Plasma vs. tissue concentration to predict antibiotic efficacy

1. Plasma vs. tissue concentration to predict antibiotic efficacy ECOLE NATIONALE VETERINAIRE T O U L O U S E PL Toutain UMR 181 Physiopathologie et Toxicologie Experimentales INRA/ENVT Fourth International conference on AAVM Prague, Czech republic 24-28, 2008

2. The inadequate tissue penetration hypothesis 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

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

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

5. Objectives of the presentation:To address some basic questions • Where are located the bugs ? • Extracellular vs. intracellular • Where is the biophase? • Interstitial space fluid vs. intracellular cytosol vs. intracellular organelles • What is a tissue and what is a tissue concentration • How to assess the biophase antibiotic concentration • Total tissular concentration vs. ISF concentration. • The issue of lung penetration • Epithelial lining fluid (ELF):???? • The hypothesis of targeted delivery of the active drug at the infection site by phagocytes • Plasma as the best surrogate of biophase concentration for PK/PD interpretation

6. Q1: Where are located the pathogens

7. Where are located the pathogens ISF Most bacteria of clinical interest • S. pneumoniae • E. coli • Klebsiella • Mannhemia ; pasteurella • Actinobacillius pleuropneumoniae • Mycoplasma hyopneumoniae • Bordetala bronchiseptia Cell (most often in phagocytic cell) • mycoplasma (some) • Chlamydiae • Brucella • Cryptosporidiosis • Listeria monocytogene • Salmonella • Mycobacteria • Rhodococcus equi

8. Q2: Where is the biophase

9. The interstitial space fluid is the biophase • Most bacterial infections are located in the extracellular compartment. • Except few cases, In acute infections in non-specialized tissues, where there is no abscess formation, interstitial space fluid (ISF) must be considered as the actual target space for anti-infective agents • ISF concentrations are of primary interest Muller et al. AAC , 2004, 48: 1441-1453

10. Q3: what is a tissue & what is a tissular concentration

11. Historical definition of a tissue drug concentration • In the past, it was used to characterize the total concentration in a homogenized biopsy sample • For vet medicine: a by-product of regulatory residue studies • It was assumed that: • tissue is homogenous • that antibiotics is evenly repartited in tissue • That bacteria are evenly repartited in tissue • Each of these assumptions is false and can be very misleading

12. Why a total drug tissue concentrations may be misleading? • Drug distributed mainly extracellularly • β-lactams and aminoglycosides, • grinding up the tissue means dilution of the drug by mixing intracellular and extracellular fluids, resulting in underestimationof its concentrations at the site of infection. • Drug accumulated by cells • fluoroquinolones or macrolides • assay of total tissue levels will lead to gross overestimation of the extracellular concentration. • The opposite is true for intracellular infections.

13. Methods for studies of target site drug distribution in antimicrobial chemotherapy

14. “Tissue concentrations” • Total tissue • homogenates • biopsies • Extracellular fluids • implanted cages • implanted threads • wound fluid • blister fluid • ISF (Microdialysis, Ultrafiltration)

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

16. 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

17. The tissue cage model : PK limitations • A foreign body • Not a physiological space • Clinical counterparts? • Ascitic fluid, effusions ( pericardial, pleural…) • Interpretation may be difficultbecause PK determined by: • diffusion capacity across the TC • TC size and geometry • surface area/volume ratio is the major determinant of peak and trough drug level

18. (C) Time The tissue cage model • 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 Drug administration Slow equilibration inoculation (C) Time

19. Microdialysis & ultrafiltration Techniques

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

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

22. Microdialysis: The Principle • the implanted MD probe is perfused with the perfusate, ie, a physiologic liquid at a very slow rate. • Substances present in the interstitial space fluid of the investigated site can diffuse into the perfusate through a semipermeable membrane at the tip of the MD probe and appear in the dialysate. • Afterward the concentration in the dialysate is chemically analyzed and the true concentration in the interstitial space fluid can be calculated. Antibiotic

23. Microdialysis materials • Introducer with CMA 60 Microdialysis Catheter • Outlet tube • Vial holder • Microvial • Inlet tube • Luer lock connection • Puncture needle. CMA60 Microdialysis

24. 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%)

25. MD need to be calibrated: 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

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

27. Microdialysis vs. Ultrafiltration Microdialysis: a fluid is pumped through a membrane; • 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

28. Marbofloxacin : plasma vs.ISFIn vivo filtration • 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) Bidgood & Papich JVPT 2005 28 329

29. What we learnt with animal and human microdialysis studies

30. Total (plasma, muscle) free (plasma) interstitial muscle interstitial adipose tissue Plasma (total, free) concentration vs interstitial concentration (muscle, adipose tissue) (Moxifloxacin) 1000 Concentration (ng/mL) 100 2 6 10 12 40 20 30 Time (h) Muller AAC, 1999

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

32. 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 • They are generally much lower than total concentrations reported from whole-tissue biopsy specimens especially for macrolides and quinolones

33. What we learnt with MD studies:Inflammation

34. Tissue concentrations of levofloxacin in inflamed and healthy subcutaneous adipose tissue Hypothesis: Accumulation of fibrin and other proteins, oedema, changed pH and altered capillary permeability may result in local penetration barriers for drugs Methods: Free Concentrations measured in six patients by microdialysis after administration of a single intravenous dose of 500 mg. Inflammation No inflammation Results:The penetration of levofloxacin into tissue appears to be unaffected by local inflammation. Same results obtained with others quinolones Bellmann & al Br J Clin Pharmacol 2004 57

35. 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

36. 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

37. The issue of lung penetration

38. 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)?

39. 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 Cefpodoxime at steady state: plasma vs. ISF (muscle & Lung) Plasma Free plasma Lung Muscle Liu et al., JAC, 2002 50 Suppl: 19-22.

40. The issue of lung penetration:Imipem • The major finding of this study was the observation of virtually superimposed free IPM concentration-versus-time profiles in the three media investigated, • This result not only is in agreement with theory but also is consistent with most of the data in the literature. imipenem distribution in muscle and lung interstitial fluids Marchand & al AAC June 2005

41. The issue of lung penetration

42. Lung infections • Uncertainty of the relevant actual location of proliferating bacteria • Alveoli, pulmonary interstitium, bronchioles, blood?? • What is the biophase?? • Epithelium lining fluid (ELF) • Lung IF, alveolar macrophages, tisue 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

44. Alveolar Alveolar Alveolar macrophage macrophage macrophage Alveolar Alveolar Alveolar space space space ISF ISF ISF AB AB AB AB AB AB Alveolar Alveolar Alveolar Epithelium Epithelium Epithelium Capillary Capillary Capillary Thigh junctions Thigh junctions Thigh junctions wall wall wall The blood-alveolar barrier • Fenestrated pulmonary capillary bed • expected to permit passive diffusion of antibiotics with a molecular weight 1,000 Epithelial lining fluid ELF (protein:<10%) The alveolar epithelial cells would not be expected to permit passive diffusion of antibiotics between cells, the cells being linked by tight junctions

45. Drug passage in the lung ELF Drug passage through the alveolar epithelial cells will depend on the lipophilicity and diffusibility of the antibiotics, similar to the drug entry into the central nervous system. Kiem & Schentag AAC 2008 Jan 24-36

46. ELF concentration: possible biais ELF • Measurement 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. Kiem & Schentag AAC 2008 Jan 24-36

47. BETA-LACTAMS ELF Measured ELF concentrations of the beta-lactams are well below serum concentrations, and their respective concentrations in AM cells were negligible The low measured ELF concentrations of betalactams in comparison to their corresponding serum levels could be the result of low capacity of their unbound free fractions for penetration through the alveolar epithelial cell barriers. Kiem & Schentag AAC 2008 Jan 24-36

48. MACROLIDES AND KETOLIDES ELF Measured ELF concentrations of macrolides and ketolides and their derived AUCs were consistently higher than serum levels by as much as 10-fold the high ratios of ELF concentration to serum concentration for macrolides and ketolides could not be explained solely on the basis of good penetration across the alveolar epithelium. The high concentrations of macrolides and ketolides in ELF might be explained by the possible contamination of intracellular antibiotics occurring during the process of BAL. Kiem & Schentag AAC 2008 Jan 24-36

49. FLUOROQUINOLONES Fluoroquinolones achieved higher ELF levels than their free serum concentrations Kiem & Schentag AAC 2008 Jan 24-36

50. Kiem & Schentag’Conclusions (1) • 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