1. Leipzig 2009 1 PK/PD approach for antibiotics: tissue or blood drug level to predict antibiotic efficacy PL Toutain National Veterinary school; Toulouse ECOLE NATIONALE VETERINAIRE T O U L O U S E

2. Leipzig 2009 2 First (scientific) consensus: The goal of PK/PD indices 1.The goal of PK/PD indices is to predict, in vivo, clinical outcomes: Cure prevention of resistance 2.Plasma free concentration is the relevant concentration for the establishment of a PKPD indice

3. Leipzig 2009 3 Second (marketing) consensus It is more easy to promote a macrolide showing its high lung concentrations than its low plasma concentrations

4. Leipzig 2009 4 PK/PD approach for antibiotics: tissue or blood drug level to predict antibiotic efficacy

5. Leipzig 2009 5 Objectives of the presentation: 1.The three PK/PD indices 2.Where are located the bugs ? Extracellular vs. intracellular 3.Where is the biophase? Interstitial space fluid vs. intracellular cytosol vs. intracellular organelles 4.How to assess the biophase antibiotic concentration Total tissular concentration vs. ISF concentration. 5.The issue of lung penetration 1.Epithelial lining fluid (ELF):? 2.he hypothesis of targeted delivery of the active drug at the infection site by phagocytes 6.Plasma as the best surrogate of biophase concentration for PK/PD interpretation

6. Leipzig 2009 6 The three PK/PD indices AntibioticsPK/PD indices GoalCritical values β LactamsTime>MICMaximize exposure time 50-100% dosage interval Quinolones 24h AUC/MIC ratio Optimize the quantity of administered AB 125 H Aminoglycosides Cmax/MIC ratio Optimize the peak concentration 10

7. Leipzig 2009 7 By essence the three PK/PD indices are hybrid parameters PK & PD

8. Leipzig 2009 8 AUC/MIC PK: PD:

9. Leipzig 2009 9 Time > MIC MIC Half-life concentrations Time (h) 24 t1 t2

10. Leipzig 2009 10 Cmax / MIC PK PD Bioavailability (%) clearance Rate of absorptione Rate of elimination Accumulation factor

11. Leipzig 2009 11 PK/PD indices are hybrid parameters For all indices: – the PD input is the MIC –The PK input is associated to plasma: why? And why not: 1.the actual concentration at the site of action (biophase) 2. the concentration of the tissue (organs) in which the infection develops

12. Leipzig 2009 12 What is an ideal concentration for a PK/PD indice A relevant concentration to serve as an input in a PK/PD model should be selective of the biophase i.e. of the fluid that bath the extracellular space namely the interstitial fluid (ISF).

13. Leipzig 2009 13 Q1: Where are located the pathogens and where is the biophase

14. Leipzig 2009 14 Where are located the pathogens ISF Most pathogens of clinical interest S. Pneumoniae, E. Coli,Klebsiella Mannhemia ; Pasteurella Actinobacillius pleuropneumoniae Mycoplasma hyopneumoniae Bordetalla bronchiseptia Cell ( most often in phagocytic cell) Mycoplasma (some) Chlamydiae Brucella Cryptosporidiosis Listeria monocytogene Salmonella Mycobacteria Rhodococcus equi

15. Leipzig 2009 15 What are Antibiotic concentrations that are considered in the veterinary literature to explain antibiotic efficacy?

16. Leipzig 2009 16 Antibiotic concentrations vs. efficacy 1.Total tissue concentrations –homogenates –biopsies 2.Extracellular fluids concentrations –implanted cages –implanted threads –wound fluid –blister fluid –ISF (Microdialysis, Ultrafiltration)

17. Leipzig 2009 17 total tissue concentrations Whole tissue concentrations are obtained by grinding up (homogenizing) tissue and subsequently determining the concentration of the antibiotic in the tissue homogenate.

18. Leipzig 2009 18 In veterinary medicine, there are many publications on tissular concentrations to promote the idea that some antibiotics having a high tissular concentration accumulate in biophase (quinolones, macrolides) and are more efficacious as suggested by their low or undetectable plasma concentrations Total tissular concentration

19. Leipzig 2009 19 Statements such as ‘concentrations in tissue x h after dosing are much higher than the MICs for common pathogens that cause disease’ are meaningless Mouton & al JAC 2007

20. Leipzig 2009 20 Q3: why a total tissular concentration has no meaning

21. Leipzig 2009 21 Two false assumptions 1.tissue is homogenous 2.bacteria are evenly distributed through tissue  spurious interpretation of all important tissue/serum ratios in predicting the antibacterial effect of AB The inadequate tissue penetration hypothesis: Schentag 1990 Schentag, 1990

22. Leipzig 2009 22 Total tissular concentration for betalactams and aminoglycosides if a compound is distributed mainly extra-cellularly (betalactams and aminoglycosides), a total tissular concentration will underestimate the active concentration at the biophase by diluting the ISF with intracellular fluids.

23. Leipzig 2009 23 Intracellular location of antibiotics Phagolysosome volume 1 to 5% of cell volume pH=5.0 Macrolides (x10-50) Aminoglycosides (x2-4) Cytosol pH=7.4 Fluoroquinolones(x2-8) beta-lactams (x0.2-0.6) Rifampicin (x2) Aminoglycosides (slow Ion trapping for weak base with high pKa value

24. Leipzig 2009 24 Total tissular concentration for macrolides & quinolones if a drug is accumulated in cells (the case for fluoroquinolones and macrolides), assays of total tissue levels will lead to gross overestimation of the extracellular biophase concentration.

25. Leipzig 2009 25 Methods for studies of target site drug distribution in antimicrobial chemotherapy

26. Leipzig 2009 26 Methods considered of limited interest for studies of target site drug distribution Tools developed to determine antibiotic concentrations in various surrogates for the ISF and having no pathophysiologic counterpart in humans. –in vitro models, – fibrin clots, – tissue chambers, –skin chambers(blister) –wound exudates, –surface fluids, –implanted fibrin clots, –peripheral lymph. Muller & al AAC 2004

27. Leipzig 2009 27 The tissue cage model for in vivo and ex vivo investigations

28. Leipzig 2009 28 Methods for studies of target site drug distribution in antimicrobial chemotherapy

29. Leipzig 2009 29 The tissue cage model Perforated hollow devices Subcutaneous implantation development of a highly vascularized tissue fill up with a fluid with half protein content of serum (delay 8 weeks) C.R. Clarke. J. Vet. Pharmacol. Ther. 1989, 12: 349-368

30. Leipzig 2009 30 PK in tissue cage in situ administration PK determined by the cage geometry (SA/V ratio is the major determinant of peak and trough drug level) T1/2 varies with the surface area / volume ratio of the tissue cage –Penicillin 5 to 20 h –Danofloxacin 3 to 30 h Greko, 2003, PhD Thesis

31. Leipzig 2009 31 The Tissue cage model: veterinary application To describe PK at site of infection (calves, dogs, horses…): NO To investigate PK/PD relationship: YES –ex vivo : Shojaee AliAbadi & Lees (exudate/transudate) –in vivo : Greko (inoculation of the tissue cage)

32. Leipzig 2009 32 Microdialysis & ultrafiltration Techniques

33. Leipzig 2009 33 What is microdialysis (MD)? Microdialysis, a tool to monitors free antibiotic concentrations in the fluid which directly surrounds the infective agent

34. Leipzig 2009 34 Microdialysis: The Principle The MD Probe mimics a "blood capillary". There is an exchange of substances via extracellular fluid Diffusion of drugs is across a semipermeable membrane at the tip of an MD probe implanted into the ISF of the tissue of interest.

35. Leipzig 2009 35 Microdialysis Technique CMA60 Microdialysis 1.Introducer with CMA 60 Microdialysis Catheter 2.Outlet tube 3.Vial holder 4.Microvial 5.Inlet tube 6.Luer lock connection 7.Puncture needle.

36. Leipzig 2009 36 Microdialysis Pump Perfusion fluid is pumped from the Microdialysis Pump through the Microdialysis Catheter into the Microvial.

37. Leipzig 2009 37 Microdialysis : Limits MD need to be calibrated Retrodialysis method –Assumption: the diffusion process is quantitatively equal in both directions through the semipermeable membrane. –The study drugs are added to the perfusion medium and the rate of disappearance through the membrane equals in vivo recovery. – The in vivo percent recovery is calculated (CV of about 10-20%)

38. Leipzig 2009 38 Ultrafiltration Excessive (in vivo) calibration procedures are required for accurate monitoring Unlike MD, UF- sample concentrations are independent on probe diffusion characteristics

39. Leipzig 2009 39 Microdialysis vs. Ultrafiltration Ultrafiltration Vacuum The 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 gradient Microdialysis : a fluid is pumped through a membrane;

40. Leipzig 2009 40 Marbofloxacin : plasma vs.ISF In vivo filtration Bidgood & Papich JVPT 2005 28 329 Microdialysis Not suitable for long term in vivo studies Ultrafiltration Suitable for long term sampling (in larger animals, the UF permits complete freedom of movement by using vacutainer collection method)

41. Leipzig 2009 41 What we learnt with animal and human microdialysis studies

42. Leipzig 2009 42 Plasma (total, free) concentration vs interstitial concentration (muscle, adipose tissue) (Moxifloxacin) Muller AAC, 1999 Time (h) Total (plasma, muscle) free (plasma) interstitial muscle interstitial adipose tissue 261012 30 40 20 100 1000 Concentration (ng/mL)

43. Leipzig 2009 43 What we learnt with MD studies: Inflammation

44. Leipzig 2009 44 Tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissue Methods: Free Concentrations measured 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 inflammation. Same results obtained with others quinolones Hypothesis: Accumulation of fibrin and other proteins, oedema, changed pH and altered capillary permeability may result in local penetration barriers for drugs Bellmann & al Br J Clin Pharmacol 2004 57 Inflammation No inflammation

45. Leipzig 2009 45 What we learnt with MD studies: Inflammation Acute 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

46. Leipzig 2009 46 The issue of lung penetration

47. Leipzig 2009 47 Animal and human studies MD: The issue of lung penetration Lung MD require maintenance under anesthesia, thoracotomy (patient undergoing lung surgery).. Does the unbound concentrations in muscle that are relatively accessible constitute reasonable predictors of the unbound concentrations in lung tissue (and other tissues)?

48. Leipzig 2009 48 Free muscle concentrations of cepodoxime were similar to free lung concentration and therefore provided a surrogate measure of cefpodoxime concentraion at the pulmonary target site Liu et al., JAC, 2002 50 Suppl: 19-22. Cefpodoxime at steady state: plasma vs. ISF (muscle & Lung) Plasma Free plasma Muscle Lung

49. Leipzig 2009 49

50. Leipzig 2009 50 The blood-alveolar barrier The alveolar epithelial cells would not be expected to permit passive diffusion of antibiotics between cells, the cells being linked by tight junctions Fenestrated pulmonary capillary bed expected to permit passive diffusion of antibiotics with a molecular weight 1,000 Epithelial lining fluid ELF

51. Leipzig 2009 51 The 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)

52. Leipzig 2009 52 Fundamentally, 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. We continue to express PK/PD parameters using serum concentration of total drug because these values do correlate with microbiological outcomes in patients. Kiem & Schentag’ Conclusions (2)

53. Leipzig 2009 53 In acute infections in non- specialized tissues, where there is no abscess formation, free serum levels of antibiotics are good predictors of free levels in tissue fluid

54. Leipzig 2009 54 PK/PD indices and tissular concentrations Currently, no equivalent recommendation has been published with tissular concentration as PK input and that, for any tissue or any type of infection including intracellular infection.

55. Leipzig 2009 55 The site of infection: Intracellular pathogens

56. Leipzig 2009 56 Intracellular location of bacteria Phagosome Lysosome Chlamydiae Listeria No fusion with lysosome Phagolysosome S.aureaus Brucella Salmonella Coxiella burneti pH=5.0 3 4 2 1 Fusion pH=7.4 B B B B B B B B Cytosol

57. Leipzig 2009 57 Intracellular location of antibiotics Phagolysosome volume 1 to 5% of cell volume pH=5.0 Macrolides (x10-50) Aminoglycosides (x2-4) Cytosol pH=7.4 Fluoroquinolones(x2-8) beta-lactams (x0.2-0.6) Rifampicin (x2) Aminoglycosides (slow Ion trapping for weak base with high pKa value

58. Leipzig 2009 58 What are the antibiotic intracellular expressions of activity Phagolysosome Macrolides Aminoglycosides Cytosol pH=7.2 Fluoroquinolones beta-lactams Rifampicin Aminoglycosides Good Low or nul

59. Leipzig 2009 59 The free plasma level is the most meaningful concentration In acute infections in non- specialized tissues, where there is no abscess formation, free plasma levels of antibiotics are good predictors of free levels in interstitial fluid

60. Leipzig 2009 60 Some statements on total tissular concentrations For 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

61. Leipzig 2009 61 Tissue concentrations According to EMEA "unreliable information is generated from assays of drug concentrations in whole tissues (e.g. homogenates)" EMEA 2000

62. Leipzig 2009 62 Conclusions: 1.In acute infections in non-specialized tissues, where there is no abscess formation, free plasma levels of antibiotics are good predictors of free levels in interstitial fluid 2.PK/PD indices predictive of antibiotic efficacy should be based on free plasma concentration 3.People who truly understand tissue concentration work in corporate marketing departments (Apley, 1999)