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

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

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

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

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

5. Objectives of the presentation: • The three PK/PD indices • Where are located the bugs ? • Extracellular vs. intracellular • Where is the biophase? • Interstitial space fluid vs. intracellular cytosol vs. intracellular organelles • How to assess the biophase antibiotic concentration • Total tissular concentration vs. ISF concentration. • The issue of lung penetration • Epithelial lining fluid (ELF):? • he 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. The three PK/PD indices

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

8. AUC/MIC PK: PD:

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

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

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: • the actual concentration at the site of action (biophase) • the concentration of the tissue (organs) in which the infection develops

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

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

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

17. Total tissular concentration 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

18. 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

19. Q3: why a total tissular concentration has no meaning

20. 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

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. Intracellular location of antibiotics Phagolysosome volume 1 to 5% of cell volume pH=5.0 Cytosol pH=7.4 Fluoroquinolones(x2-8) beta-lactams (x0.2-0.6) Rifampicin (x2) Aminoglycosides (slow Macrolides (x10-50) Aminoglycosides (x2-4) 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 overestimationof the extracellular biophase concentration.

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

25. 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

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

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

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 cagein 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 investigate PK/PD relationship: YES • ex vivo : Shojaee AliAbadi & Lees (exudate/transudate) • in vivo : Greko (inoculation of the tissue cage)

31. 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". • 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

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

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 • 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%)

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

38. 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

39. 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

40. What we learnt with animal and human microdialysis studies

41. 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

42. What we learnt with MD studies:Inflammation

43. 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 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

44. 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

45. The issue of lung penetration

46. 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)?

47. 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.

49. 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 The alveolar epithelial cells would not be expected to permit passive diffusion of antibiotics between cells, the cells being linked by tight junctions

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