Potential of Microdialysis in Evaluation of Topical Drug Products

Transcription

1 Potential of Microdialysis in Evaluation of Topical Drug Products Eva Benfeldt MD, PhD Consultant dermatologist, Ass. professor, lecturer University of Copenhagen Department of Dermatology, Roskilde Hospital Denmark

2 45 minutes what to do? 2

3 Today s talk Microdialysis: the principle Microdialysis for use in the skin All research areas Skin penetration Impact of disturbed skin barrier function on penetration Bioequivalence of topicals Which skin penetration situations are suitable for microdialysis methodology and which aren t Recent developments Topically applied corticosteroids (revisited) Probe depth matters! Conclusion 3

4 Microdialysis technique 4

5 Microdialysis principle 5

6 Probe material: an example 6

7 Probe types: Linear and concentric 7

8 Probe types for use in the skin and subcutaneous tissue Linear or concentric design Linear (laboratory made) Low flux 2-10 kda High flux kda Plasmapheresis membranes 3000 kda Concentric design (fixed area) Commercially available 20 and 100 kda 8

9 Analysis of microdialysates separation techniques HPLC gas chromatography electrophoresis direct assays RIA REA enzyme methodology LC-MS, LC-MS/MS 9

10 Topical drug administration 10

11 Factors affecting recovery I Substance specific MW Configuration Lipophilicity Protein binding inverse relation MW~RR Related to choice of instrumentation Probe design linear, concentric Probe material Probe surface area Perfusate Perfusate flow rate cut-off; pore size protein content, lipids inverse relation flow~rr 11

12 Factors affecting recovery II In vitro recovery >> in vivo recovery Tortuosity factor Blood flow Tissue metabolism Temperature Important to standardize all parameters accessible to standardization! 12

13 Calculations of absolute ( true ) tissue concentrations Limited to situations where tissue levels are at steady state No net flux Stop flow / low flow Retrodialysis by drug* Retrodialysis by calibrator Relative values (study design with multiple probes in each animal/person) will often suffice The in vitro performance of the probe and the in vivo loss (retrodialysis) should be established prior to a human study 13

14 Research areas employing microdialysis sampling in the skin Allergy, inflammation Histamine Substance P IL 6 IL 8 Bradykinin Prostaglandin E2 Protein extravasation Metabolism glucose (up to 3 w) Plastic surgery Flap survival Pharmacokinetics Systemic drugs Antibiotics Corticosteroids Transdermal drug delivery Effect of ischaemia, diabetes Drug penetration in tumour processes Pharmacokinetics Topical drugs Effect of formulation Effect of iontophoresis Effect of alterations in blood supply Effect of barrier perturbation Comparison in vitro-in vivo Ex vivo-in vivo 14

15 Microdialysis in the skin: Issues! The impact of the probe depth in the skin Low variability (e.g ± 0.16 mm) Intended variation in probe depth if impact to be demonstrated The invasiveness of microdialysis Insertion trauma lasts 60 minutes (Groth, 1998) Bruising at the site of insertion, infections very rare Sources of variability The feasibility of sampling very lipophilic, low concentration drugs in a formulation The competent skin barrier and more LATER 15

16 Variability: Systemic administration study Investigation of dermal drug levels by MD technique 10 healthy volunteers 4 MD probes in volar forearm 2 g. acetyl salicylic acid (ASA) p.o. Sampling every 20 min for 4 h (Benfeldt et al. Acta Derm-Venereol 1999) 16

17 SA, ASA in dialysate (µg/ml) Variability I: Systemic administration study Time (min) 17

18 Recovery (%) Variability II: Where variability occurs Time (h) 14 C-Salicylate (nmol/ml) Time (h) Retrodialysis by calibrator: Recovery of 3 H-salicylic acid added to the perfusate. Concentration of 14 C-Salicylate in dialysates sampled after topical application of 14 C-Butyl salicylate. (L Simonsen et al., Eur J Pharm Sci 2004) 18

19 Topical drug administration 19

20 Barrier perturbation study 18 healthy human volunteers Barrier perturbation Quantification by non-invasive methods Local anesthesia 8-10 MD probes in the dermis Topical salicylic acid 5% w/v in ethanol Sampling every 20 min for 4 h (Benfeldt, Serup and Menné. Br J Derm 1999; 140: 739) 20

21 Old school microdialysis 21

22 Salicylic acid (µg/ml) The effect of barrier perturbation on cutaneous drug penetration 100,00 10,00 1,00 0,10 0,01 0, Time (min) Mean Tape Mean SLS 1 % Mean SLS 2 % Mean Acetone Mea n Normal 22

23 Bioequivalence of topicals Topical formulations are considered bioequivalent when the active ingredient and its strength is the same in both formulations the inactive ingredients in the generic topical product are the same as the innovator product the new product is labelled for the same condition of use as the existing product both formulations produce comparable drug concentration-time curves (AAPS report 1998) 23

24 MD in bioequivalence studies 24

25 MD in bioequivalence studies 25

26 I The penetration of lidocaine 5% cream vs. ointment formulation, evaluated by microdialysis (Benfeldt E, Honoré Hansen S, Vølund Aa, Menné T, Shah VP. J Invest Dermatol. 2007) 26

27 Lidocaine study 8 healthy volunteers (4 male, 4 female) Participation on 2 separate study days DAY 1: Lidocaine 5% cream DAY 2: Lidocaine 5% ointment Left: 2 probes in 2 areas (duplicate) Probe insertion without local anesthesia Sampling every 20 min for 5 h Right: 3 areas, strip at 30 min, 2 h, control (Benfeldt E, Honoré Hansen S, Vølund Aa, Menné T, Shah VP. J Invest Dermatol. 2007) 27

28 to formulation applied 2-5 mg/cm 2 28

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37 Ultrasound scanning measuring skin thickness and probe depth 37

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43 RESULTS FROM ONE VOLUNTEER Lidocaine concentration in dialysates 43

44 Lidocaine penetration from the 2 formulations 1000 Lidocain (ng/ml) AUC lidocaine min (mean, SEM) (ng/ml)min C ream Ointment Time (min) 1000 Cream Ointment mdmlogcs.wmf 44

45 MD: Pharmacokinetic comparison AUC t-5 ng/ml*min C max ng/ml Lag time min T max min Lidocaine 5% cream Lidocaine 5% ointment P value

46 Analysis of MD variables Subjects 1 Areas A B Probes 1 2 Positions a b c 8 A B 3 4 a b c 46

47 Where is the variability introduced: Relative size of coefficients of variation 100 (%) Between probes Between areas Between subjects 20 0 Cream Ointment Cream Ointment Cream Ointment AUC C max Rate varkin5.wmf 47

48 Sources of variability: conclusion The following factors did not significantly increase variability (MD): Age Gender Room temperature and humidity Dose applied Probe depth in the skin Skin thickness Main source of variability identified as individual skin barrier properties 48

49 Study size estimates for BE studies of topical formulations 49

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51 Conclusion from lidocaine study Different pharmacokinetics depending on formulation Intersubject (skin barrier associated) variability dominated overall variability BE studies feasible in 18 subjects (best case) 51

52 Metronidazole study 14 healthy volunteers (7 & 7 ) 3 metronidazole cream formulations: Flagyl 1%, Metronidazole 1%, Rozex 0.75% Non-invasive measuring methods: TEWL Erythema Ultrasound: Probe depth Skin thickness (García Ortiz P, Hansen SH, Shah VP, Sonne J, Benfeldt E. Are Marketed Topical Metronidazole Creams Bioequivalent? Evaluation by in vivo Microdialysis Sampling and Tape Stripping Methodology. Skin Pharmacol Physiol 2011;24:44 53) 52

53 Study I: Set up Left forearm Dermal microdialysis 3 penetration areas 3 probes/area = 9 probes Flow rate: µl/min Sampling every 20 min for 5 hours 53

54 Metronidazole in dialysates 54

55 BE evaluation by DMD: variability issue 55

56 Conclusions from metronidazole study Variability was higher than anticipated May depend on formulation? Composition of formulations as well as concentration of active was not the same Methodology still relevant But mid-study analysis for correction of no of subjects needed Or in vivo pilot study 56

57 Ketoprofen gel study. (Tettey-Amlalo RN, Kanfer I, Skinner MF, Benfeldt E, Verbeeck RK. Eur J Pharm Sci 2009) 57

58 Conclusion from ketoprofen study Gel formulation applied in excess gave very low variability Perhaps infinite dose is better than finite? 58

59 The effect of a disturbed skin barrier function (Ortiz PG, Hansen SH, Shah VP, Menne T, Benfeldt E. Contact dermatitis 2008;59:23-30) (Ortiz PG, Hansen SH, Shah VP., Menne T and Benfeldt E. Acta Derm Venereol. 89: ). Using the same probe, perfusate and topical MTZ formulation (Flagyl 1%) 59

60 Study II: Set up Left forearm Dermal microdialysis 2 penetration areas 2 probes/area = 4 probes Flow rate: µl/min Sampling every 20 min for 3 h 60

61 Study III: Set up Left forearm Dermal microdialysis 2 penetration areas 2 probes/area = 4 probes Flow rate: µl/min Sampling every 20 min for 3 hours 61

62 Concentration (ng/µl) Study II and III: Dermal concentration of MTZ Dermal Concentration of Metronidazole sampled by Microdialysis Results from study II and III Healthy skin Irritant Dermatitis 10 Uninvolved skin Atopic Dermatitis Time (minutes) 62

63 Concentration (ng/µl*min) Study II and III: AUC sampled by microdialysis Dermal concentration of metronidazole sampled by Microdialysis Results from Study II and III p= p=0.004 Healthy skin Irritant Derm atitis Uninvolved skin Atopic Derm atitis 63

64 Study II and III: Conclusion Microdialysis sampling in irritant dermatis as well as atopic dermatitis was perfectly feasible Variability even decreased in irritant dermatitis The location of endogenous skin disease may add extra variability due to variability between anatomical regions 64

65 The competent skin barrier (Morgan, Renwick and Friedmann, Br J Derm 2003;148:434) 65

66 the competent skin barrier (Morgan, Renwick and Friedmann, Br J Derm 2003;148:434) 66

67 ..still something to worry about! (Klimowicz, Farfal and Bielecka-Grzela, J Clin Pharm Ther 2007; 32: 143) 67

68 ..but there is good news (Wei HL et al, Drug Dev and Industrial Pharm 2012;38(7):785-91) 68

69 Microdialysis and topical formulations II Many topically active drugs are Very lipophilic (corticosteroids)(log P ) Very very lipophilic (tacrolimus, pimecrolimus)(log P 6.8+) Some of them are Very potent and/or hormones - thus present in low (0.25%, 0.1%, 0.03%) concentration in the formulation So if a formulation contains a very very lipophilic drug of interest at a low concentration 69

70 Mission Impossible 70

71 Recent developments Remakes of challenging studies and unfinished business 71

72 Concentration [μg/ml] CP sampled by DMD in humans in vivo Time [hrs] Figure 3. Mean concentration of CP in dermal microdialysates following application of the CP solution at t=0 (n= 10). (Au WL, Skinner MF, Benfeldt E, Verbeeck RK, Kanfer I. Application of dermal microdialysis for the determination of bioavailability of clobetasol propionate applied to the skin of human subjects. In press, Skin Pharmacol Physiol 2012) 72

73 Ex vivo microdialysis 73

74 Feasibility study: ex vivo microdialysis 74

75 Impact of probe depth (Holmgaard R, Benfeldt E, Bangsgaard N, Sorensen JA, Brosen K, Nielsen F, Nielsen JB. Skin Pharmacol Physiol 2012;25:9-16) 75

76 Impact of probe depth (Holmgaard R, Benfeldt E, Bangsgaard N, Sorensen JA, Brosen K, Nielsen F, Nielsen JB. Skin Pharmacol Physiol 2012;25:9-16) 76

77 Overall conclusion: Microdialysis for in vivo (human) studies of topicals The methodology is not suitable for all drugs Study design should acknowledge intersubject variability Variability may be different between studies due to drug or formulation characteristics A thorough pre-study work-up and modifications of the set-up can overcome partial unsuitability Variability and validation are still important issues 77

78 Regulatory outlook on microdialysis methodology 78

79 Regulatory outlook I FDA has atively sought information, presentations and co-operation with MD researchers Little is known about how much MD is used in preapplication work in industry Submission of MD studies is encouraged by the FDA European counterpart slower to act 79

80 Regulatory outlook II 21 CFR 320. Bioavailability definition: and becomes available at the site of action Approval of topical drug products Clinical study Surrogate markers HPA axis Vasoconstriction DPK method rejected as FDA standard for BE 80

81 Regulatory outlook III Microdialysis has been used as a part of a formulation optimization programme MD cannot replace clinical trial as yet FDA has requested both pre- and post approval MD data to support in vivo bioavailability trials MD is in line with the Critical Path Initiative (FDA 2004) 81

82 Regulatory outlook IV: EMA Microdialysis, which does measure drug in solution in skin, can supply in vivo data in terms of absorption and, should there be sufficient absorption, pharmacokinetic studies may also be performed. The usefulness of data produced in this manner will be evaluated on a case by case basis by the relevant regulatory authority. The sponsor developing a topical formulation using these techniques should seek scientific advice from the concerned regulatory authority before conducting such trials. (Mugglestone C, Mariz S and Lane ME. The development and registration of topical pharmaceuticals. Int J Pharm 2012:435;22 26) 82

83 Key papers I 83

84 Key papers II 84

85 Key papers III 85

86 Key papers IV 86

87 Key papers 87

88 Thank you for your attention 88