PierreLouis Toutain Visiting Professor Royal Veterinary College London 5 July 2018 Australian amp New Zealand College of Veterinary Scientists Q T Gold Coast Surfers Paradise Australia ID: 1033656
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1. PK/PD approach for antibiotics: tissue, biophase or blood drug level to predict antibiotic efficacyPierre-Louis Toutain Visiting Professor Royal Veterinary College , London 5 July 2018Australian & New Zealand College of Veterinary Scientists QT Gold Coast, Surfers Paradise Australia
2. Objectives of the presentationTo demonstrate that:A total tissular concentration has no meaningThe best surrogate marker to predict efficacy for a (systemic) infection is the plasma free antibiotic concentration
3. 1-What is PK/PD approach for antibiotics?
4. What is PK/PD for AMD?4PK/PD is an attractive alternative to dose-titration studies to discover an optimal dosage regimen:No infectious model (ethical)Dose(s) for different indicationsDosing intervalexpected duration of efficacy
5. EMA guideline 20155
6. 2-Why plasma concentration for PK/PD
7. Dose titrationDoseResponseclinicalBlack boxPK/PDDoseResponse (surrogate)PKPDPlasmaconcentrationBodyPathogenDose titration for antibiotic using infectious model
8. A fundamental PK/PD relationshipA dose can be determined rationally using a PK/PD approach provided to know the therapeutic concentration!For all antibiotics, the in vivo MIC is directly related to the Therapeutic concentrations throughout the so-called PK/PD indexMIC
9. In order to use the MIC to determine a dose, It has been developed 3 surrogate indices (predictors) of antibiotic efficacy taking into account MIC (PD) and exposure antibiotic metrics (PK) Practically, 3 indices cover all situations:fAUC/MIC (more or less universal) fTime>MIC fCmax/MIC (no longer use)
10. Computation of a dose for a fluoroquinolone e.g. if the objective is an AUC/MIC=125h, the therapeutic concentration should be 125h/24h≈5XMIC
11. Q3: And now why tissular concentrations in this story?
12.
13. Prague 2008 13The case of macrolides (azalides)MICThulathromycin disposition in pigs
14. MIC vs Cmax for a selection of AMD
15. Prague 2008 15Explanation 1: Non-antibibacterial properties of macrolides: anti-inflammatory & immunomodulatoryThere is evidence that macrolide antibiotics have a variety of biological actions in addition to their antimicrobial properties. For instance, erythromycin and roxithromycin inhibit cytokine production and chloride secretion from airway epithelial cells), expression of adhesion molecules on neutrophils , and neurotransmission in vagal nerve fibers .
16. Prague 2008 16Explanation2: The tissular lung concentration is the relevant concentration , no plasma concentrationsMICThulathromycin disposition in pigsLung-to-plasma AUC ratio=60
17. Prague 2008 17Intracellular location of antibioticsPhagolysosomevolume 1 to 5% of cell volumepH=5.0Macrolides (x10-50)Aminoglycosides (x2-4)CytosolpH=7.4Fluoroquinolones(x2-8)beta-lactams (x0.2-0.6)Rifampicin (x2)Aminoglycosides (slowIon trapping for weak base with high pKa value
18. Prague 2008 18What are the antibiotic intracellular expressions of activityPhagolysosomeMacrolides AminoglycosidesCytosolpH=7.2Fluoroquinolonesbeta-lactamsRifampicin AminoglycosidesGood Low or nul at pH=5
19. Explanation 3: The lung is a reservoir controlling local concentrationActually Lung is a sink
20. Prague 2008 20The hypothesis of targeted delivery of active drug at the active site by the phagocytes
21. Explanation 4: the small trucks (macrophages) transportationBy living macrophages: release rate too low regarding transit timeBy dying macrophagesMass balance considerationsAMDRelease rate (hours)
22. Why macrolides works despite « high » MIC
23. Prague 2008 23Why macrolides works despite « high » MICMICThulathromycin disposition in pigs
24. MICs for M. haemolytica (calf isolates) in biological fluid (calf serum) versus MH broth (n=6) MICserum / MICbroth ratiosSerum MIC 50 times lowerthan MIC in brothLees et al 2016 JVPT
25. Prague 2008 25The case of macrolides (azalides)MIC in serum Thulathromycin disposition in pigs
26.
27. PK/PD cut-offs (ng/mL in serum) for tulathromycin in calves, Intervals (h)Percentiles (%)0-240-480-720-960-1200-1440-1680-1920-2160-240951421321201099991847872679017216014513212211110396898380210200183167153140129119111104702442292091921761621501391291216027826324222220418817416115014050316300277255234217201186172161PK/PD cut off=83ng/mL in serum or 4.15µg/mL in MHBPK/PD index over 10 days : fAUC/MIC – recommended dose
28. How to explain broth vs. serum differences?We tested many factors but none was able to explain the serum effect
29. Clinical Infectious Diseases 2012 55 , 534“Macrolides show antimicrobial activity against P. aeruginosa in eukaryotic media through increased uptake and reduced efflux. These data may help explain the clinical efficacy of macrolides against pseudomonas Infections”.29
30. MIC of azithromycin (mg/L):CA-MBH vs. RPMI1640 medium30Buyck et al 2012 CID 55, 534Macrolides are considered intrinsically inactive against P. aeruginosa, with high (⩾256 mg·L−1) minimal inhibitory concentrations (MICs) measured in the recommended conventional broth but not in RPMI1640
31. Outer Membrane Permeability :CA-MBH vs. RPMI 31Buyck et al 2012 CID 55, 534Macrolides have low MICs against P. aeruginosa when tested in the presence of serum, bronchoalveolar lavage fluid or culture media used for eukaryotic cell cultures. Enhanced macrolide activity in these media results from an increased permeability of the bacterial outer membrane (no overexpression of efflux pump)
32. Clinical consequences of (1) ignoring the true potency of macrolides against bacteria and (2) seeking to explain macrolide efficacy by an anti-inflammatory activity 32The risk of selecting macrolide-resistant P. aeruginosa
33. Two false assumptionsBacteria are evenly distributed through tissueTissue is homogenous spurious interpretation of all important tissue/serum ratios in predicting the antibacterial effect of ABThe inadequate tissue penetration hypothesis: Schentag 1990Schentag, 1990
34. Where are located the pathogensExtra Cellular Fluid (ISF)Most bacteria of clinical interest- respiratory infection- wound infection- digestive tract inf.Cell(in phagocytic cell most often)Legionnella sppmycoplasma (some)chlamydiaeBrucellaCryptosporidiosisListeria monocytogeneSalmonellaMycobacteriaMeningococciRhodococcus equiMost pathogens of veterinary interest are extracellularExcept few cases, interstitial space fluid (ISF) must be considered as the actual target space for anti-infective agents
35. Prague 2008 35Intracellular location of bacteriaPhagosomeLysosomeChlamydiaeListeriaNo fusion with lysosomePhagolysosomeS.aureausBrucellaSalmonellaCoxiella burnetipH=5.03421FusionpH=7.4BBBBBBBBCytosol
36. Prague 2008 36The free concentration paradigm in pharmacokineticsBlood/PlasmaInterstitial fluidTotal tissue concentrationTotalPlasma boundFree ISF boundFreeFreeTissue boundBUGEliminationPlasma clearance (not tissue concentration or protein binding ) control free plasma concentrationTissular spaceTissue compartment should be conceptualized as a sink not a reservoir with respect to local ISF concentration
37. Free drug concentration is the driving force controlling AMD concentrations at the biophase level
38. When there is no barrier to penetration, free antibiotic plasma concentration reflects antibiotic concentration at the site of infection
39. Microdialysis: The Principle Diffusion of drugs is across a semipermeable membrane at the tip of an MD probe implanted into the ISF of the tissue of interest.
40. UltrafiltrationMD: Excessive (in vivo) calibration procedures are required for accurate monitoring , Not suitable for long term in vivo studies UF-sample concentrations are independent on probe diffusion characteristics, Suitable for long term sampling (in larger animals, the UF permits complete freedom of movement by using vacutainer collection method)Molecular weight cut-off value of e.g. 30 kd,
41. Microdialysis vs. UltrafiltrationUltrafiltrationVacuumThe driving force is a pressure differential (a vacuum) applied across the semipermeable membrane The analyte cross the membrane by diffusion The driving force is a concentration gradientMicrodialysis :a fluid is pumped through a membrane;
42. What we learnt with animal and human microdialysis studiesPrague 2008 42
43. Plasma (total, free) concentration vs interstitial concentration (muscle, adipose tissue) (Moxifloxacin)Prague 2008 43Muller AAC, 1999Time (h)Total (plasma, muscle)free (plasma)interstitial muscleinterstitial adipose tissue2610123040201001000Concentration (ng/mL)
44. Plasma (total, free) concentration vs muscle (free) concentrationPrague 2008 44Total (plasma)free (muscle)free (plasma)Liu J.A.C. 2002cefpodoxinecefixime
45. What we learnt with animal and human MD studiesMD studies showed that: the concentrations in ISF of selected antibiotics correspond to unbound concentrations in plasma and are much lower than concentrations reported from whole-tissue biopsy specimens.Concentrations of beta –lactams and aminoglycosides in ISF are mostly in the range of free concentrations in serum Concentration of quinolones and macrolides at their target site are considerably lower than those predicted from tissue biopsy specimens
46. Influence of protein binding on tissue penetrationPrague 2008 46Muller & al AAC 2004Cefodizime: Binding=74-89%CefpiromeBinding=10% Plasma ISF muscle------: Free predicted from TOT
47. Prague 2008 47Tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissueMethods: Free Concentrations measured in six patients by microdialysis after administration of a single intravenous dose of 500 mg. Results:The penetration of levofloxacin into tissue appears to be unaffected by local nflammation.Same results obtained with others quinolonesHypothesis: Accumulation of fibrin and other proteins, oedema, changed pH and altered capillary permeability may result in local penetration barriers for drugsBellmann & al Br J Clin Pharmacol 2004 57InflamedControl
48. What we learnt with MD studies: InflammationAcute inflammatory events seem to have little influence on tissue penetration. These observations are in clear contrast to reports on the increase in the target site availability of antibiotics by macrophage drug uptake and the preferential release of antibiotics at the target site a concept which is also used as a marketing strategy by the drug industry (Muller & al AAC May 2004)Prague 2008 48
49. Influence of disease on piperacilline distributionPrague 2008 49Healthy volonteersHeart operationMuller & al AAC 2004
50. Prague 2008 50Marbofloxacin : plasma vs. ISFIn vivo filtrationBidgood & Papich JVPT 2005 28 329During the steady state period there was nostatistical difference between unbound plasma and ISF concentrations for marbofloxacin ISFFree plasma concentration
51. Enrofloxacin UF in pigsMessnger et al JVPT 2011 Tissue penetration, as measured by a ratio of area-under-the curve (AUC), was 139% (±69%). Plasma protein binding was 31-37% This study demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasmaThe explanation for such a high drug concentration in tissue is elusive,
52. The issue of lung penetrationPrague 2008 52
53. Lung infectionsWhat is the best choice of an accessible specimen for determining the representative AB concentration?ELF, lung IF, alveolar macrophages, tissue biopsies, blood, bronchial secretion, sputum??ELF seems the most relevant specimen but potential sources of error: dilution, release of AB from alveolar macrophage in the sample Prague 2008 53
54. Prague 2008 54
55. The blood-alveolar barrierPrague 2008 55The alveolar epithelial cells would not be expected to permit passive diffusion of antibiotics between cells, the cells being linked by tight junctionsFenestrated pulmonary capillary bed expected to permit passive diffusion of antibiotics with a molecular weight 1,000 Epithelial lining fluidELFISFCapillarywallAlveolarEpitheliumThigh junctionsspaceAlveolarmacrophageABABISFCapillarywallAlveolarEpitheliumThigh junctionsspaceAlveolarmacrophageABABISFCapillarywallAlveolarEpitheliumThigh junctionsspaceAlveolarmacrophageABABAlveolarAlveolarAlveolar
56. ELF concentration: possible biaisMeasurement problems may confound the interpretation of the ELF concentrations of antibiotics.Cells, especially AM cells (that constitute 3.8 to 10.0% of ELF volume) are included in the composition of ELF The cells may be lysed during the measurement of antibiotic concentration in BAL-derived fluids. Prague 2008 56Kiem & Schentag AAC 2008 Jan 24-36
57. Prague 2008 57The high ELF concentrations of some antibiotics, which were measured by the BAL technique, might be explained by possible contamination from high achieved intracellular concentrations and subsequent lysis of these cells during the measurement of ELF content. This effect is similar to the problem of measuring tissue content using homogenization Kiem & Schentag’ Conclusions (1)
58. Prague 2008 58Fundamentally, ELF may not represent the lung site where antibiotics act against infection. In view of the technical and interpretive problems with conventional ELF and especially BAL, the lung microdialysis experiments may offer an overall better correlation with microbiological outcomes. Kiem & Schentag’ Conclusions (2)
59. The tissue cage modelPerforated hollow devicesSubcutaneous implantationdevelopment of a highly vascularized tissuefill up with a fluid with half protein content of serum (delay 8 weeks)
60. Prague 2008 60The tissue cage modelDrug administrationSlow equilibrationinoculationTime(C)Time(C)T1/2 varies with the surface area / volume ratio of the tissue cagePenicillin 5 to 20 hDanofloxacin 3 to 30 hGreko, 2003, PhD Thesis
61. Prague 2008 61The tissue cage modelFluidRepresentative of ExamplesSerumShallow compartmentISF of well perfused tissuesTransudateDeep peripheral compartmentPenetration impeded by diffusion barrier (CNS, prostate)ExsudateDeep peripheral infection sitesPenetration impeded by pathological barriers (abcess, inflammatory debris)Adapted from P Lees
62. Prague 2008 62Some statements on total tissular concentrationsFor veterinary medicine (Apley, 1999)people who truly understand tissue concentration work in corporate marketing departments For human medicine (Kneer, 1993)tissular concentrations are inherently inaccurate tissular concentrations studies little contribute to the understanding of in vivo efficacy and optimal dosing
63. Prague 2008 63Statements such as ‘concentrations in tissue x h after dosing are much higher than the MICs for common pathogens that cause disease’ are meaninglessMouton & al JAC 2007
64. Tissue concentrationsAccording to EMEA"unreliable information is generated from assays of drug concentrations in whole tissues (e.g. homogenates)"EMEA 2000
65. ConclusionsPlasma free concentration is the driving force controlling extracellular concentration for tissues with no specific barriersBarrier: BBB, prostate, eye (posterior segment)Most pathogens are extracellular thus free plasma concentration is the relevant concentration for PK/PD indexA different story for intracellular pathogens