1. PK/PD approach for antibiotics: tissue or blood drug levels to predict antibiotic efficacy PL Toutain National Veterinary school; Toulouse Wuhan 8 October 2015

2. 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

3. 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

4. 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

5. For pulmonary infection, plasma free antibiotic concentration, not the epithelial Lining Fluid (ELF), is the best surrogate of biophase concentration

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

7. MIC distribution for M haemolytica & P multocida (2004-20010) for tulathromycin

8. The PK/PD issue for macrolides (triamilides): plasma concentration lower than MICs Good clinical efficacy and bacteriological cure with macrolides is achievable with plasma concentrations (much) lower, than the in vitro MICs for major lung pathogens Cmax=0.5µg/mL ≤ to MIC 90 MIC

9. MIC in MHB vs. calf serum 25%,50%,75% and 100% 25% 50% 75% 100 % MIC in MHB MIC in serum 9

10. The case of tulathromycin See presentation entitled “ how to establish a dosage regimen for a sustainable use of antibiotics 10

11. By essence the three PK/PD indices are hybrid parameters PK & PD (see previous presentation)

12. Leipzig 2009 12 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

13. Why plasma is the relevant fluid to consider for PK/PD integration? For mechanistic reasons –Extracellular location of pathogen For practical reasons –A dosage regime is able to directly control a plasma concentration

14. 1: Where are located the pathogens and where is the biophase

15. 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

16. 2: What are Antibiotic concentrations that are considered in the veterinary literature to explain antibiotic efficacy?

17. Leipzig 2009 17 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)

18. 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.

19. Leipzig 2009 19 3: why a total tissular concentration has no meaning

20. 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

21. 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.

22. Leipzig 2009 22 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

23. 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.

24. 4: what are the methods for studies of target site drug distribution in antimicrobial chemotherapy

25. Prague 2008 25 Muller & al AAC 2004 PET images following administration of 18 f-trovafloxacine

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. The tissue cage model for in vivo and ex vivo investigations

28. Leipzig 2009 28 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

29. 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

30. The Tissue cage model: veterinary application To describe PK at site of infection (calves, dogs, horses…): NO To assess the influence pf inflammation by comparing exudate and transudate concentration To investigate PK/PD relationship: YES –ex vivo : killing curves (exudate/transudate) –in vivo : Greko (inoculation of the tissue cage)

31. 5-Microdialysis & ultrafiltration Techniques

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

33. 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.

34. 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.

35. Microdialysis Pump Perfusion fluid is pumped from the Microdialysis Pump through the Microdialysis Catheter into the Microvial.

36. Microdialysis : Limits MD need to be calibrated Retrodialysis method –tedious. – The in vivo percent recovery is calculated (CV of about 10-20%)

37. A small experimental error in recovery estimate results in a relatively larger error in drug concentration estimates which is probably responsible for the greater interanimal variability observed in lung tissue than in the other media Marchand & al AAC June 2005 MD need to be calibrated:

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

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. MD vs UF Plock :Eur J Pharm Sci 2005 25 1-24

41. 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)

42. 42 This study’s objectives were to determine intestinal antimicrobial concentrations in calves administered enrofloxacin or ceftiofur sodium subcutaneously, and their impact on representative enteric bacteria Ultrafiltration devices were implanted in the ileum and colon of 12 steers,

43. Enrofloxacin (SQ, 7.5mg/kg) 43 AUC (enro+cipro) Plasma=19 (total) ISF=25 (free) Ileaum=21 (free) Spiral colon =36 (free) AUC (enro+cipro) Plasma=19 (total) ISF=25 (free) Ileaum=21 (free) Spiral colon =36 (free)

44. Ceftiofur SQ (2.2mg/kg) 44 AUC Plasma=137 (total) ISF=15 (free) Ileum=40 (free) Spiral Colon =34 free) AUC Plasma=137 (total) ISF=15 (free) Ileum=40 (free) Spiral Colon =34 free)

45. 6-What we learnt with animal and human microdialysis studies

46. 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)

47. Plasma (total, free) concentration vs muscle (free) concentration Total (plasma) free (muscle) free (plasma) Liu J.A.C. 2002 cefpodoxine cefixime

48. What we learnt with animal and human MD studies MD 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

49. What we learnt with animal and human microdialysis studies Free plasma concentration is a good surrogate of most interstial fluid (ISF) concentration

50. MIC measured in MHB is homogeneous to a “free concentration”

51. Effect of protein binding on antimicrobial activity MICs of Staphylococcus aureus (Data from Kunin et al 1973 ) MIC (µg/mL) fbfb 0.22 0.37 0.650.90 0.93 0.95

52. The free concentration paradigm in pharmacokinetics is supported by MD findings Blood/PlasmaInterstitial fluid Total Measured by analytical technique Plasma bound Free ISF bound Free Tissue bound BUG Elimination Tissular space

53. What we learnt with MD studies: Inflammation

54. 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

55. 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

56. 7-The issue of lung penetration

57. 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)?

58. 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

59. Possible confounding factors in interpreting ELF concentrations of antibiotics measured by BAL ELF: Epithelial lining Fluid BAL:bronchoalveolar lavage

60. 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

61. 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)

62. 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.. Kiem & Schentag’ Conclusions (2)

63. 8-The site of infection: Intracellular pathogens

64. 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.

65. Key questions for intracellular antibiotherapy Where are the bacteria ? Which antibiotics accumulate in cells ? Where are antibiotics located ? What is the intracellular expression of activity ? What is the bacterial responsiveness ? Cooperation with the cell own defenses and cytokines ? Tulkens - Bangalore

66. 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

67. Cellular penetration of antibiotics and action on the intracellular bugs  Lactames Lincomycine Macrolides Fluoroquinolones Penetration + +++ Intracellular localisation Cytosol Cytosol, lysosomes Ubiquitaire Activity - ++ +++

68. Leipzig 2009 68 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

69. Which antibiotics do accumulate in cells ? beta-lactams :  1 x aminoglycosides: <1 to 2 x ansamycins:2-3 x tetracyclines:2-4 x fluoroquinolones:10-20 x macrolides:4 to > 100 x

70. Prague 2008 70 Lysosomes and distribution of weak bases (macrolides, Lincosamides ) Lysosomes are small membranes organelles containing hydrolytic enzymes Liver, Kidney, spleen, leucocytes, macrophages, lung pH: 4-5; volume 1 to 5% of cell volume Ion trapping for weak base with high pKa value (energy dependent process; inhibition by monensin, a proton-cation exchanging ionophore that abolish pH gradient and the drug uptake )

71. Leipzig 2009 71 What are the antibiotic intracellular activity Phagolysosome Macrolides Aminoglycosides Cytosol pH=7.2 Fluoroquinolones beta-lactams Rifampicin Aminoglycosides Good Low or nul

72. Conclusions

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

74. 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

75. 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

76. Never use tissue concentrations to determine an antibiotic dose According to EMEA "unreliable information is generated from assays of drug concentrations in whole tissues (e.g. homogenates)" EMEA 2000