S. aureus - Cellular and Molecular Pharmacology

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S. aureus - Cellular and Molecular Pharmacology

Maritza Barcia-Macay (Patterson)Unité de Pharmacologie cellulaire et moléculaireActivity of antibiotics againstextracellular and intracellular formsof Staphylococcus aureusPharmacodynamic studies in vitrousing a modelof human THP-1 macrophages


I. INTRODUCTION


Staphylococcus aureusBacteria with thegreatestpathogenic potentialin humaninfections.A major causeof nosocomialandcommunity-acquiredinfections.


S. aureus infections1. skin and soft tissue infectionsimpetigoerysipelascellulitisfuruncleabscess


2. food-poisoningS. aureus infections


S. aureus infections3. Deep-organ infectionsendocarditispneumoniaosteomyelitis


S. aureus infectionsAll these infections are difficult to treat :• recurrence / persistencein relation with the intracellular character of S. aureus selection of antibiotics based onpharmacokinetic / pharmacodynamics properties• resistance to currently available antibiotics need of drugs acting on multiresistant strains


S. aureus infectionsAll these infections are difficult to treat :• recurrence / persistencein relation with the intracellular character of S. aureus selection of antibiotics based onpharmacokinetic / pharmacodynamics properties• resistance to currently available antibiotics need of drugs acting on multiresistant strains


Intracellular infection by S. aureusNormal process of phagocytosis1) attachment tophagocytemembrane2) ingestion(phagosome)3) fusion lysosomeand phagosome4) bacterialdestruction5) digestion and releaseof microbial debris


Intracellular infection by S. aureusIncomplete phagocytosis of S. aureus1) attachment tophagocytemembrane2) ingestion(phagosome)3) fusion lysosomeand phagosomeX4) bacterialdestructionX5) digestion and releaseof microbial debris


Intracellular infection by S. aureusBacteria able to survive in different host cells :phagocytes and non-phagocytesNeutrophilsMacrophagesMammary epithelial cellsEnterocytesKeratinocytesOsteoblastsFibroblastsEndothelial cellsBrouillette et al, Vet Microbiol (2004) 101:253-262; Microb Pathog. (2003) 35:159-68.


S. aureus infectionsAll these infections are difficult to treat :• recurrence / persistencein relation with the intracellular character of S. aureus selection of antibiotics based onpharmacokinetic / pharmacodynamics properties• resistance to currently available antibiotics need of drugs acting on multiresistant strains


Antibioticpharmacokinetics / pharmacodynamicsgeneral conceptDosageregimenConcentrationversus timein serumabsorptiondistributioneliminationConcentrationversus timein tissues andother body fluidsConcentrationversus timeat siteof infectionPharmacologicor toxicologiceffectAntimicrobialeffect versustimePHARMACOKINETICSPHARMACODYNAMICSCraig CID (1998) 26:1-10


Antibioticpharmacokinetics / pharmacodynamicsintracellularlyPHARMACOKINETICSCarryn et al, Infect Dis Clin North Am. (2003) 17:615-34PHARMACODYNAMICS


S. aureus infectionsAll these infections are difficult to treat :• recurrence / persistencein relation with the intracellular character of S. aureus selection of antibiotics based onpharmacokinetic / pharmacodynamics properties• resistance to currently available antibiotics need of drugs acting on multiresistant strains


S. aureus resistanceThe world first commercially available antibiotic appeared in 19411941 1960 YEAR OF REPORTED RESISTANCE 2000Introduction ofPenicillinAminoglycosides, 1950OxazolidinonesMacrolides, 1952Glycopeptides, 1956Methicillin, 1960Quinolones,Streptogramins, 1962


S. aureus resistanceMost worrying resistance phenotypes having emerged over timeyear phenotype first description1960 HA-MRSA England1967 MDR HA-MRSA Europe, Australia, Japan1980 Genta-R MRSA USA, Ireland, UK1993 CA-MRSA Australia1997 VISA Japan2002 VRSA USAGrundmann et al., Lancet (2006) 368:874-85


S. aureus resistancePercentage of MRSA resistance in Europe in 2004:S. aureus proportion of invasive isolates MRSA in 2004Data from the European antimicrobial resistant surveillance system, EARSS.


S. aureus resistancePrevalence of resistance to other antibiotic classesMSSAMRSA10080604020% resistanceamoxiamoxi-clavglycopeptidesmacrolideslincosamidessynergistinsaminoglycosidestetracyclinesrifampicinfluoroquinolones0Fluit et al., J Clin Microbiol (2001) 39: 3727-3732


S. aureus infectionsAll these infections are difficult to treat :• recurrence / persistencein relation with the intracellular character of S. aureus selection of antibiotics based onpharmacokinetic / pharmacodynamics properties• resistance to currently available antibiotics need of drugs acting on multiresistant strains


Need for new antistaphylococcal agentsA lot of drugs in the pipeline …recent and novel agents for S. aureusrecentlybrought on theBelgian marketon the market;not yetin Belgium(late) stage ofclinicaldevelopmentinvestigationalmoxifloxacinlinezolidsynerciddaptomycintigecyclinetelavancinoritavancindalbavancinCS-023/PZ-601MX-2401API-1252ceftobiproleDK-619iclaprimretapamulinWCK-771new oxazolidinonesnew ketolides...What will be your choice ?Sympo – S. aureus – 11/01/07 - 8F. Van Bambeke, symposium on S. aureus – Brussels, 11-1-2007


II. AIM OF THE STUDY


• recurrence / persistencein relation with the intracellular character of S. aureus selection of antibiotics based onpharmacokinetic / pharmacodynamics propertiesTo develop an intracellular model allowing to comparethe activity of antibiotics on a pharmacodynamic basis• resistance to currently available antibiotics need of drugs acting on multiresistant strainsTo evaluate the cellular pharmacokineticsand the intracellular activity towards multiresistant strainsof a new antibiotic in development


III. METHODOLOGY


Setting-up of the intracellular modelMethod1) opsonization of S. aureus with human serum2) phagocytosis of the bacteria by THP-1 macrophages(ratio 4 bacteria vs 1 macrophage)3) elimination of extracellular S. aureus(gentamicin 100 X MIC).Rinse of infected macrophages (time-zero)4) intracellularly infected macrophages,ready to test antibiotic activity5) maintenance of gentamicin at its MICduring the whole incubation period for controlsto avoid extracellular contamination


Setting-up of the intracellular modelcell line ?THP-1= many features of human monocytes/macrophagesparameterTsuchiya, Int. J. Cancer (1980) 26:171-176; Auwerx, Experientia (1991) 47:22-31morphological featurescytochemical featuressurface antigens andreceptorssecreted proteinsfunctional featurescharacteristicsmorphology resembling that of monocytic leukemia cells:- diameter 12-14 µm- moderate basophile cytoplasm- small azurophiles granules, few vacuoles- nuclei irregular in shape- positive for α-naphthyl butyrate esterase- negative reaction with periodic acid-Schiff and Sudan black B- diploid (46,XY) chromosome numberCD4, CD30, Factor X receptor, Factor Xa receptor, FcRI, FcRII,GM-CSF-receptor, HDL receptor, LDL receptor, TNF receptor,C3b receptor, LFA-1 receptor, Fibronectin receptor, Leu M1, LeuM2, Leu M3, HLA-DR antigens, scavengers receptors- hormones, cytokines: TNF-α, IL-1, IL-1b, CSF-1, M-CSF,erythrocyte differentiation factor, PDGF-1 and –2, thymosinB4, killer T cell activating factor, monocyte chemotactic factor- enzymes: lipoprotein lipase, lyzozyme- binding proteins: apoprotein E- phagocytosis- production of lyzozyme- capacity to restore the lympocyte T mitogenic responsiveness


Setting-up of the intracellular modelbacterial strain ?ATCC 25923• clinical isolate from 1976• fully susceptible• widely used as a standard for microbiological testing ofantibioticsuseful to compare all antibiotics towards a single strain,but may differ in virulence with current strains …


Setting-up of the intracellular modelantibiotics ?• Beta-lactams: first choice for susceptible strains• Aminoglycosides: highly bactericidal extracellularly• Rifampicin: considered as first choice for intracellularinfections• Macrolides, quinolones: high cellular accumulation• Glycopeptides: alternative for MRSA• Linezolid: recently introduced, active on MRSA


S. aureus intracellular modelgentamicinpreventsextracellularcontaminationBacteria multiply in phagolysosomeswhere pH is acidicmembrane bond vacuole


General protocolAssessment of antibiotic extracellular and intracellularactivityEXTRACELLULARINTRACELLULAR4 bacteria/cell10 6 CFU/ml10 6 CFU/mg proteinIncubation over 24 h with antibioticsColony counting(CFU/ml)Collectionand lysis of cells(sonication)CFU/protein contentAntibiotic concentration(microbiological, radiochemical,fluorimetric assays)


IV. RESULTS


First goal of this thesisHuman macrophagesFully susceptible strain


Antibiotics accumulate to variablelevels in THP-1 macrophagesHigh level:macrolidesoritavancinrifampinmoderate level:aminoglycosideold glycopeptidesquinoloneslow level:linezolidβ-lactams


influence of time on activitylow to moderateaccumulationmoderate to highaccumulationGEN, RIF, ORIquickly cidalOXA, MXF, ORIslowly cidal• in all cases, intracellular activity


concentration-effect relationships• sigmoidal relationships


concentration-effect relationshipsEmaxEmaxEmaxEmax• sigmoidal relationships• Emax intra


concentration-effect relationshipsEC50EC50EC50EC50• sigmoidal relationships• Emax intra


intracellular killing is visible !controlOXAORI


extracellular versus intracellular activitygrowthkilling-5intracellularΔ log CFU from time 0-4-3-2-100AZM-1LNZTEL-2RIFCIPAMPTEC VANNAF PEN VGEN-3ORIGRN MXFLVX TLVOXA-4-5killinggrowthPoorly activeagainstextra and intraS. aureusextracellularΔ log CFU from time 0


extracellular versus intracellular activitygrowthkilling-5intracellularΔ log CFU from time 0-4-3-2-10AZMLNZTELORIGRN MXFLVX TLVOXARIFCIPAMPTEC VANNAF PEN VGENkillinggrowthActiveagainst extraS. aureus0-1-2-3-4-5extracellularΔ log CFU from time 0


extracellular versus intracellular activitygrowthkillingintracellularΔ log CFU from time 0-5-4-3-2-10AZMLNZTELORIGRN MXFLVX TLVOXARIFCIPAMPTEC VANNAF PEN VGENkillinggrowthBest choicefor activityagainst extraandintra S. aureus0-1-2-3-4-5extracellularΔ log CFU from time 0


conclusionmodel of infection of human macrophagesby S. aureus over 24 h allowing for the study of• influence of time and concentration on antibiotic activity• relation between activity and accumulationintracellular activity


Second goal of this thesisNew drug in developmentDifferent phenotypesof resistance


Telavancin, a new glycopeptideHemi-synthetic derivative of vancomycin,with new mode of action and new pharmacokinetic profile• permeabilization of bacterial membrane• prolonged half-life• dimerization andcooperative bindingto peptidoglycanprecursors• shortening of half-life


MIC and MBC against S. aureuswith different resistance phenotypesMIC and MBC of vancomycin and telavancin against the S. aureus strains used.vancomycintelavancinphenotype strain MIC MBC MIC MBCMSSA ATCC25923 1 1 0.5 0.5ATCC29213 1 1 0.5 0.5MRSA ATCC33591 2 4 0.5 1ATCC43300 2 2 0.5 0.5VISA NRS23 4 4 0.5 0.5NRS52 4 4 0.5 0.5VRSA VRS1 >128 >256 4 8VRS2 16 64 2 8• more active than VAN against VISA and VRSA• bactericidal against all strains


Influence of timeon EXTRACELLULAR activityVAN vs TLV – MSSA ATCC 25923Telavancin is more rapidly cidal than vancomycin


Influence of concentrationon EXTRACELLULAR activityVAN vs TLV – MSSA ATCC 25923• at 3 h, TEL shows bimodal conc-dependent effects• at 24 h, both drugs are bactericidal at high concentrations


EXTRACELLULAR activity of telavancin:comparison of different strainsTLV versus MSSA, MRSA, VISA, VRSAat 3 h, TEL shows bimodal conc-dependent effectstowards MSSA and MRSA


INTRACELLULAR activity of vancomycin andtelavancin towards different strainsVAN and TLV versus MSSA, MRSA, VISA, VRSATEL shows bimodal conc-dependent effects towards MSSA / MRSAVAN is only static intracellulalry


Why bimodal effects for telavancin ?VAN and TLV: inhibitionof peptidoglycan synthesisIn MSSA and MRSA,telavancin can exert multiplemodes of actionΔ log CFU from time 03210-1-2-3-4-5vancomycintelavancin-2 -1 0 1 2 3log concentration (µg/ml)TLV:membrane permeabilizationHiggins et al., AAC (2004) 49:1127-34


Telavancincellular pharmacokinetic datarationalizing its intracellular activity(studies with J774 macrophages)


subcellular distribution of telavancinSame distributionas a lysosomal enzymeHigh concentrationin the compartmentwhere S. aureus sojourns !


conclusionmodel of infection of human macrophagesby S. aureus over 24 h applicable to multiresistant strainsvancomycin• slowly bactericidal extracellularly (MSSA and MRSA)• poorly or not active on VISA and VRSA• static intracellularlytelavancin• bactericidal extra- and intracellularly,including against resistant strains• bimodal effect against MSSA and MRSA couldbe related to multiple modes of action• high accumulation in the infected compartment


V. GENERAL CONCLUSION:can we do better ?


Limitations of the model andperspectives for future workConstant concentrations(pharmacokinetic variations not taken into account):‣ develop dynamic modelsProtein binding(free fraction is active and able to accumulate)‣ develop in vivo modelsPhagocytic cells(S. aureus also infects non phagocytic cellswhere its fate may be different)‣ develop models of infection in non-phagocytic cellsTesting of antibiotics alone(combinations often used in the clinics to cope with resistance)‣ testing of drug combinations


THANKYOU !


THANKS TO :Prof. M.-P. Mingeot-LeclercqDr. C. Seral (Spain)Prof. J.- P. HervegProf. V. PréatProf. M. DelméeProf. Y. GlupczynskiProf. B. GallezProf. A. Pascual (Spain)Prof. M. Struelens (ULB)Theravance (USA)


HURRA FACM !!!Special thanks to O. Meert and M.-C. Cambier


THANKS TO YOU ALL

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