ⁇ agonist (80 ⁇ M), or IFN- ⁇ (10 ⁇ g/ml). Bacteria are then grown to an OD 60O of 0.5, 1.0, and 2.0, representing three stages of growth: exponential phase, late exponential phase, and stationary phase, respectively. These three time points will permit the capture of genes that are expressed early in the PA-I signaling pathway as well as during time points of high cell density.
  • RNA is isolated from bacterial cells (treated and non-treated with morphine, K and ⁇ opioid receptor agonists, and IFN- ⁇ ) at the three designated points in the growth phase.
  • cDNA synthesis, fragmentation, labeling, and hybridization, as well as P. aeruginosa GeneChip genome array processing, are performed as described herein or as known in the art. Each experiment is preferably performed in triplicate.
  • Genes showing at least a 2.5-fold change in expression resulting from exposure to morphine, K and ⁇ opioid receptor agonists, and/or IFN- ⁇ are individually tested for their specific role in PA-I protein expression by screening mutant strains from a PAOl transposon library (University of Washington Genome Center, see below) using dot blot analysis. Briefly, strains are grown in sequential runs using 384-well microliter plates at 2 separate bacterial cell densities (OD 600 of 1.0 and 2.0) predetermined to respond to the inducing compound (opioids, IFN- ⁇ ).
  • Dose-response curves are generated with varying doses of the PA-I inducing compounds at different bacterial cell densities in wild-type strains and in several mutant strains to determine the optimal conditions for screening.
  • Experiments are performed separately for morphine, U-50488, BW373U86, and IFN- ⁇ . Briefly, either morphine, U-50488, BW373U86, or IFN- ⁇ are added to the wells containing mutant strains at the predetermined dose. All runs are performed with the wild-type strain as a control. The PA-I-inducing compound is added to the well for a predetermined time. Next, the supernatant is removed and the bacterial cell pellet is lysed by the addition of lysis solution directly into the well.
  • membrane biosensors are constitutively expressed and therefore gene expression will not change in response to opioids or IFN- ⁇ . If this is the case, then the entire transposon library will be screened for PA-I expression in response to opioids or IFN- ⁇ , approaches that are feasible given the high-throughput nature of the dot-blot technique.
  • gene expressions can be triggered at different times during culturing and can respond to an exogenous compound(s) to varying degrees depending on the concentration of compound.
  • the genomically sequenced strain PAOl makes abundant PA-I and the anti-PA-I lectin/adhesin antibodies are highly specific.
  • the genes that control PA-I expression are dependent on two key global regulatory systems that activate hundreds of virulence genes in P. aeruginosa.
  • the activation of these interconnected systems of virulence gene regulation are directly influenced by membrane biosensors that recognize elements of host cells and include, but are not limited to, CyaB and GacS, via a hierarchical cascade involving the transcriptional regulators Vfr and Gac A.
  • Genes that are differentially expressed in response to opioids and IFN- ⁇ will be identified using an unbiased transcriptome analysis approach. This approach was chosen instead of pursuing individual candidate genes involved in known pathways of PA-I expression because all previous studies have been performed only at high cell densities and in the absence of any host cell elements. Accordingly, previously described gene expression patterns may not be applicable in the physiologic models. The goal of this study is to identify and functionally validate the genes that are involved in PA-I expression in response to morphine, K and ⁇ opioid receptor agonists, and IFN- ⁇ .
  • membrane proteins of P. aeruginosa strain PAOl are solubilized using mild detergents.
  • the binding capacity of solubilized protein fractions for IFN- ⁇ or morphine is then determined using simple ELISA binding assays.
  • Protein fractions are then imrrmnoprecipitated using the respective antibody and proteins are identified, e.g., by Maldi-MS.
  • Confirmation of the identity of a binding protein(s) is achieved by determining that a transposon knockout of the gene encoding the candidate protein(s) does not respond to IFN- ⁇ or morphine with an increase in PA-I, using the techniques described herein. In order to confirm the function of candidate proteins showing fidelity in these two analyses, candidate proteins are re-expressed in the corresponding transposon knockout to verify that the PA-I response is re-established. Additionally, receptor antagonists may also be developed.
  • membrane receptors for morphine and IFN- ⁇ can be identified by identifying proteins from solubilized membranes.
  • a potential limitation using this technique is that morphine could diffuse directly into the bacterial cytoplasm and interact with a downstream target and not a membrane protein. This possibility is consistent with results demonstrating that morphine does not change the transcript profiles of any genes encoding membrane proteins, but the data for IFN- ⁇ disclosed herein is inconsistent with this interpretation.
  • Li addition, morphine binding to a solubilized bacterial membrane protein was demonstrated using fluorescent imaging and analysis. Also, there is the possibility that transmembrane proteins or proteins that bind host stress-derived BSCs could be secreted into the culture medium and not be present within bacterial membranes.
  • bacterial iron binding proteins are the bacterial iron binding proteins (enterochelin), which are released by bacteria into the culture medium and then re-enter the bacterial cells.
  • the screening of cytosolic fractions and inner and outer membrane preparations are contemplated, along with iterative experiments probing for binding proteins with specific antibodies. Any discordance between the transposon mutant experiments and the proteins purified from bacterial membranes will be reconciled by analyzing IFN- ⁇ - membrane protein or morphine- membrane protein interactions directly using surface plasmon resonance and mass spectrometry.
  • PA-I knockout strains (lecA ⁇ ) do not decrease the TEER of cultured epithelial cells.
  • the lethality of strains of P. aeruginosa exposed to opioid agonists and IFN- ⁇ can be defined in vivo using the well-characterized invertebrate, Caenorhabditis elegans, and the established model of gut-derived sepsis in mice.
  • opioids or IFN- ⁇ can activate P. aeruginosa to express a lethal phenotype against an epithelium, as judged by an increase in exotoxin A flux across epithelial cell monolayers, through the action of its PA-I lectin/adhesin.
  • MDCK cells are grown to confluence to maintain a stable TEER in transwells.
  • Cells are apically inoculated with P. aeruginosa strain PAOl (10 cfu/ml) in the presence and absence of varying doses of morphine (about 20 ⁇ M), K agonist (about 80 ⁇ M), ⁇ agonist (about 80 ⁇ M), or IFN- ⁇ (about 10 ⁇ g/ml).
  • morphine about 20 ⁇ M
  • K agonist about 80 ⁇ M
  • ⁇ agonist about 80 ⁇ M
  • IFN- ⁇ about 10 ⁇ g/ml
  • dose and time response curves are generated.
  • TEER is measured using chopstick electrodes hourly for 8 hours.
  • the apical to basolateral flux of exotoxin A using Alexa-594- labeled exotoxin A is determined in iterative experiments performed at each hourly time point in order to correlate the decrease in TEER to exotoxin A flux for each condition.
  • the specific role of PA-I is defined by performing iterative experiments in the presence and absence of 0.3% GaINAc (N-acetylgalactoside) and 0.6% mellibiose, two oligosaccharides that specifically bind to PA-I 78 . Irrelevant sugars (heparin/mannose) are used as negative controls.
  • PA-I knockout mutant strains alter TEER and exotoxin A flux in response to opioids or IFN- ⁇ , then this will indicate that PA-I alone may not be responsible for the virulence of P. aeruginosa against the intestinal epithelium.
  • Data from these studies are directly compared and correlated to worm and mouse lethality studies (see below) to determine if these in vitro assays accurately predict a lethal phenotype in vivo, as expected.
  • Wild-type N2 Caenorhabditis elegans worms are grown to the L4 larval stage on normal growth medium (NGM) with E. coli OP50 as a nutrient source.
  • NGM normal growth medium
  • E. coli OP50 E. coli OP50
  • Specialized agar plates are prepared onto which the PA-I-inducing compounds (vehicle (negative control)), opioids (morphine, K and ⁇ agonist), IFN- ⁇ , and C4-HSL (positive control)) will be added and adsorbed into the agar as described for ethanol.
  • PA-I-inducing compounds vehicle (negative control)
  • opioids morphine, K and ⁇ agonist
  • IFN- ⁇ IFN- ⁇
  • C4-HSL positive control
  • aeruginosa wild type PAOl and PA-I knockout PAOl (lecA-)
  • aeruginosa wild type PAOl and PA-I knockout PAOl (lecA-)
  • aeruginosa wild type PAOl and PA-I knockout PAOl (lecA-)
  • Worms from the NGM medium are transferred onto the prepared culture dishes and killing dynamics assessed over time at temperature conditions of 25 0 C. Experiments are performed at different doses and re-dosing schedules to establish the optimum conditions under which a killing effect for each of the PA-I-inducing compounds can be demonstrated.
  • mice are fasted for 24 hours and are subjected to general anesthesia, a 30% surgical hepatectomy, and cecal instillation of 10 6 cfu/ml of wild-type PAOl or PAOl (lecA-) via direct puncture.
  • Dose-response curves for P. aeruginosa in this model have been established and show that 10 6 cfu/ml of P. aeruginosa induces a 50% mortality rate at 48 hours.
  • opioid agonists or IFN- ⁇ enhance the lethality of P.
  • mice used in the study include two strains (wild-type + PA-I knockout) and, with 6 groups of 10 mice per group, a total of 120 mice is suitable.
  • mice studies to confirm results obtained with C. elegans preferably includes verification that luminally delivered PA-I-inducing compounds are efficacious in up-regulating PA-I as a general measure of enhanced virulence.
  • experiments are performed to show that the PA-I-inducing compounds injected into the small bowel enhance PA-I expression in the mouse cecum.
  • One approach involves the use of quantitative RT-PCR for PA-I and exotoxin A on freshly isolated RNA from cecal contents 24 hours following cecal instillation of P. aeruginosa.
  • An alternative approach to delivering opioids and IFN- ⁇ directly into the cecum is to engineer nonpathogenic E. coli strains that produce both morphine and IFN- ⁇ .
  • the "microbial soup" typical of a critically ill patient consists of highly pathogenic and resistant strains of bacteria that compete for nutrients in a highly adverse environment. Therefore, not only will the use of morphine- and/or IFN- ⁇ -producing E. coli constitute a feasible alternative approach to obtaining in vivo mouse data, it may also recapitulate actual events in vivo. Finally, C. elegans normally feed on E. coli strains that do not induce mortality. The availability of morphine- and/or IFN- ⁇ - producing E. coli strains could also be used in the C. elegans assays.
  • mice have shown the feasibility of this approach is feasible in mice, as shown by delivering IL-10 into the intestinal mucosa of mice using direct intestinal instillation of bacteria that produce recombinant IL-10.
  • the use of the C. elegans assay is expected to result in the rapid identification of therapeutics and prophylactics that modulate expression of a virulence phenotype by microbial pathogens in contact with, or proximity to, a mammal.
  • the virulence phenotype is amenable to assessment using a variety of measures, many of them indirect, e.g., measurement of epithelial cell barrier function.
  • Endogenous morphine concentrations in the blood of humans and animals increase in direct response to the degree of surgical stress.
  • the neural network of the mammalian intestine contains the most abundant concentration of opioid receptors in the body.
  • Morphine has been recently shown to enhance the release of nitric oxide in the mammalian gastrointestinal tract via the ⁇ 3 opiate receptor subtype.
  • the nematode, Ascaris suum produces and liberates morphine in the gut.
  • IFN- ⁇ has been shown to be released by the gut from intestinal intraepithelial lymphocytes in response to a variety of stressors, including bacterial challenge and ischemia/reperfusion injury (I/R).
  • worms are grown permissively at 2O 0 C in massive cultures in liquid medium to 1 x 10 6 worms using conventional culturing techniques. Stock cultures are treated with antibiotics 24 hours prior to the imposition of stress conditions. Worms are separated from any remaining bacteria by sedimentation and sucrose flotation as known in the art. Worms are then exposed to either heat stress (35 0 C for 1 hour) followed by 2 hours of recovery, or hypoxic stress (0.3% O 2 for 45 minutes) followed by 1 hour of normoxic recovery, as described. Control worms are maintained at 20 0 C and 21 % O 2. Both the growth medium and the supernatant of homogenized C.
  • elegans are preferably assayed for morphine by HPLC/ GC/MS using conventional techniques.
  • HPLC/ GC/MS aqueous cytoplasmic cytoplasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic plasmic ma fibroblasts, fibroblasts, and fibroblasts, and fibroblasts, and fibroblasts, and fibroblasts, and fibroblasts, and fibroblasts, and fibroblasts, and hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematoma hematom
  • the ischemia reperfusion model involves isolation of a 10 cm segment of distal ileum that is luminally cannulated and subjected to 10 minutes of ischemia (segmental artery clamp) followed by 10 minutes of reperfusion.
  • Luminal perfusion with 2 ml of Ringers solution is performed to collect the luminal contents before and after I/R.
  • Luminal contents, the homogenized intestinal segment, and blood are assayed for morphine by HPLC and GC/MS; IFN- ⁇ is assayed by ELISA using a specific anti-IFN- ⁇ antibody.
  • a suitable number of mice for such assays is 30-50 mice. Release of significant amounts of morphine and/or IFN- ⁇ into the gut following surgical stress confirms that P.
  • aeruginosa has been exposed to highly active compounds capable of activating or enhancing its virulence phenotype during host stress, hi addition, a better understanding of the precise concentration of morphine and/or IFN- ⁇ to which P. aeruginosa are exposed in vivo can be determined by these experiments. Whether morphine is released in high concentration in the lumen versus within the intestinal tissues is amenable to experimental determination. If luminal levels of morphine are elevated in hepatectomy versus controls, mice can be decontaminated with antibiotics (e.g., ciprofloxacin, metronidazole). Following such decontamination, the extent to which the luminal flora contribute to the opioid level can be determined using conventional techniques.
  • antibiotics e.g., ciprofloxacin, metronidazole
  • opioids and cytokines may be released from microbial pathogens such as P. aeruginosa that actively participate as host stress-derived BSCs. It is also possible that both opioids and IFN- ⁇ are enzymatically degraded in the intestinal lumen.
  • An alternative approach would be to use quantitative immuno-fluorescence of stained tissues to assess morphine and IFN- ⁇ presence in tissues as antibodies specifically recognizing these compounds are readily available. Notwithstanding the preceding observations, these compounds have been measured by others from luminal contents without difficulty.
  • IFN- ⁇ is a key immune element that actively participates in both the local and systemic clearance of bacteria during acute infection.
  • Animal models have shown that IFN- ⁇ knockout mice have higher mortality rates following infectious challenge at local tissue sites (lung) compared to IFN- ⁇ -sufi ⁇ cient mice in association with diminished ability to clear bacteria.
  • Virtually all of the studies that have assessed the role of IFN- ⁇ on P. aeruginosa infection in vivo have been performed in non-stressed mice where the infectious challenge has been instilled into the lung, and not in stressed mice, such as surgically stressed mice.
  • PPA Pseudomonas isolation agar
  • aeruginosa is attenuated in IFN- ⁇
  • a GFP PA-I reporter strain is injected directly into the cecum of mice subjected to a 30% hepatectomy and bacterial strains are recovered 24 hours later to determine fluorescence.
  • the results of these experiments guide the performance of complementary studies using the segmental mesenteric ischemia model. Briefly, the lumena of 10 cm ileal segments subjected to sham ischemia (no clamp), 10 minutes of ischemia, and 10 minutes of reperfusion is perfused with Ringers solution and the timed aliquots of the perfusates is collected from both EFN- ⁇ knockout mice and their wild-type cohorts.
  • Use of the GFP-PA-I reporter strains facilitates the determination of the extent to which each perfusate induces PA-I promoter activity.
  • a suitable number of mice for such studies is 50 mice, divided into five groups with ten mice in each group.
  • IFN- ⁇ knockout mice The display of attenuated lethality by P. aeruginosa in IFN- ⁇ knockout mice is consistent with IFN- ⁇ playing a role as a host stress-derived bacterial signaling compound, or protein, during stress (e.g., surgical stress). IFN- ⁇ may be only one of many signals necessary to orchestrate a fully lethal virulence repertoire for P. aeruginosa under the circumstances of catabolic stress, however. It is noted that IFN- ⁇ knockout mice subjected to hepatectomy may develop an overcompensated and excessive inflammatory response to intestinal P. aeruginosa, resulting in increased mortality that is based more on immune response than enhanced microbial virulence.
  • Tissue and blood culture results from these studies are used to determine whether mortality is due, in part, to such overcompensation.
  • An alternative approach to distinguish between these possibilities is to perform studies in IFN- ⁇ knockout mice and their matched wild-type cohorts (with and without surgical hepatectomy) to determine if there is a mortality difference when groups of mice are systemically inoculated (e.g., intraperitoneal, intravenous, lung instillation) with P. aeruginosa.
  • the data disclosed herein establishes that i) filtered luminal contents from the cecum of mice subjected to hepatectomy, or from the small bowel lumen of intestinal segments subjected to mesenteric arterial occlusion, induce a strong signal in P. aeruginosa to express PA-I; and ii) media and membrane preparations from hypoxic or heat-shocked Caco-2 cells induce PA-I expression.
  • Intestinal epithelial hypoxia is a common consequence of critical illness following surgical stress and is often an inadvertent consequence of its treatment.
  • hyperthermia often develops during the acute stress response to injury and infection.
  • hypoxic or hyperthermic stress to cultured intestinal epithelial cells causes the release of soluble P A-I-inducing compounds into the cell culture medium.
  • This example discloses the isolation and identification of P A-I- inducing compounds that are released by Caco-2 cells exposed to hypoxia and hyperthermic stress.
  • Two sets of experiments are preferably performed.
  • Caco-2 cells grown to confluence in cell culture plates (150 cm ) are exposed to either normoxia (21% O 2 ,) or hypoxia (0.3% O 2 for 2 hours followed by 1 hour of normoxic recovery).
  • Caco-2 cells are exposed to normothermic (37 0 C) or hyperthermic (immersed in water bath at 42°C for 23 minutes followed by 3 hours recovery) conditions. Paired samples from each set of experiments are then processed to identify the specific host stress-derived bacterial signaling compound(s) using GFP-PA-I reporter strains as a detection system.
  • Media from Caco-2 cells is collected, filtered through a 0.22 ⁇ m filter (Millipore) and separated by molecular weight using centricones with a MW cutoff of 3, 10, 30, 50, 100 KDa ( ⁇ 3, 3-10, 10-30, 30-50, 50-100, >100 KDa). All fractions are preferably tested in 96 well plates to determine fractions that activate PA-I expression using PA-I GFP reporter strains. Two preferred approaches are contemplated for use in identifying the proteins that activate PA-I in the stress-conditioned media (hypoxia, hyperthermia).
  • the first approach subjects bioactive fractions (i.e those that induce PA-I), and their molecular weight-matched control fractions (non-P A-I-inducing), to Maldi-Mass Spectrometry (MS) analysis. Spectra from the control media fractions are compared to the fractions of stress-conditioned media to determine the appearance of possible protein molecular ions present only in the samples that induce PA-I. This will allow us to subtract proteins that are present in both non-P A-I-inducing and P A-I-inducing fractions. In order to separate the molecular ion protein peaks that are present only in the PA-I-inducing fractions, bioactive fractions are loaded onto an HPLC equipped with a Vydac C4 column.
  • Eluted samples are collected as fractions and individual fractions are tested for the ability to induce PA-I expression using the GPF-PA-I reporter strain. Proteins are then further separated, preferably by MW, hydrophobicity, and charge using stepwise well-controlled physico- chemical separation techniques in the HPLC system. Samples pre-fractionated in this manner should simplify the observed mass spectra and increase the likelihood of observing any putative protein(s) that induce PA-I expression. For any such proteins, identification using bottom-up proteomics techniques is performed.
  • protein-containing fractions are digested by using trypsin and digested fractions are analyzed with a LC/MSD XCT ion trap mass spectrometer system (Agilent Technologies, Santa Clara, CA). Data analysis for the data from the mass spectrometer is carried out using the SpectrumMill software platform (Agilent Technologies, Santa Clara, CA). Confirmation of the ability of identified proteins to induce PA-I expression is conveniently achieved in the PA-I:EGFP reporter strain by measuring fluorescence, and in P. aeruginosa strain PAOl by immunoblot analysis.
  • lipid assays involve adjusting fraction pH to 3.5, followed by HPLC using, e.g., a Sep-Pak C 18 column. Eluted samples are trapped on a fraction collector, evaporated to dryness, and re-suspended in PBS for PA-I reporter assays.
  • the structure of the active compound is preferably identified with IT/LC/MS/MS.
  • relevant fractions are resolved by IT/LC/MS/MS using a C 18 column and a quadrapole-time of flight mass spectrometer and NMR.
  • Individual compounds are determined by their mass-fragmentation spectra, isolated, and tested for PA-I inducing activity using GFP reporter strains.
  • Alternative approaches such as 2D-SDS-PAGE electrophoresis for protein separation and TLC for non-protein separation, are also contemplated.
  • Proteins separated by 2D-SDS-PAGE are typically transferred to a polyvinylidene difluoride transfer protein membrane for automated Edman degradation N-terminal sequence determination using an ABI 477A protein sequencer (Applied Biosystems). Protein identification is further facilitated by sequence comparison to database(s).
  • the invention contemplates any assay for a modulator of the expression of a virulence phenotype by a microbe in association with, or proximity to, a mammal such as a human.
  • the invention comprehends a wide variety of assays for modulators of, e.g., eukaryotic cell barrier function, such as epithelial cell barrier function (e.g., epithelial cells of the intestine, lung, and the like).
  • the invention further comprehends numerous assays for modulators of PA-I lectin/adhesin activity, whether due to a modulation of the specific activity of PA-I or a modulation of the expression of PA-I of constant specific activity, or both.
  • the invention contemplates any assay known in the art as useful for identifying compounds and/or compositions having at least one of the above-described characteristics.
  • Candidate PA-I inducer compounds that are released into the extracellular milieu following epithelial stress include ATP, lactate, cAMP, cytokines, and heat shock proteins.
  • the aforementioned candidate modulators, and other candidate modulators found in properly conditioned media, are identified using screening methods that constitute another aspect of the invention. Screens for such modulators are conveniently conducted in 96-well plates that contain the GFP-PA-I reporter strain PA27853/PLL-EGFP (see Example 24, below).
  • the reporter strain is exposed to varying concentrations of candidate host stress BSCs including, but not limited to, heat shock proteins (HSP 25, 72, 90, 110), extracellular nucleosides and nucleotides (adenosine, ATP, cAMP) and cytokines (IL-1-18). Agents are added to the wells and dynamic assessment of bacterial fluorescence is carried out over 12 hours. Positive results are preferably verified by Western blot analysis of PA-I expression.
  • the invention further comprehends assays to identify the receptors on P. aeruginosa to which such proteins bind.
  • the identified protein inducer of PA-I activity is used as a probe to screen, e.g., a comprehensive library of P. aeruginosa by dot blot analysis. Confirmation of the screen results is available by assaying the protein-binding capacity of a lysate from a corresponding clone from a P. aeruginosa transposon library in which the relevant coding region has been disrupted by insertional inactivation.
  • Identified modulators are then subjected to additional in vitro and in vivo virulence assays to refine the understanding of the role in virulence expression played by such modulators.
  • Caco-2 cells and MDCK cells are well-differentiated epithelial cell lines that maintain a stable TEER when grown in confluent monolayer. Apical to basolateral exotoxin A flux across monolayers is assessed with Alexa 594-labeled exotoxin A using standard flux measurements.
  • P. aeruginosa strain PAOl was obtained from the University of Washington Genome Center and is preferably used in the procedures disclosed herein, where appropriate.
  • Antibodies to PA-I are generated using conventional techniques. Preferably, such antibodies are purified by affinity chromatography. IFN- ⁇ and morphine antibodies are commercially available.
  • ImmunoDot Blot assays for the detection of bacterial proteins in large matrix systems are known in the art. The technique has been validated as highly sensitive and accurate.
  • RNA is isolated from bacterial cultures exposed to opioids and/or IFN- ⁇ as described herein at optical densities of 0.5, 1.0, 2.0. Between 1 X 10 9 and 2 X 10 9 cells are then mixed with RNA Protect Bacteria reagent (Qiagen) and treated as recommended by the manufacturer's mechanical disruption and lysis protocol. RNA is purified by using RNeasy mini columns (Qiagen), including the on-column DNase I digestion described by the manufacturer, hi addition, the eluted RNA is preferably treated for 1 hour at 37°C with DNase I (0.1 U per ⁇ g of RNA). DNase I is then removed by using DNA-Free (Ambion) or by RNeasy column purification.
  • Qiagen RNA Protect Bacteria reagent
  • RNA is purified by using RNeasy mini columns (Qiagen), including the on-column DNase I digestion described by the manufacturer, hi addition, the eluted RNA is preferably treated for 1 hour at 37°C with D
  • RNA integrity is monitored by agarose gel electrophoresis of glyoxylated samples. Further sample preparation and processing of the P. aeruginosa GeneChip genome arrays are then done as described by the manufacturer (Affymetrix). For cDNA synthesis 12 ⁇ g of purified RNA is preferably combined with semirandom hexamer primers with an average G+C content of 75%, and Superscript II reverse transcriptase (Life Technologies). Control RNAs from yeast, Arabidopsis, and Bacillus subtilis genes are added to the reaction mixtures to monitor assay performance. Probes for these transcripts are tiled on the GeneChip arrays. RNA is removed from the PCR mixtures by alkaline hydrolysis.
  • the cDNA synthesis products are purified and fragmented by brief incubation with DNase I, and the 3' termini of the fragmentation products are labeled with biotin-ddUTP. Fragmented and labeled cDNAis hybridized to an array by overnight incubation at 50° C. Washing, staining, and scanning of microarrays is performed with an Affymetrix fluidic station.
  • the Affymetrix Microarray Software suite (MAS) (version 5.0) is a suitable software choice for determining transcript levels and whether there are differences in transcript levels when different samples are compared.
  • Affymetrix scaling is used to normalize data from different arrays.
  • a scale factor is derived from the mean signal of all of the probe sets on an array and a user-defined target signal. The signal from each individual probe set is multiplied by this scale factor. For any given array, between 18 and 28% of the mRNAs are considered absent by MAS, indicating that the corresponding genes are not expressed at levels above background levels. Furthermore, it is known in the art that the average changes in control transcript intensities are less than twofold for any comparison of array data.
  • the log 2 ratio for absolute transcript signals obtained from a given pair of arrays will be calculated by using MAS.
  • a statistical algorithm of the software is also assigned a change call for each transcript pair, which indicates whether the level of a transcript is significantly increased, decreased, or not changed compared to the level for the baseline sample.
  • the baseline samples are those derived from cultures of P. aeruginosa PAO-I without any added opioids or IFN- ⁇ .
  • transcripts with significant increases or decreases compared to the baseline in one or more samples those that showed at least a 2.5-fold change are subjected to further analysis.
  • GeneSpring software (Silicon Genetics, Redwood City, Calif.) is contemplated as a suitable choice.
  • the fold change values for each gene will be normalized independently by defining the half-maximal value for the gene as 1 and representing all other values as a ratio that includes that value. This scaling procedure will allow direct visual comparison of gene expression patterns within an experiment, as well as between experiments.
  • GeneSpring is also contemplated for use in sorting genes according to the P. aeruginosa genome project.
  • P. aeruginosa cells are washed with PBS and re-suspended in PBS containing a protein inhibitor cocktail.
  • P. aeruginosa cells are disrupted by French pressure and centrifuged at lOOOOg ⁇ 30 minutes to eliminate debris. The supernatant is recentrifuged at 50000gx60 minutes. The pellet is solubilized in 4% CHAPS at 37°C for 3 hours. After being recentrifuged at 50000gx60 minutes, the supernatant is spun through a IOOK centricone and dialyzed against PBS. The binding capacity of the solubilized protein to ⁇ -IFN is confirmed by ELISA binding assay.
  • Samples (0.5 ⁇ L) are mixed with an equal volume of a 5 mg/mL solution of ⁇ - cyanohydroxycinnamic acid in 30% acetonitrile in water with 0.1 % TFA and are then manually spotted onto a 192 spot target plate (Applied Biosystems, Foster City, CA).
  • the plate is inserted into a 4700 MALDI TOF/TOF (Applied Biosystems, Foster City, CA) operated in linear mode. Samples are desorbed by a 200 Hz YAG laser.
  • the acquisition program is set to acquire a summed spectrum (200-1000) shots across the range 5000 to 100000 Thompsons.
  • the protein extract sample is diluted in 50 mM ammonium carbonate buffer, pH 8.5, containing 0.1 % Rapigest SF acid labile detergent (Waters Corp, Millford, MA). The sample is heated to 100°C for 10 minutes to completely denature the proteins. Ten ⁇ L of 10 mM TCEP is added to reduce disulfide bonds and the sample is incubated for 10 minutes at 37°C. The pooled sample is digested with Lys-C (12.5 ng/ ⁇ L) at a mass ratio of 1:100 for 3 hours at 37°C and then digested with trypsin (12.5 ng/ ⁇ L) at a mass ratio of 1 :50 (trypsinrprotein) for 3 hours at 37°C. Digestion is halted by adding PMSF to final concentration of 1 mM. After digestion, 10 ⁇ L of TFA is added to the solution and the sample is incubated for 45 minutes at 37 0 C to destroy the acid labile Rapigest detergent.
  • a digested protein sample is injected (10 ⁇ L) onto an HPLC (Agilent Technologies 1100) containing a Cl 8 trapping column (Agilent Technologies, Santa Clara, CA) containing Zorbax 300SB-C18 (5 x 0.3 mm).
  • HPLC Alkaline
  • Cl 8 trapping column Alkaline
  • Zorbax 300SB-C18 5 x 0.3 mm.
  • the column valve is switched to its secondary position 5 minutes after injection and the trapped peptides are then eluted onto a 75 ⁇ m id Zorbax Stablebond (300 A pore) column and chromatographed using a binary solvent system consisting of A: 0.1% formic acid and 5% acetonitrile and B: 0.1% formic acid and 100% acetonitrile at a flow rate of 300 nL/minute.
  • a gradient is run from 15% B to 55% B over 60 minutes on a reversed-phase column (75 ⁇ m id Zorbax Stablebond (300 A pore), and the eluting peptides are sprayed into a LC/MSD XCT ion trap mass spectrometer system (Agilent Technologies, Santa Clara, CA), equipped with an orthogonal nanospray ESI interface.
  • the mass spectrometer is operated in positive ion mode with the trap set to data dependent auto-MS/MS acquisition mode.
  • Source conditions are: Vcap -4500V, drying gas flow 8 L/minute, drying gas temperature 230°C and CapEx 65V.
  • the instrument is set to complete a mass scan from 400-2200 Thompsons in one second.
  • the instrument's dynamic ion exclusion filter is set to allow the instrument to record up to 2 MS/MS spectra for each detected ion to maximize the acquisition of qualitative data from peptides (by preventing high abundance peptides from dominating the subsequent MS/MS experiments) and the excitation energy is set to "smart frag" mode to insure the generation of useful product ion spectra from all peptides detected. Data files that result are then transferred to a file server for subsequent data reduction.
  • SpectrumMill is derived from the MS-Tag software package and is contemplated as a suitable software platform.
  • Raw data is extracted from the MS data files using the data extractor module and the data is then subjected to protein library search and de Novo spectral interpretation by the Sherenga module.
  • SpectrumMill is designed to minimize spurious identifications obtained from the MS/MS spectra of peptides by careful filtering and grouping of related MS and MS/MS data during extraction from the raw data file.
  • the library searching and de Novo interpretation identify the detected proteins form the individual peptides.
  • the results for all proteins detected are collected and listed by protein name, detected peptide sequence(s), and search score.
  • the reports are exported to an Excel spreadsheet file for inclusion in a result database.
  • data extracted from the raw data files from the ion trap are preferably submitted to the Mascot (MatrixScience Lie, London, UK) search program and searched against both the NCBI non-redundant protein database and the SWISSPROT protein database. The identifications from these two systems are correlated to arrive at a final consensus list of identified proteins.
  • Fractions are pH adjusted to 3.5, and run across a Sep-Pak C 18 column on a HPLC system (Millipore corp., Milford, MA). The columns are washed with ddH 2 O, and compounds are eluted with increasingly polar mobile phases (hexane-methyl formate- methanol). Fractions are concentrated under a stream of nitrogen and reconstituted in either 1 ml PBS (for PA-I reporter assay) or 100 ul of methanol (for UWHPLC).
  • Active fractions from Sep-Pak are preferably further resolved by a C 18 reversed-phase HPLC column (150 mm x 5 mm, Phenomenex, Torrance, CA) with acidified (0.1% acetic acid) MetOH:H 2 O (60:40 vol/vol) at 1 ml/minute on a 1050 series HPLC using ChemStationTM software (Hewlett Packard, Palo Alto, CA).
  • Example 23 The separate effects of both tertiary and peripheral ⁇ -opioid receptor antagonists on morphine-induced PA-I lectin/adhesin expression in Pseudomonas aeruginosa were investigated.
  • the P. aeruginosa strain used for the study was the PA-I lectin/adhesin reported strain 27853/PLL-EGFP, described above.
  • PA-I lectin/adhesin assays were performed as described herein except where specifically indicated.
  • the reporter strain was incubated in wells of a 96-well plate, and fluorescence and cell density were measured using conventional techniques. Results presented in Fig. 7 represent fluorescence data normalized to cell densities after 20 hours of incubation.
  • Bars represent median of twelve values ⁇ stdv.
  • Apparent from Fig. 7 is the effect of 20 ⁇ M morphine on PA-IL expression, as well as the separate inhibitory effects of each of 20 ⁇ M methylnaltrexone and 20 ⁇ M naloxone on the morphine-induced expression of PA-I lectin/adhesin.
  • these opioid-induced increases in PA-I lectin/adhesin are significantly attenuated by either of the ⁇ -opioid receptor antagonists, naloxone or methylnaltrexone.
  • the effects on opiate-mediated virulence may be mediated through classical mu opioid receptors or in subtypes of opioid receptors or splice variants. Without wishing to be bound by theory, this effect may be mediated by MAPK/ERR phosphorylation similar to or related to VEGF.
  • tertiary ⁇ -opioid receptor antagonists e.g., naloxone
  • peripheral ⁇ -opioid receptor antagonists e.g., methylnaltrexone
  • the aim of the study described in this Example was to determine whether intestinal epithelial hypoxia, a common response to surgical stress, could activate PA-I expression. Because splanchnic vasosconstriction and intestinal epithelial hypoxia are a common consequence of surgical injury, the aim of the experiments described herein was to determine the specific role of the intestinal epithelium in signaling to P. aeruginosa by examining the effect of epithelial cell hypoxia and reoxygenation on PA-I expression. A fusion construct was generated to express green fluorescent protein downstream of the PA-I gene, serving as a stable reporter strain for PA-I expression in P. aeruginosa, as described herein.
  • Polarized Caco-2 monolayers were exposed to ambient hypoxia (0.1—0.3% O 2 ) for 1 hour, with or without a recovery period of normoxia (21% O 2 ) for 2 hours, and then inoculated with P. ae?-uginosa containing the PA-I reporter construct.
  • Hypoxic Caco-2 monolayers caused a significant increase in PA-I promoter activity relative to normoxic monolayers (165% at 1 h; P ⁇ 0.001).
  • Similar activation of PA-I was also induced by cell- free apical, but not basal, media from hypoxic Caco-2 monolayers.
  • PA-I promoter activation was preferentially enhanced in bacterial cells that physically interacted with hypoxic epithelia. As shown below, the virulence circuitry of P. aeruginosa is activated by both soluble and contact-mediated elements of the intestinal epithelium during hypoxia and normoxic recovery.
  • Caco-2 ⁇ B e cells expressing SGLTl were maintained in DMEM with 25 mM glucose (high-glucose DMEM) with 10% fetal calf serum, 15 mM HEPES, pH 7.4, and 0.25 mg/ml geneticin, as previously described (Turner JR et al., Am J Physiol 273: C1378-1385, 1997).
  • Caco-2 cells were plated on 0.33-cm 2 collagen-coated, 0.4- ⁇ m pore size polycarbonate membrane Transwell supports (Corning-Costar, Acton, MA) for 20 days, and media were replaced with identical media without geneticin at least 24 h before bacterial inoculation.
  • GFP fusion constructs of wild-type P. aeruginosa are wild-type P. aeruginosa.
  • P. aeruginosa (ATCC-27853, American Type Culture Collection, Manassas, VA) was transformed with the plasmid pUCP24/PLL-EGFP.
  • This construct harbors a PA27853 chromosomal DNA fragment containing an upstream regulatory region of PA-I followed by the entire PA-I gene fused at the COOH terminal with an enhanced green fluorescent protein (EGFP) gene excised from the pBI-EGFP plasmid (Clontech, Palo Alto, CA).
  • EGFP enhanced green fluorescent protein
  • Caco-2 cells were grown to confluence on collagen-coated 96-well fluorimetry plates (Becton Dickinson Labware, Bedford, MA) and maintained in a 37°C incubator with 5% CO 2 and 21% O 2 . The day before experiments, media were removed and replaced with 150 ⁇ l of antibiotic-free media. Three experimental conditions were created using a modification of the methods previously described by Xu et al. J Trauma 46:280-285, 1999). In control conditions, Caco-2 cells were maintained in a 5% CO2-21% 02 incubator for 2 h. Hypoxic conditions were achieved by placing Caco-2 cells in a humidified hypoxia chamber at 37 0 C with 5% CO-95% N2 for 2 h. Measured 02 in the chambers varied between 0.1 and 0.3%.
  • TER monolayer transepithelial electrical resistance
  • PA-I expression was confirmed using Northern blot analyses.
  • GFP reporter strain PA27853/PLL-EGFP would display increased PA-I promoter activity when added to Caco-2 cells exposed to hypoxia (2 h at ⁇ 0.3% 02) and normoxic recovery (hypoxia followed by 2 h of recovery in normoxic conditions)
  • reporter strains were added to the media of Caco-2 cells exposed to the two conditions.
  • GFP reporter strains demonstrated significantly more PA-I promoter activity, as measured by fluorescence, within 1 h of incubation with Caco-2 cells exposed to either hypoxia or normoxic recovery.
  • the media pH in all experimental conditions was measured at all time points and demonstrated no significant difference among control, hypoxia, and normoxic recovery groups because all media were buffered (data not shown).
  • Caco-2 cells were imaged by fluorescent microscopy following exposure to hypoxia and apical inoculation with PA27853/PLL-EGFP. Fluorescence imaging demonstrated that PA27853/PLL-EGFP exposed to hypoxic Caco-2 monolayers appeared markedly more fluorescent than bacteria exposed to normoxic monolayers at the 120-min time point. Multiple images of the bacterial/Caco-2 cell coculture demonstrated that more bacteria were located near or within the plane of the cell monolayers exposed to hypoxia than in nonhypoxic cells.
  • TER was measured in Caco-2 cells apically inoculated with either PA27853 or purified PA-I following exposure to hypoxia and normoxic recovery.
  • the TER of Caco-2 cells exposed to these conditions were unchanged in response to a P. aeruginosa inoculated with purified PA-I exhibited an attenuated drop in TER compared with normoxic cells.
  • Soluble factors present in the media of hypoxic Caco-2 cells induce increased barrier resistance in normoxic cells.
  • normoxic Caco-2 cells were induced to increase their resistance to barrier dysregulation by P. aeruginosa through signals present in hypoxic cell media
  • Normoxic Caco-2 cells exposed to media from hypoxicepithelia displayed a prolonged resistance to barrier dysregulation induced by P. aeruginosa, suggesting that normoxic epithelia may be activated to enhance their barrier function in the presence of soluble mediators produced during hypoxia.
  • P. aeruginosa is not considered to be an intestinal pathogen in the classic sense, it induces one of the most rapid and profound decreases in intestinal epithelial TER of any bacteria reported to date.
  • P. aeruginosa PA27853
  • P. aeruginosa PA27853
  • P. aeruginosa is among the most pathogenic organisms to the intestinal epithelium yet described. The observation that as many as 5% of the normal population harbor this pathogen within their intestinal tracts, coupled with our animal studies demonstrating that control mice do not develop any symptoms of infection following the direct introduction of large quantities of P.
  • aeruginosa into the cecum suggest that this organism behaves like a classic opportunist, switching virulence genes on and off in response to selected environmental cues.
  • environmental cues such as pH, redox state, and nutrient composition can activate virulence gene expression in bacteria through a variety of membrane- bound biosensor kinases, there are no previous reports suggesting that bacterial signaling compounds are released by host cells following physiological or ischemic stress.
  • a pathogen might recognize the biochemistry of host cell stress, because possessing a system that recognizes host susceptibility would allow for a more accurate assessment of the costs versus benefits of host invasion.
  • intestinal pathogens can communicate directly with the cells to which they adhere, that such a molecular dialogue might be bidirectional is poorly described.
  • the PA-I lectin is under tight regulatory control of two key systems of virulence gene regulation in P. aeruginosa: the quorum-sensing signaling system and the alternative sigma factor RpoS.
  • the quorum-sensing signaling system and RpoS are interconnected systems of virulence gene regulation in P. aeruginosa that control the expression of hundreds of virulence genes in this pathogen.
  • PA-I expression is dependent on the function of both quorum sensing and RpoS, it serves as a relevant biological readout for generalized virulence gene activation in P. aeruginosa.
  • the finding that soluble elements of intestinal epithelial cells and, in particular, adenosine can activate PA-I expression suggests that specific host cell-derived compounds may be released that signal colonizing pathogens such as P. aeruginosa to a weak and susceptible host. That adenosine alone can activate PA-I expression is an important finding given that adenosine is released and can accumulate in the extracellular milieu of hypoxic tissues at high concentrations.
  • 5'-AMP derived from migrating polymorphonuclear leukocytes is converted to adenosine by the apical surface epithelium of the intestine. Strohmeier et al. (14) have demonstrated that under normal conditions, the human intestinal epithelial cell line T-84 can convert substantial amounts of 5'-AMP that accumulate to as much as 5 mM adenosine in the apical media within 30 min.
  • activation of PA-I promoter activity in P. aeruginosa required what appeared to be an unphysiological dose of adenosine, the precise concentration of adenosine to which P.
  • aeruginosa might be exposed within the intestinal tract during prolonged hypoxia and reoxygenation is unknown. In addition, adenosine exposure required 6 h before PA-I promoter activity was observed, whereas with hypoxic media PA-I promoter activity was observed at 4 h.
  • an opportunistic organism like P. aeruginosa may require an inordinately potent and prolonged host-derived signal for it to invest the resources and energy required to mount a toxic offensive against the intestinal epithelium. Under such circumstances, P. aeruginosa might "sense" that the host on which its survival depends is subjected to an extreme degree of inflammation and vulnerability and hence represents a liability to its survival.
  • HIF-I- ⁇ activation mediates the release of soluble compounds that activate P. aeruginosa virulence as judged by expression of the PA-I lectin/adhesin.
  • the media from three groups of Caco-2 cells were examined, namely, control cells, Caco2 cells exposed to hypoxia, and Caco2 cells with forced expression of HIF-I ⁇ .
  • Media fractions were separated into 4 molecular weight fractions which were added to the microtiter plates containing the PA-I/GFP reporter strains and evaluated by dynamic fluorimetry.
  • hypoxia or Forced expression of HIF-I - ⁇ in Caco-2 cells results in the extracellular release of soluble compounds that activate the virulence circuitry of P. aeruginosa.
  • the data presented herein show that adenosine and inosine may play an important role in this response.
  • This Example provides data establishing that a mu opioid receptor antagonist in the form of MNTX inhibits opiate-, thrombin- and LPS-induced endothelial cell barrier disruption by mu opioid receptor (mOP-R)-dependent, and -independent, mechanisms.
  • the mOP-R-independent mechanisms of MNTX-induced endothelial cell barrier regulation include activation of receptor-like protein tyrosine phosphatase mu (RPTP ⁇ ) and inhibition of thrombin- and LPS-induced, Src-dependent, SlP 3 receptor transactivation (tyrosine phosphorylation).
  • RPTP ⁇ receptor-like protein tyrosine phosphatase mu
  • tyrosine phosphorylation tyrosine phosphorylation
  • Reagents for SDS-PAGE electrophoresis were purchased from Bio-Rad (Richmond, CA), Immobilon-P transfer membranes were from Millipore (Millipore Corp., Bedford, MA), and gold microelectrodes were from Applied Biophysics (Troy, NY).
  • Rabbit anti-mu opioid receptor antibody was purchased from Abeam (Cambridge, MA).
  • Rabbit anti- Si P 1 receptor antibody was purchased from Affinity Bioreagents (Golden, CO).
  • Mouse anti- SlP 3 receptor antibody was purchased from Exalpha Biologicals (Watertown, MA).
  • Mouse anti-RPTP ⁇ antibody was purchased from Cell Signaling Technologies (Danvers, MA).
  • Mouse anti-phospho-tyrosine antibody, mouse anti-pp60src antibody and recombinant active Src were purchased from Upstate Biotechnologies (Lake Placid, NY). PP2 was purchased from Calbiochem (San Diego, CA). Mouse anti- ⁇ -actin antibody, rabbit anti-phospho- tyrosine (418) Src antibody, naloxone, DAMGO, thrombin, LPS and ionomycin were purchased from Sigma (St. Louis, MO). Secondary horseradish peroxidase (HRP)-labeled antibodies were purchased from Amersham Biosciences (Piscataway, NJ).
  • HRP horseradish peroxidase
  • IP buffer 50 niM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl 2 , 1% Nonidet P-40 (NP-40), 0.4 mM Na 3 VO 4 , 40 mM NaF, 50 ⁇ M okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, 1 :250 dilution of Calbiochem protease inhibitor mixture 3).
  • IP buffer 50 niM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl 2 , 1% Nonidet P-40 (NP-40), 0.4 mM Na 3 VO 4 , 40 mM NaF, 50 ⁇ M okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, 1 :250 dilution of Calbiochem protease inhibitor mixture 3).
  • siRNA sequence(s) targeting human mOP-R, SlP 1 , SlP 3 , RPTP ⁇ were generated using mRNA sequences from Gen-BankTM (gi:56549104, gi:87196352, gi:38788192, and gi:18860903, respectively). For each mRNA (or scramble), two targets were identified. Specifically, mOP-R target sequence 1 (5'-
  • mOP-R target sequence 2 (5'- AATGTCAGATGCTCAGCTCGG-3'; SEQ ID NO:15), SlPi target sequence 1 (5'- AAGCTACACAAAAAGCCTGGA-S 1 ; SEQ ID NO:16), SlP 1 target sequence 2 (5 T - AAAAAGCCTGGATCACTCATC-3'; SEQ ID NO: 17), SlP 3 target sequence 1 (5 1 - AACAGGGACTCAGGGACCAGA-3'; SEQ ID NO: 18), SlP 3 target sequence 2 (5 1 - AAATGAATGTTCCTGGGGCGC-3'; SEQ ID NO: 19), RPTP ⁇ target sequence 1 (5'- AATCTGAAGGTGATGACTTCA-S '; SEQ ID NO:20), RPTP ⁇ target sequence 2 (5'- AACACCTTGACTAAACCGACT-3'; SEQ ID NO:21), scrambled sequence 1 (5'- AAGAGAG
  • Sense and antisense oligonucleotides were provided by the Johns Hopkins University DNA Analysis Facility or were purchased from Integrated DNA Technologies (Coralville, IA).
  • siRNA a transcription-based kit from Ambion was used (SilencerTM siRNA construction kit).
  • Human lung endothelial cells were then transfected with siRNA using siPORTamineTM as the transfection reagent (Ambion, TX) according to the protocol provided by Ambion.
  • Cells (about 40% confluent) were serum-starved for 1 hour followed by incubated with 3 ⁇ M (1.5 ⁇ M of each siRNA) of target siRNA (or scramble siRNA or no siRNA) for 6 hours in serum- free medium.
  • the serum-containing medium was then added (1% serum final concentration) for 42 hours before biochemical experiments and/or functional assays were conducted.
  • Tyrosine Phosphatase Activity Assay Treated or untreated HPAEC lysates and/or immunoprecipitated RPTP ⁇ were analyzed for tyrosine phosphatase activity using the fluorometric RediplateTM 96 EnzChek Tyrosine Phosphatase Assay Kit (Invitrogen (Molecular Probes), Eugene, OR). Briefly, cellular materials were incubated in reaction buffer at 3O 0 C and then added to a 96-well plate coated with 6,8-difluoro-4- methylumbelliferyl phosphate (DiFMUP). Tyrosine phosphatase activity cleaves DiFMUP into DiFMU with excitation/emission maxima of 358/452 nm.
  • DiFMUP 6,8-difluoro-4- methylumbelliferyl phosphate
  • SlP 3 Receptor Phosphorylation/Dephosphorylation The SlP 3 receptor phosphorylation/dephosphorylation reaction was carried out in 50 ⁇ l of the reaction mixture containing 40 mM Tris-HCl (pH 7.5), 2 niM EDTA, 1 mM dithiothreitol, 7 mM MgCl 2 , 0.1% CHAPS, 100 ⁇ M ATP, purified enzymes ⁇ i.e.
  • Cell barrier properties were measured using a highly sensitive biophysical assay with an electrical cell- substrate impedance sensing system (Applied Biophysics Inc., Troy, NY), as described previously in Garcia et al., Am. J. Physiol. 273 :LI 72-Ll 84 (1997); J. Appl. Physiol. 89:2333- 2343 (2000); J. Clin. Invest. 108:689-701 (2000).
  • the cells were cultured to confluence in polycarbonate wells containing evaporated small gold microelectrodes (10 '4 cm 2 ) and culture medium was used as electrolyte.
  • the total electrical resistance was measured dynamically across the monolayer and was determined by the combined resistance between the basal surface of the cell and the electrode, reflective of focal adhesion, and the resistance between the cells. As cells adhered and spread out on the microelectrode, TER increased (maximal at confluence), whereas cell retraction, rounding, or loss of adhesion was reflected by a decrease in TER.
  • the small gold electrode and the larger counter electrodes (1 cm 2 ) were connected to a phase-sensitive Ion-in amplifier with a built-in differential preamplifier (Applied Biophysics). A I- V, 4000-Hz alternating current signal was supplied through a MQ resistor to approximate a constant-current source. Voltage and phase data were stored and computer processed using conventional techniques.
  • mice Male C57BL/6J mice (8-10 weeks, Jackson Laboratories, Bar Harbor, ME) were anesthetized with intraperitoneal ketamine (150 mg/kg) and acetylpromazine (15 mg/kg) before exposure of the right internal jugular vein via neck incision. LPS (2.5 mg/kg) or water (control) were instilled intravenously through the internal jugular vein. Four hours later, mice received methylnaltrexone (MNTX, 10 mg/kg) or water control through the internal jugular vein. The animals were allowed to recover for 24 hours after LPS before bronchoalveolar lavage protein analysis and/or lung immunohistochemistry.
  • MNTX methylnaltrexone
  • the sections were then histologically evaluated by either anti-mu opioid receptor, anti-RPTP ⁇ or anti-SlP 3 receptor antibody and secondary HRP-labeled polymer with DAB staining (Dako En VisionTM + System, HRP (DAB) (DakoCytomation, Carpinteria, CA)), followed by hematoxylin QS counterstaining (Vector Laboratories, Burlingame, CA). Negative controls for immunohistocheniical analysis were done by the same method as above but without primary antibody. Immunostained sections were photographed (10Ox) using a Leica Axioscope (Bannockbura, IL).
  • Bronchoalveolar lavage was performed by an intratracheal injection of 1 cc of Hank's balanced salt solution followed by gentle aspiration. The recovered fluid was processed for protein concentration (BCA Protein Assay Kit; Pierce Chemical Co., Rockford, IL).
  • methylnaltrexone (MNTX) in agonist-induced endothelial cell barrier disruption. Endothelial cell barrier disruption is a causative factor in a variety of pathologies, including atherosclerosis and acute lung injury.
  • MNTX methylnaltrexone
  • mOP-R charged peripheral mu opioid receptor
  • TER transendothelial resistance
  • Figure 9-A,B indicate that ligands for the mOP-R (i.e., morphine sulfate (MS) and DAMGO) induced endothelial cell barrier disruption in a dose-dependent manner.
  • Methylnaltrexone is a charged molecule that cannot cross the blood-brain barrier (BBB). This property allows MNTX to selectively block peripheral mOP-R activity.
  • BBB blood-brain barrier
  • naloxone another mOP-R antagonist
  • naloxone did not display the same endothelial cell barrier-protective effects as MNTX with thrombin and LPS challenge ( Figure 11).
  • silencing (siRNA) mOP-R or SlP receptor subtypes were investigated ( Figures 12 and 18). Silencing mOP-R expression had little effect on MNTX protection from thrombin- and LPS-induced endothelial cell barrier disruption indicating potential mOP-R-independent effects of MNTX.
  • Endothelial cells express both SlP 1 and SlP 3 receptors with SlP 1 receptor activating Racl -mediated signaling, while SlP 3 receptor activates RhoA-mediated signaling.
  • the silencing Of SlP 1 receptor had previously been shown to completely eliminate the barrier-protective effects of SlP (1 ⁇ M). At higher concentrations (10 to 30 ⁇ M), SlP- induced barrier disruption is likely due to SlP 3 receptor activation. In contrast to SlP 1 receptor, silencing SlP 3 receptor inhibited thrombin- and LPS-induced, and MNTX protection from, endothelial cell barrier disruption (Figure 12-B,C; Figure 18).
  • SlPi receptor transactivation is important in agonist-induced endothelial cell barrier enhancement.
  • SlP 3 receptor transactivation would be an important regulatory mechanism in endothelial cell barrier disruption.
  • Figure 13 provides data indicating that barrier disrupting, but not barrier enhancing (i.e. SlP at 1 ⁇ M), agents promoted Src activation and Src family kinase- mediated SlP 3 receptor transactivation (tyrosine phosphorylation). Further, inhibition of Src family kinases by PP2 blocked agonist-induced barrier disruption but did not affect S IP- mediated endothelial cell barrier enhancement.
  • RPTP ⁇ receptor tyrosine phosphatase mu
  • silencing RPTP ⁇ (but not mOP-R or SlP 3 receptor) protein expression significantly attenuated the MNTX- mediated increase of total endothelial cell tyrosine phosphatase activity ( Figure 16-A).
  • silencing RPTP ⁇ inhibited the protective effects of MNTX of, and enhanced the thrombin- and LPS-induced effects on, endothelial cell barrier disruption ( Figure 16-B,C).
  • MNTX methylnaltrexone
  • mOP-R selective peripheral mu opioid receptor
  • SlP 3 receptor transactivation is an important regulator of agonist-induced endothelial cell barrier disruption.
  • MNTX stimulated mOP-R-independent receptor tyrosine phosphate mu (RPTP ⁇ ) activity, which is important in inhibiting agonist-induced SlP 3 receptor transactivation (Src-mediated tyrosine phosphorylation).
  • RPTP ⁇ mOP-R-independent receptor tyrosine phosphate mu
  • Src-mediated tyrosine phosphorylation agonist-induced SlP 3 receptor transactivation
  • MNTX is a quaternary derivative of the pure narcotic antagonist naltrexone. The addition of the methyl group to naltrexone at the amine in the ring forms the compound N-methylnaltrexone with greater polarity and lower lipid solubility.
  • MNTX does not cross the blood-brain barrier and thus could play a therapeutic role in reversing the peripheral effects of opiates in palliative care, especially for patients taking high doses of opiates for analgesia.
  • MNTX is expected to have a clinical role in the perioperative period, in the ICU (e.g., patients with burns), or with advanced medical illness. Because this population is most at risk for defects in cell barrier function, particularly pulmonary dysfunction, these work disclosed herein focused on MNTX rather than the tertiary opiate antagonists.
  • Peak plasma concentrations of intravenous or intramuscular morphine in normal therapeutic doses are 80 ng/ml.
  • analgesia in cancer patients was associated with steady-state concentrations of morphine in plasma ranging from 6 to 364 ng/ml.
  • a metaanalysis of dose-adjusted peak plasma concentrations of morphine revealed a C ma ⁇ of 1-10 nM/L per mg of morphine, although there were some differences between single- and multiple-dosing and populations.
  • the plasma concentration of morphine and MNTX in patients after parenteral or oral administration is consistent with the levels that regulated endothelial cell barrier function in the in vitro model.
  • concentrations of MNTX in the in vitro study were similar to those achieved in clinical trials of the drug.
  • methadone maintenance patients who received mean doses of 0.1 mg/kg MNTX intravenously the mean plasma levels of MNTX were 162 ng/ml. After repeated IV doses of MNTX in volunteers, levels of MNTX in plasma were maintained well above the range in which we observed an effect on endothelial cell barrier function.
  • MNTX but not naloxone, provided protection from both thrombin- and LPS- induced endothelial cell barrier disruption.
  • Thrombin induced rapid, transient endothelial cell barrier disruption through activation of PAR (Protease- Activated Receptors), with consequent Ca 2+ , RhoA and Ras/MAP kinase signaling, hi contrast, LPS induced a delayed endothelial cell barrier-disruptive response by activating a receptor complex of TLR4, CD 14 and MD2, with consequent NF- ⁇ B activation and cytokine production.
  • PAR Protease- Activated Receptors
  • MNTX is expected to provide cell barrier protection (including endothelial cell barrier protection) from a wide range of disrupting agents.
  • SlP 1 receptor transactivation is a key component in agonist-induced endothelial cell barrier enhancement.
  • these findings have been extended to show that transactivation (Src- mediated tyrosine phosphorylation) of the SlP 3 receptor played an important role in agonist- induced endothelial cell barrier disruption.
  • SlP 3 receptor signaling activated the small G- protein, RhoA, which is involved in actin cytoskeletal reorganization.
  • RPTP ⁇ was established herein as playing an important role in regulating endothelial cell barrier integrity. RPTP ⁇ is highly expressed in the lung vasculature, where it is localized to endothelial cell-cell junctions. Consistent with the results disclosed herein, researchers have shown that silencing RPTP ⁇ expression in HPMVEC inhibited barrier function. RPTP ⁇ can associate with various cell surface receptors, including VE-cadherin, N-cadherin, c-Met and the VEGF receptor. These findings were extended to show that RPTP ⁇ regulated Sl P 3 receptor transactivation. RPTP ⁇ further interacted with signaling molecules including IQGAPl, cdc42, RACKl, ⁇ -catenin, ⁇ -catenin and PKC ⁇ .
  • signaling molecules including IQGAPl, cdc42, RACKl, ⁇ -catenin, ⁇ -catenin and PKC ⁇ .
  • MTNX is expected to be a useful therapeutic treatment (including preventative and ameliorative treatments) for diseases involving cell barrier disruption or dysfunction, such as endothelial cell barrier dysfunction.

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Abstract

L'invention concerne des procédés prophylactiques et thérapeutiques d'administration d'un antagoniste des récepteurs des µ-opioïdes pour traiter les maladies et les affections de la barrière cellulaire, par exemple les maladies et les affections de la barrière des cellules endothéliales et des cellules épithéliales. Les maladies ou affections concernés par les procédés sont l'inflammation, par exemple les lésions aiguës des poumons, l'athérosclérose, la septicémie d'origine viscérale, les lésions par brûlure, l'entérocolite nécrotique néonatale, la neutropénie grave, la colite toxique, les maladies inflammatoires des viscères, l'entéropathie, le rejet des transplants, la pouchite, l'entérite nécrotique, les infections ophtalmiques médiées par Pseudomonas, les infections otologiques médiées par Pseudomonas et les infections cutanées médiées par Pseudomonas. De manière plus générale, les procédés prophylactiques et thérapeutiques concernés peuvent être envisagés pour traiter les affections de la barrière des cellules épithéliales. Les maladies et les affections peuvent être induits par des pathogènes microbiens, notamment des pathogènes bactériens tels que Pseudomonas aeruginosa. L'invention concerne en outre des procédés prophylactiques et thérapeutiques qui inhibent l'expression de la lectine/adhésine PA-I bactérienne et d'inhibition des niveaux d'activité des MvfR bactériens. L'invention concerne également un procédé utilisant un antagoniste des récepteurs des µ-opioïdes pour la fabrication d'un médicament destiné à être utilisé dans les procédés qu'elle décrit.
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EP2012790A4 (fr) * 2006-04-19 2009-07-01 Smith Jill P Traitement de maladies inflammatoires et ulcéreuses du côlon à l'aide d'antagonistes opioïdes
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US9879024B2 (en) 2007-03-29 2018-01-30 Progenics Pharmaceuticals., Inc. Crystal forms of (R)-N-methylnaltrexone bromide and uses thereof
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