clinical microdialysis in skin and soft tissues - an update.full

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2008 48: 351J Clin PharmacolStephan Schmidt, Rebecca Banks, Vipul Kumar, Kenneth H. Rand and Hartmut Derendorf

Clinical Microdialysis in Skin and Soft Tissues: An Update

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REVIEW/METHODS

J Clin Pharmacol 2008;48:351-364 351

Traditionally, plasma or serum drug concentrations havebeen used for the assessment of bioavailability and bioe-quivalence. Since in the majority of cases the site of drugaction is in the tissue rather than the blood, the use of cor-responding free, unbound concentrations in the tissue is amuch more meaningful approach. This can become espe-cially important for topical drug administrations, wherelocally active drug concentrations can significantly exceedfree concentrations in plasma. The ability to measurethese free concentrations at the site of drug action overtime makes microdialysis a very valuable tool for theassessment of bioavailability and bioequivalence. This

has been recognized by industry and regulatory authori-ties, resulting in a recommendation of the microdialysistechnique as a tool for bioequivalence determination oftopical dermatologic products. The aim of this article is toprovide an updated review of the microdialysis technique,its applications in skin and soft tissues, and the resultingimpact on clinical drug development.

Keywords: Microdialysis; skin; soft tissues; pharmaco-kinetics

Journal of Clinical Pharmacology, 2008;48:351-364© 2008 the American College of Clinical Pharmacology

Clinical Microdialysis in Skin andSoft Tissues: An Update

Stephan Schmidt, BS, Rebecca Banks, BS, Vipul Kumar, PhD,Kenneth H. Rand, MD, and Hartmut Derendorf, PhD, FCP

Historically, pharmacokinetic/pharmacodynamic(PK/PD) approaches link plasma drug concen-

trations to observed effects. However, most PDeffects are mediated by interaction with enzyme,transporter, or receptor systems that are located inthe tissues.1,2 Consequently, linkage of PD effects totissue drug concentrations at the site of action is amore accurate approach to characterize exposure-effect relationships.2 Closer evaluation of concentra-tion-effect relationships have shown that onlyfree, unbound drug at the target site is responsiblefor PD efficacy.3,4 Therefore, continuous sampling offree drug in the tissues is the most rational approachto estimate active drug profiles at the site of action.

Microdialysis (MD) is currently the most appro-priate sampling technique that can provide thisinformation.2,5

The MD principle was first employed in the early1960s in order to sample free amino acids and otherelectrolytes in the extracellular fluid of animalbrains.6 Further technical advancement resulted inthe development of the “dialytrode” in 1972, thefirst simple version of today’s MD probe.7 In 1974,Ungerstedt and Pycock discussed the use of “hollowfibers” as a superior in vivo sampling technique.8

Steady improvement of both MD catheters andmethodology allowed not only measurement of neu-rotransmitters and metabolites in animals, but alsoapplication in humans.9 In early clinical trials, glu-cose levels were determined in subcutaneous adi-pose tissue.10-12 The approval of MD catheters for usein humans by the US Food and Drug Administration(FDA) and the European Union ConformiteEuropeene (CE) has opened the door to further stud-ies in virtually every human tissue, including mus-cle, skin, lung, myocardium, brain, and eventumors.2 Consequently, in recent years, considerableexperience has been gained in clinical studies of

From the Department of Pharmaceutics, College of Pharmacy (MrSchmidt, Ms Banks, Dr Kumar, Dr Derendorf), and the Department ofPathology, Immunology, and Laboratory Medicine, College of Medicine,(Dr Rand) University of Florida, Gainesville, Florida. Submitted for pub-lication July 25, 2007; revised version accepted November 9, 2007.Address for correspondence: Hartmut Derendorf, PhD, FCP, Departmentof Pharmaceutics, College of Pharmacy, Box 100494, University ofFlorida, Gainesville, FL 32610; e-mail: [email protected]: 10.1177/0091270007312152



352 • J Clin Pharmacol 2008;48:351-364

both healthy volunteers and patients, resulting inmore than 2000 publications as of today.

RATIONALE FOR THIS REVIEW

In 2004, the FDA issued its Critical Path Document,Innovation and Stagnation? Challenge and Opportu-nity on the Critical Path to New Medical Products.13

In this report, the FDA critically evaluated reasonsfor the recent decline of drug launches onto themarket despite the increased investment of time andresources. One of the key issues identified was thelack of sufficient safety and efficacy measures fornew drugs and drug formulations.13 In the FDA’sview, a new product development toolkit containingpowerful new scientific and technical methods (i.e.,animal or computer-based predictive models, bio-marker for safety and effectiveness, and new clinicalevaluation techniques) is urgently needed.13

Recently, the FDA published Critical PathOpportunities for Generic Drugs.14 MD is one of thesenew clinically applicable evaluation techniquesspecifically mentioned in this list. Its capability ofdetermining free, active local drug concentrationsqualifies it for site-specific safety and efficacyassessment. This has become of particular interest toindustry and regulatory authorities for the evalua-tion of bioavailability (BA) and bioequivalence (BE)of topically applied drug formulations, especially ofdermatologic products.14-16 For systemic drugs, usu-ally the serum concentrations are used in BA and BEstudies. However, based on the respective defini-tions of BA and BE, measurements of exposure atthe site of action would be more meaningful.17 Thegoal of this review is to give an overview of newerclinical BA and BE studies in skin and soft tissuesusing MD.

METHODS

The MD catheter (probe) consists of a small, semi-permeable hollow fiber membrane that is connectedto inlet and outlet tubing.12 It is constantly perfusedwith a physiological solution (perfusate) at flowrates of approximately 0.1 to 5 μL/min.2 After inser-tion into a selected tissue or (body) fluid, solutes cancross the membrane by passive diffusion dependingon their concentration gradient.1,2,12 Hence, theprobe can be used as a sampling tool as well as adelivery tool.2 The solution leaving the probe(dialysate) is collected at certain time intervals foranalysis.

Calibration Methods

Since the MD probe is continuously perfused withfresh perfusate, a total equilibrium across the mem-brane cannot be established. Rather, a steady-staterate of exchange across the MD membrane is rapidlyreached. This steady-state exchange rate is describedby the extraction efficiency (EE). The EE is the ratiobetween the loss/gain of analyte during its passagethrough the probe (Cperfusate-Cdialysate) and the differ-ence in concentration between perfusate and thesample of interest such as tissue fluid, in vitro ana-lyte, etc (Cperfusate-Csample), as shown in Equation 1.

EE =Cperfusate − Cdialysate

Cperfusate − Csample(1)

At steady state, EE has the same value for allCperfusate, no matter if the analyte is being enriched ordepleted in the perfusate.2 For this reason, MDprobes can be calibrated with either drug-containingperfusate or sample solutions. While various cali-bration techniques are available (eg, low–flow ratemethod, zero-net flux method,18 extended zero-netflux method,19 etc), retrodialysis by drug20 is themost commonly employed method in humans.During retrodialysis, the probe is perfused withdrug-containing perfusate prior to or after drugadministration, without the drug in the tissue. Sinceabsence of drug in the tissue is required for retro-dialysis, this calibration technique cannot beapplied to endogenous compounds.2

The proper selection of an appropriate calibrationmethod is critically important for the success of anMD experiment, so supportive in vitro experimentsprior to use in animals or humans are recom-mended.2 The recovery determined in vitro mightdiffer from the recovery in humans; therefore, itsactual value needs to be determined in every in vivoexperiment.21

Strengths of the Microdialysis Technique

Depending on the molecular cut-off of the MD probemembrane, larger molecules (such as proteins) areprevented from diffusing into the dialysate.21 Thisallows the analysis of protein-free, active drug con-centrations to be performed frequently without fur-ther sample preparation.1

While MD selectively determines free, unboundconcentrations in the interstitial fluid (ISF) of a

SCHMIDT ET AL



particular living tissue, other sampling techniqueshave limited capabilities to distinguish between dif-ferent sites within the tissue or between free and bounddrug. For example, tape stripping is a commonlyused method that is well established for evaluatingthe penetration of topically applied compounds intothe upper part of the skin.22 However, this method islimited to the stratum corneum and cannot, there-fore, be used for the assessment of free, active drugconcentrations in deeper tissues such as the dermis.In fact, continuous tape stripping does disturb thebarrier function of the skin and can result in artificiallyincreased drug levels in the skin.23 Other tissue-sampling approaches also have major limitations.While, for example, broncho-alveolar lavage (BAL)pools data from large segments throughout the lung,concentrations obtained from homogenized tissue,positron emission tomography (PET), and scintigra-phy will include drug that is bound to interstitialand intracellular proteins or to intra- and intercellu-lar membrane structures.1,2,21

In comparison, the skin blister technique is capa-ble of target-site specific sampling, yet it reflectsconcentrations in experimentally induced secretoryfluids. The concentrations in these fluids might varywith blister size and surface-to-volume ratio due toprotein and chemokine content and might not becomparable to the ISF.1,21,24-27 It was shown, forexample, that ciprofloxacin and moxifloxacin accu-mulated preferably in blister fluid, whereas analmost complete equilibration of the free unboundantibiotic plasma fraction with the ISF was observedusing MD.1,28,29

Another limitation of most of these techniques isthat they are usually not capable of continuous mea-surement of the concentration-time profile. Hence,investigators using, for example, epithelial liningfluid (ELF) and tissue biopsies are forced to pooldata from different subjects in order to receive con-centration-time profiles.30,31 In contrast, continuoussampling via MD allows the generation of PK pro-files from individual subjects.

Limitations of the Microdialysis Technique

Usually, MD probe insertion is associated with min-imal tissue damage. However, some tissue sites,such as brain,32,33 lung,34,35 bone,36 heart,37 liver,38 orthe peritoneal cavity39 are not readily accessible tothe MD procedure. The MD probe must then be sur-gically implanted into these tissues.

Another limitation of the MD technique isits dependency on flow rate and sensitivity of the

analytical assay. As the analyte concentration decreaseswith increasing flow rate, assays with small samplevolumes are restricted to highly sensitive analysistechniques (ie, high performance liquid chromatog-raphy [HPLC], liquid chromatography tandem massspectrometry [LC/MS/MS], capillary electrophore-sis).2 However, if very low flow rates are used, thetime resolution might be compromised.2 For this rea-son, flow rate and analytical procedures requireextensive fine-tuning.

MD has emerged as the method of choice to monitordrug concentrations in the extracellular space. If thesite of action is located intracellularly, MD is not ableto measure that concentration directly. Even in thesecases, the respective extracellular concentrationresides closer to the site of interest than the respec-tive plasma or blood concentrations.

Bioavailability at the Site of Action

In the FDA Guidance for Industry, BA is defined as“the rate and extent to which the active ingredient oractive moiety is absorbed from a drug product andbecomes available at the site of action. For drug prod-ucts that are not intended to be absorbed into thebloodstream, BA may be assessed by measurementsthat reflect the rate and extent to which the activeingredient or active moiety becomes available at thesite of action.”17 Local site drug levels can becomeimportant in, for example, anticancer therapy, becausetumors might show altered physiology and/or limiteddrug access compared to normal tissue.40-42 Knowledgeof the active drug concentrations inside the tumor istherefore critically important for the selection of anappropriate drug and/or dosing regimen.

Even though metastatic malignant melanomas(MMMs) respond poorly to drugs due to resistance ata molecular level and impaired transcapillary drugtransfer, dacarbazine is an effective treatment ofMMMs.43 Transcapillary transfer rates of dacar-bazine and its active metabolite, 5-aminoimidazole-4-carboxamide (AIC), into the tumor weredetermined after intravenous (IV) administration ofdacarbazine at doses of 200 mg/m2 to 1000 mg/m2

body surface area in 7 MMM patients using MD.Dialysates (tumor and healthy adipose tissue) andplasma (ultrafiltered) samples were collected for 240min and analyzed using HPLC for free dacarbazineand AIC concentrations. Results indicated that forall doses, area under the curve (AUC) measurementsfor dacarbazine and AIC were not significantly dif-ferent between plasma (free concentration) and tumorinterstitium. It was concluded that dacarbazine and

MICRODIALYSIS ASSESSMENT OF BIOAVAILABILITY AND BIOEQUIVALENCE

REVIEW/METHODS 353



354 • J Clin Pharmacol 2008;48:351-364

AIC showed significant tumor penetration character-istics after intravenous administration. The lack ofresponse to antineoplastic therapy with dacarbazinemight be explained by resistance at the molecularlevel rather than by inability of dacarbazine and AICto penetrate into the interstitium of MMM.43

Once the drug reaches the systemic circulation,there are no more differences in distribution, metab-olism, and elimination between the IV and the non-IV administration route. Depending on the value ofthe oral bioavailability (F), oral doses will have to beincreased in comparison to the respective IV dose inorder to achieve similar therapeutic drug levels.

As a case in point, MD was employed to comparefree, active ciprofloxacin concentrations in the ISFof skeletal muscle and subcutaneous adipose tissue,after IV or oral ciprofloxacin administration, respec-tively.28 Each of 8 healthy volunteers was studiedtwice and randomly assigned to initial ciprofloxacintreatment with either 500 mg orally or 400 mg intra-venously, with a washout period of at least 7 days.Free ciprofloxacin concentrations were determined inthe ISF of skeletal muscle and subcutaneous adiposetissue, saliva, and cantharis-induced skin blister,as well as capillary plasma, and compared to totalvenous plasma concentrations. Samples were ana-lyzed using HPLC, and respective AUCs were calcu-lated. In order to predict the antimicrobial activity ofciprofloxacin, PK profiles, determined in the ISFafter oral and IV dosing, were simulated in an invitro PD model against Enterobacter, K pneumoniae,and S aureus. Results showed that after oral and IVadministration, mean fAUCs (± SD) of both muscleand subcutaneous adipose tissue were statisticallysignificantly lower than the corresponding AUC forplasma.28 Whereas fAUCmuscle IV (7.43 ± 1.40 mg.h/L)was significantly higher than fAUCmuscle oral (4.49 ±1.41 mg.h/L), no significant differences could bedetected between fAUCsubcutis IV (4.13 ± 1.63 mg.h/L)and fAUCsubcutis oral (3.85 ± 2.26 mg.h/L).28 However, acloser look at the continuously increasing ƒCmuscle oral/Cplasma ratio indicates that steady-state conditions havenot yet been reached. While a Cskin blister/Cplasma ratio>4 is an indicator that ciprofloxacin preferably accu-mulates in inflamed lesions, saliva and capillaryblood concentrations were similar to total plasma.28

In addition, results from the in vitro PD modelshowed that a comparable outcome was achievedagainst selected test strains with ciprofloxacin giveneither as 400 mg IV or 500 mg orally.28 The aboveinformation led to the conclusion that single IV infu-sion of 400 mg and single oral administration of500 mg of ciprofloxacin result in different skeletal

muscle and subcutaneous adipose tissue concentra-tions. Yet, these differences in PK might not be pro-nounced enough to result in clinically significantlyaltered PD outcome.28

In some cases, on the other hand, the absorption ofactive drug molecules into the blood stream is unde-sirable, especially when drug-specific systemic adverseevents are induced. Topical administration of thesecompounds might, therefore, be considered as an alter-native. For example, nonsteroidal anti-inflammatorydrugs (NSAIDs) such as diclofenac are widely pre-scribed for the treatment of rheumatic diseases.44

Although they are among the most commonly pre-scribed drugs worldwide, they are also responsible forapproximately one quarter of all adverse drug reac-tions, such as increased risk of severe gastrointestinal(GI) complications.44 Topical diclofenac administrationmight therefore be superior to oral or IV applicationfor the treatment of inflammatory diseases.

MD was used in the following study to determinethe relative BA of diclofenac in plasma, subcuta-neous adipose, and skeletal muscle tissue afterapplication of a novel diclofenac spray gel formula-tion (4%), or oral dosing with enteric-coateddiclofenac tablets, respectively.45 In one study, 12healthy male volunteers received 2 dosing regimenswith a 14-day washout period in between. Duringthe first regimen, a diclofenac spray gel formulation(48 mg) was applied topically TID for 3 days (10doses total). In the second regimen, enteric-coateddiclofenac tablets (50 mg) were administered orallyTID for 3 days (10 doses total). In both cases, afteradministration of the 10th dose, blood and MD (fromsubcutaneous adipose and skeletal muscle tissue)were collected in 1-h intervals at 10 h postdose andat 48 h. Diclofenac concentrations in dialysate andplasma samples were determined using LC/MS/MSand the respective AUCs calculated. While the rela-tive BA of diclofenac in skeletal muscle (209%) andsubcutaneous adipose tissue (324%) was higherafter topical compared to oral administration, therelative BA in plasma was 50-fold lower (Table I).45

In addition, results showed that maximum plasmaconcentrations (Cmax,plasma) after topical administra-tion were approximately 250-fold lower than theCmax,plasma values after oral administration. These dataled to the conclusion that the spray gel formulationis a good alternative to oral diclofenac formulationsfor the treatment of inflammatory soft tissue condi-tions due to the favorable penetration characteristicsand low systemic availability.45

Although the results of the previous study indi-cate topical diclofenac formulations are appropriate

SCHMIDT ET AL



for treatment of rheumatic diseases, there is stilluncertainty whether these concentrations are suffi-cient in the target tissues.46-48 The aim of anotherMD study was to investigate transdermal penetrationof diclofenac after local topical administration intosuperficial and deep tissue layers.49 Two MDcatheters were inserted into the tissue of 20 healthymale volunteers, one into the superficial layer (3.9 ±0.3 mm) and one into the deep layer (9.3 ± 0.5 mm)of the skin. The correct position (distance betweenskin surface and MD tip) was determined by high fre-quency ultrasound scanning. Diclofenac gel (approx-imately 300 mg/100 cm2) was applied onto skinabove the inserted MD membrane. Dialysate wascollected every 30 min for up to 4 h, and respectivefree AUCs were calculated. Results showed thatdiclofenac concentrations could be determined inboth sampling sites in just 7 volunteers and could notbe correlated to the insertion depth. Linkage to theIC50 (0.5 μg/mL) of cyclooxygenase 2 (COX-2) showedthat effective levels in underlying tissue layers werereached in only 8 out of 20 subjects.49,50 In contrast tothe previous study, it was therefore concluded thatdue to insufficient deep tissue penetration, a general-ized use of transdermal diclofenac formulations, atleast single doses, is often not justified and may begreatly dependent on individual skin properties.49

The effect of NSAIDs is triggered by inhibition ofkey enzymes (COX-1 and COX-2) of the prostaglandin(PG) synthesis. Prostaglandins are mediators involvedin a number of physiological and pathophysiologicaleffects, including the evolvement of pain.51 However,

it is still debated whether antihyperalgesic effects ofNSAIDs include both peripheral (inflammation site)and central sites of action.51

MD was used to determine PG levels in the skinafter topical and oral diclofenac administration. Allof the 10 healthy volunteers were treated in 3 con-secutive treatment periods. Each treatment periodwas randomly assigned and could contain the fol-lowing combinations: (a) oral formulation (93 mg)plus topical formulation (placebo); (b) oral formula-tion (placebo) plus topical formulation (65 mg); or(c) oral formulation (placebo) plus topical formula-tion (placebo), respectively. While MD samples weretaken every half hour after administration for up to6 h, blood samples were drawn for 24 h and ana-lyzed using LC/MS/MS. In addition, antihyperal-gesic action of diclofenac was assessed usingan inflammatory model of cutaneous hyperalgesia(freeze lesion). The response was quantified by esti-mating mechanical pain threshold (MPT) before andafter dosing at half-hour intervals for up to 6 h.Study results showed that both topical and oraldiclofenac formulations are significantly more effec-tive than placebo. While higher tissue levels weremeasured for topical treatment during the first hourafter the application compared to oral administration(46.1 ng/mL vs 11.4 ng/mL), oral administrationresulted in higher tissue levels at time points 2 and2.5 h (Figure 1).51 However, after topical administration,diclofenac could not be detected in plasma. Eventhough the AUC in tissue (fAUCtissue) after oral dosingwas lower than after topical application (32.2 ng⋅h/mL


REVIEW/METHODS 355

Table I Pharmakokinetic Parameters for Diclofenac in Plasmaa

and Subcutaneous and Skeletal Muscle Tissueb

Topical Oral

Parameters Median 95% CI Median 95% CI

Plasma (n=12)AUC∞, AUCτ, ng⋅h⋅mL–1 32.8 22.7-52.9 1569.7 1255.8-1849.8Cmax, ng/mL 4.9 3.4-7.7 1240.2 787.0-1388.9

Subcutaneous tissue (n=12)AUC∞, AUCτ, ng⋅h⋅mL–1 21.5 19.4-50.5 8.6 7.0-10.6Cmax, ng/mL 13.1 9.3-33.6 1.9 1.6-2.5

Skeletal muscle (n=12)AUC∞, AUCτ, ng⋅h⋅mL–1 18.2 11.8-28.1 8.8 7.8-12.3Cmax, ng/mL 12.3 6.2-22.0 2.6 2.0-4.0

Data from 12 healthy males after multiple dose regimens of either topical (diclofenac spray gel 4%) or oral (50 mg enteric-coated tablets) diclofenac.45

CI, confidence interval; AUC∞, AUCτ, area under the plasma or tissue concentration vs time curve of diclofenac approximated to infinity (AUC∞) orevaluated in the last dosage interval (0-8 h; AUC); Cmax, maximum plasma or tissue concentration.a. Total drug.b. Free drug.



356 • J Clin Pharmacol 2008;48:351-364

vs 40.7 ng⋅h/mL), the overall pain relief was 1.7-foldhigher after oral administration during the first 3hours. Accordingly, the authors suggested that anadditional centrally mediated antihyperalgesiceffect is involved in the analgesic effect of systemi-cally administered diclofenac.51

Insufficient BA at the tissue site, in addition to theoccurrence of adverse events, might limit the use oforal or IV formulations.52 Instead, topical administra-tion can overcome these limitations and is employedto deliver drugs at, or close to, the point of applica-tion.53 Topical drug formulations can be applied to theeye, nose and throat, ear, vagina, lung, etc; however,the vast majority of topical medications are appliedto the skin.53 Dermal drug formulations are thus com-monly used for the treatment of skin diseases such asurticaria, psoriasis, or skin cancer.

In the area of skin cancer treatment, photody-namic therapy (PDT) has shown promising resultsfor the treatment of basal cell carcinoma (BCC), themost common type of nonmelanotic skin cancer.54

During PDT therapy, an intravenously or topicallyadministered photosentisizer such as protopor-phyrin IX accumulates in the cancer tissue.54 Afterexposure to light of a specific wavelength, this pho-tosensitizer releases cytotoxic singlet oxygen.

MD was used to assess the BA ofD-Aminolevulinic acid (δ-ALA), a prodrug, that isspecifically metabolized by tumor cells to protopor-phyrin IX, in BCC (n = 14) and normal skin (n = 4)

after dermal application.54 In addition, the skinblood flow was mapped and skin amino acid contentdetermined using laser Doppler perfusion imagingand MD, respectively. Results indicated that intersti-tial ALA concentration in BCC increased from 0 to3.1 ± 1.7 mmol/L (mean ± SEM) within 15 min ofapplication, whereas no ALA could be detected inhealthy skin. In contrast, amino acid levels werefound to be similar in both healthy and BCC tissue,and blood flow was increased 2.5-fold in BCC com-pared to normal skin during treatment.54 It was con-cluded that MD is an appropriate technique for thedetermination of ALA PK in the skin. However, therapid penetration of ALA into tumor tissue andincreased blood flow in BCC might lead to fasterelimination from the tumor and warrants furtherinvestigation.

Factors Affecting Bioavailability

Despite great interest in the skin as an applicationroute for therapeutic agents, the availability ofclinical BA studies evaluating the mechanisms oftransdermal absorption and factors affecting the dis-position of topically applied drugs is limited.23,55,56

In order to address this lack of information, anincreasing number of human studies have been per-formed evaluating factors (eg, skin barrier function,blood flow, degree of ionization, etc) that can alterthe BA after dermal drug application. The ability ofMD to continuously monitor the change of free,unbound drug in the ISF of different layers of theskin or subcutaneous adipose tissue has made ita valuable tool for investigating these factors. MDhas, for example, been used to study the effect ofskin barrier perturbation by repeated tape stripping,treatment with 1% sodium lauryl sulfate (SLS) or2% SLS, and treatment with acetone on the penetra-tion of salicylic acid into the skin.23 Findings showthat there was an approximate 150-fold differencebetween unmodified and tape-stripped/ SLS(2%)–treated skin, indicating a massive disturbance ofthe skin barrier function (Figure 2). In comparison,penetration increased approximately 46-fold afterSLS (1%) and 2.2-fold after acetone treatment,respectively.

The BA of topically applied drugs in skin and softtissue is not only dependent on the integrity of theskin barrier but also highly correlated to the localblood flow.2,55,57-61 An increase, as well as a decrease,in local blood flow can be due to physiological ordrug-induced changes. Whereas an increase in bloodflow enhances the penetration of a compound into

SCHMIDT ET AL

Figure 1. Mean concentration-time profile of diclofenac after oraland topical administration in subcutaneous tissue and plasma.Within 0 to 4 h after topical administration, no detectable levelsof diclofenac acid could be detected in plasma (detection limit,2.5 ng/mL).51



the respective tissue, a decrease slows down its tis-sue uptake.2,55,60-62

This situation was demonstrated in an MD studyevaluating the BA of penciclovir (PCV) in the skinafter a single oral dose (250 mg) of its prodrug, fam-ciclovir.61 Three MD probes were implanted into theleft forearm of 7 healthy volunteers. While all 3probes were perfused with Ringer solution, vasocon-striction was additionally induced in the tissuesaround probes #2 and #3 by either supplementationwith adrenaline (0.2 mg/mL) or cooling (20oC),respectively.61,63 Results (Figure 3) indicate that pen-etration of PCV into untreated skin was statisticallysignificantly higher than into skin with decreasedmicrocirculation due to either adrenaline or cooling,respectively.61 In comparison, local warming of thedermis (40oC) was shown to enhance the microcir-culation in soft tissues and was correlated withan increased penetration of ciprofloxacin into thesetissues.60

However, physiological or pathophysiologicalconditions determine not only the BA of a drug inthe soft tissue, but the physicochemical properties ofthe drug itself. It was shown in both in vitro and invivo experiments that unionized drug penetrates moreefficiently through the stratum corneum than ionizeddrug.64 Whereas chemical modifications of the drugmolecule (eg ester, ion pairs, etc) are frequently

employed to increase the lipophilicity of the parentcompound, the molecule’s charge can also beactively used (eg in iontophoresis) for active drugdelivery.56,65 Yet, these chemical modifications canresult in different penetration behavior and conse-quently alter relative BA.56 Altered BA can becomean issue when 2 different drug formulations of the sameparent compound are used and can be addressed inBE studies.

Bioequivalence

In the case of systemically active drugs, althoughboth BA and BE evaluate the release of a drug sub-stance from a drug product and the subsequentabsorption into the systemic circulation, BE is amore formal comparative test between 2 drug prod-ucts using specified criteria. In the FDA’s Guidancefor Industry, BE is defined as “the absence of a sig-nificant difference in the rate and extent to whichthe active ingredient or active moiety in pharmaceu-tical equivalents or pharmaceutical alternativesbecomes available at the site of drug action whenadministered at the same molar dose under similarconditions in an appropriately designed study.”17

However, the determination of BE for locally actingand targeted delivery products has confronted bothindustry and regulatory authorities with problemsduring the approval process since plasma concentra-tions are usually inappropriate surrogates of phar-macological activity.14


REVIEW/METHODS 357

Figure 2. Concentration (mean ± SD)–time plots demonstratingdifferences in dermal salicylic acid (SA) penetration sampled byMD probes, inserted in the 4 barrier-perturbed skin area (n = 16).In the interval from 50 to 120 min, mean SA concentration levelsare significantly different between all treatment groups (P < .05,shown by solid horizontal line), except between tape-stripped and2% SLS–pretreated skin.23

Figure 3. Median concentration-time profiles for penciclovir(PCV) in skin for control, solution perfused with adrenaline, andcold skin tissue following single oral administration of 400 mgfamciclovir (prodrug) in healthy volunteers (n = 4).61



358 • J Clin Pharmacol 2008;48:351-364

A recent study compared the applicability of MDfor BE determination of 2 topical lidocaine formula-tions (cream 5%, ointment 5%) to the tape-strippingmethod.66 Multiple MD and tape-stripping sampleswere taken at 2 different application sites. Results ofboth methods showed that these 2 formulationswere not bioequivalent, and consequently, not inter-changeable. In addition, further statistical analysisof the applied study design indicated that BE stud-ies (90% confidence interval [CI] and 80%-125% BElimits) using 2 formulations in each subject need aminimum of 27 subjects (when 2 probes are used perapplication site), or 18 subjects (when 3 probes areused per application site), respectively.66 In compar-ison, it has been estimated that approximately 40 to50 subjects are required for BE studies using thetape-stripping method and up to 300 subjects usingthe current clinical BE study design.2,66

PK/PD Indices

In addition to assessment of BA and/or BE, theactual information on free drug concentrationsobtained from MD experiments can be further usedto predict treatment outcome. This approach is fre-quently employed during drug development of anti-infective agents. In the following clinical MDstudies, measured free, active antibiotic concentra-tions were linked to the respective PD outcome para-meters of the most prevalent skin and skin structurepathogens in order to predict their clinical efficacy.

Infections of skin and soft tissue can be causedby a variety of gram-positive and gram-negativepathogens and are routinely treated with antibiotics.Whereas penicillins and cephalosporins are drugsof first choice, agents of different classes (eg oxazo-lidinones, glycopeptides, macrolides, tetracyclines,etc) have to be used in case of adverse events oremergence of β-lactam resistance. In order toincrease the chances of clinical success and todecrease the likelihood of toxic side effects as wellas resistance development, selection of an appropri-ate antibiotic dosing regimen is extremely impor-tant.67 The most rational approach is to link activedrug concentrations to the respective PD outcome.However, outcome predictions based on totalplasma concentrations might be misleading, as mostinfections are not located in the bloodstream butrather in the ISF of tissues.29 In fact, it is free,unbound drug in the ISF that is responsible forantimicrobial efficacy.25 Once free antibiotic concen-trations have been determined at the infection site,outcome should be predictable from the respective

susceptibility breakpoints of the infection-causingpathogens.68

To date, the minimum inhibitory concentration(MIC) has served as a well-established and routinelydetermined susceptibility breakpoint parameter forantibiotics. According to the Clinical and LaboratoryStandards Institute (CLSI, formerly National Committeefor Clinical Laboratory Standards NCCLS), the MIC isdefined as the lowest concentration of drug that com-pletely inhibits visible growth of the organism asdetected by the unaided eye after an 18- to 24-h incu-bation period with a standard inoculum of approxi-mately 5 × 105–106 CFU/mL.67,69 Combinations of thisPD marker with free (ƒ), unbound PK parameters toMIC-based PK/PD indices such as ƒT>MIC, ƒAUC/MICand ƒCmax/MIC have led to a much better understand-ing of antibiotic dosing.67

The first PK/PD index was developed for peni-cillins. It correlates in vivo efficacy with the amountof time free drug levels stay above the MIC of the tar-get organism (ƒT>MIC).67 Although a further index dif-ferentiation within the drug class was suggested, acommon threshold of ƒT>MIC ≥ 40% seems to be suf-ficient for the clinical efficacy of β-lactam antibi-otics.67 While ƒCmax/MIC index values of 10 to 12seem to be a good predictor for aminoglycosides, themagnitude of the fluoroquinolone index is stillcontroversial.70-73 Nonetheless, target ƒAUC24/MICvalues of 100 to 125 (gram-negatives), 25 to 35 (gram-positives), and ƒCmax/MIC index values of 10 havebeen identified for fluoroquinolones.73-75 In compar-ison, ƒAUC24/MIC values 50 to 100 or ƒT>MIC ≥ 85%were good outcome predictors for oxazolidinones.76

Once these MIC-based PK/PD indices are identified,they can support the identification of optimizeddosing regimens and the prediction of treatmentoutcome.67

Knowledge of the free antibiotic concentration-time course in the ISF is necessary in order to estab-lish the respective ƒT>MIC, ƒCmax/MIC and ƒAUC24/MIC index values. Whereas various techniques areavailable for determination of free, unbound con-centrations, they are not all capable of characterizingdynamic changes in free ISF concentrations. OnlyMD combines these 2 properties. Consequently, it istherefore a very valuable sampling tool and hasbecome an inherent part of evaluation and establish-ment of PK/PD indices.

ƒT>>MIC

Although clinical studies have demonstrated theeffectiveness of ertapenem in skin and skin-structure

SCHMIDT ET AL



infections (SSSI) treatment, few studies on the invivo penetration of ertapenem into ISF of soft tissues,such as skeletal muscle and subcutaneous adiposetissue, and resulting free, active concentrations,have been available.20,77,78

In a single-center, prospective, open-label study,free, unbound ertapenem concentrations in the ISFof skeletal muscle and subcutaneous adipose tissuewere measured using MD.20 After determination ofthe individual probe recoveries, 6 healthy volun-teers received 1-g ertapenem as a single 30-minshort-term IV infusion. Plasma and MD sampleswere collected during 12-hour postdosing and ana-lyzed using LC/MS/MS. Results indicate that free,unbound ertapenem profiles in the ISF of both skele-tal muscle and subcutaneous adipose tissue arelower than corresponding total plasma concentra-tions as shown in Figure 4. While free ISF concen-trations of the skeletal muscle correlated well withfree, unbound concentrations in plasma (4%-16% oftotal plasma concentration), they were comparablyhigher than free ISF concentrations in subcutaneousadipose tissue. This phenomenon was observed inother studies as well and might be explained by dif-ferences in blood flow in these 2 tissues.79 Freeertapenem concentrations of 1.13 ± 0.68 mg/L in themuscle, observed 12 h after single-dose IV infusionof 1 g ertapenem, exceeded the MIC90s of methicillin-susceptible S aureus (0.25 mg/L), Streptococcus spp(0.5 mg/L), extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae (0.03-0.06 mg/L),Bacteroides fragilis, and other anaerobic bacteria(≤ 1.0 mg/L) for at least 50% of the entire dosinginterval. In comparison, free levels of 0.31 ± 0.16 mg/Lin the subcutaneous adipose tissue (at 12 h) exceededthe MIC90 of the same SSSI pathogens for at least30% of the dosing interval. The authors concludedthat free, active ertapenem concentrations reachedsufficient levels in noninfected ISF of muscle andsubcutaneous adipose tissue and that previous clin-ical findings were supported by this data.20

ƒAUC24/MIC

Linezolid, the first oxazolidinone, is approved bythe FDA for the treatment of nosocomial pneumoniaand complicated SSSIs. It shows good antimicrobialactivity against various resistant gram-positive bacteria,including methicillin- and glycopeptide-resistant Saureus.80 Despite the fact that (1) only free, unbounddata is considered for antimicrobial efficacy and(2) most relevant pathogens are located in the ISF,most of the available linezolid PK data is based on

total plasma concentrations.81-83 Therefore, a clinicalMD study was performed that evaluated the pene-tration of linezolid into soft tissues of 10 healthyvolunteers after single- and multiple-dose adminis-tration.81 On day 1 of this study, MD catheters wereplaced into the subcutaneous adipose tissue and theskeletal muscle of each volunteer. After calibrationand baseline determination, 600 mg linezolid wereinfused intravenously for 30 min. MD and bloodsamples were taken for up to 8 h. After withdrawalof the MD probes, volunteers were started on orallinezolid (600 mg) BID for 5 consecutive days.81 Thesecond set of MD experiments was started simulta-neously with the last oral dose. This time, probeswere calibrated after the 8-h sampling period. TheAUC0-8 was calculated by using the trapezoidal rule,the AUC24 by extrapolation to 24 h. In addition,AUC24/MIC ratios were calculated for pathogenswith MICs of 2 mg/L and 4 mg/L, respectively.Results show that after single IV administration oflinezolid, ƒAUC0-8 of both skeletal muscle (65.3 ±18.2 mg.h/L) and subcutaneous adipose (75.8 ± 24.2mg.h/L) tissue were statistically significantly higherthan the ƒAUC0-8 of plasma (53.0 ± 11.6 mg.h/L).However, at steady state, no significant differencesbetween concentrations in the ISF of skeletal muscleand subcutaneous adipose tissue could be detected.81


REVIEW/METHODS 359

Figure 4. Ertapenem concentration (mean ± SD)–time profiles in totalplasma, skeletal muscle fluid, and interstitial adipose tissue (sub-cutis) following 1 g infusion for 30 min in healthy volunteers (n =6). Horizontal lines indicate MIC90 values for methicillin-suscepti-ble S aureus (. . .), Streptococcus spp (− . . −), extended-spectrumb-lactamase (ESBL)–producing Enterobacteriaceae (. . . . . . . . . . ),Bacteroides fragilis, and other anaerobic bacteria (——).20



360 • J Clin Pharmacol 2008;48:351-364

The findings further indicated that steady-stateconcentrations in both muscle (ƒAUC24 muscle/MIC58.9 ± 33.0 mg.h/L) and adipose subcutaneous tissue(ƒAUC24 tissue/MIC 46.6 ± 15.9 mg.h/L) were sufficientto treat infections that are caused by pathogens withMICs of up to 4 mg/L.81

ƒCmax/MIC

According to the Centers for Disease Control (CDC),surgical site infections (SSI) include skin and sub-cutaneous tissue infections and are the second mostcommon cause of serious nosocomial infections.84,85

These SSI can be caused by various pathogens (egS aureus, P aeruginosa, Klebsiella ssp) and are fre-quently treated with β-lactam antibiotics.85,86 Inpatients with confirmed allergy or adverse reactionto β-lactam antibiotics, gentamicin can be used incombination with either clindamycin or metronida-zole.87 Gentamicin is a broad-spectrum antibioticthat shows good antimicrobial activity against gram-positive S aureus and gram-negative bacteria, includ-ing Enterobacteriaceae and Pseudomonaceae.88 Itsshort half-life of 2 to 3 h in patients with normalrenal function requires a well-designed dosing regi-men in order to prevent concentrations from drop-ping rapidly to subtherapeutic levels.87 Because it isoften unclear whether surgical procedures changefree, active drug levels in the ISF, the most rationalapproach is to measure respective free concentra-tions directly at the surgical site using MD.

After insertion of the MD catheter into the subcu-taneous fat layer of the abdominal wall (10 cm lat-eral to the umbilicus), 7 healthy volunteers withnormal renal function received 240 mg gentamicinas an IV bolus.88 For the first hour, plasma and MDsamples were taken every 20 min, followed by60-min sampling intervals for up to 6 h. Samples wereanalyzed using a spectrophotometric immunoassay.Pharmacokinetic parameters were determined bycompartmental analysis. Free peak concentrationsin tissue (ƒCmax tissue) of 6.7 ± 2.0 mg/L, which isequivalent to 39.1% of the total peak serum concen-tration (Cmax), were reached within 10 to 30 min ofadministration.88 Since the ƒCmax/MIC ratio wasidentified as the best predictor of aminoglycosideefficacy, ƒCmax/MIC values were calculated forcommon SSI pathogens such as P aeruginosa (7.4:1,MIC: 0.9 mg/L), S aureus (33.5:1, MIC: 0.2 mg/L),and Klebsiella ssp (4.8:1, MIC: 1.4 mg/L), respec-tively.88 The authors concluded that (1) MD could besuccessfully used to measure gentamicin concentra-tions in subcutaneous tissue, and (2) gentamicin

concentrations were sufficient to treat infectionswith the most common SSI pathogens.88

In some cases, the antibiotic dosing regimen doesnot result in sufficient concentrations in the ISF. Ifthey fail to outreach the MIC thresholds of respectiveSSSI pathogens, they cannot be used for treatment.Telithromycin, for example, is typically used as areserve antibiotic for the treatment of respiratory tractinfections as it shows high concentrations in inflam-matory fluids, bronchopulmonary tissues, tonsillartissue, and saliva.89-91 However, its good activityagainst some Streptococci ssp has lead to speculationon its applicability in the treatment of SSSI.89,92

Hence, an MD study was designed to evaluate the PKprofile of telithromycin in the ISF of soft tissues aftersingle-dose administration. In this study, commer-cially available 800 mg telithromycin tablets weregiven orally to 10 healthy male volunteers.92 Bloodand dialysate concentrations were determined. Areasunder the concentration-time curve from 0 to 8 h(AUC0-8) were calculated for free concentrations in theISF of muscle and subcutaneous adipose tissue aswell as free and total plasma concentrations, respec-tively. Results showed that there were no statisticallysignificant differences between areas under the meanfree concentration-time curve from 0 to 8 h (ƒAUC0-8)in muscle (0.6 ± 0.3 mg⋅h/L), subcutaneous adiposetissue (0.9 ± 0.6 mg⋅h/L), and plasma (0.5 ± 0.2mg⋅h/L). However, ƒAUC0-8 of muscle and subcuta-neous adipose tissue were significantly lower thanthe mean AUC0-8 of total plasma (4.1 ± 1.5 mg⋅h/L) as

SCHMIDT ET AL

Figure 5. Telithromycin concentration-time profiles in plasma(total and free), muscle, and subcutaneous adipose tissue after asingle 800-mg dose in healthy volunteers (n = 10), respectively.92



shown in Figure 5. Since antimicrobial efficacy ofketolides correlates best with ƒAUC0-24/MIC ratio,the ƒAUC0-24 was additionally calculated. Using theMIC where the growth of 90% of the SSSI-causingpathogens is inhibited (MIC90), the ƒAUC0-24/MIC90ratios indicated that bacteria highly susceptibleto telithromycin such as S pyogenes might be eradi-cated from tissues and plasma.93,94 Nevertheless, theseƒAUC0-24/MIC90 ratios further indicated thattelithromycin shows insufficient concentrationsagainst other prevalent SSSI pathogens such as Saureus or bite pathogens such as Prevotella canis. Theauthors concluded that telithromycin shows limitedactivity against common SSSI-causing pathogens andshould therefore not be used for their treatment.92

CONCLUSION

Despite the fact that most pharmacologic eventstake place in tissues, PK data is usually still basedon blood or serum concentrations.2 However, free,unbound drug concentrations can differ significantlybetween blood and tissues. Measurement of free,active drug concentrations in the tissue of interest isconsequently the most rational approach but has fre-quently been restricted by insufficient sampling/analysis techniques. With MD, a well-accepted, rela-tively inexpensive, semi-invasive sampling tech-nique that is able to sample free, active drug directlyfrom the extracellular space fluid of tissues hasbecome available. The resulting tissue concentra-tion-time profiles reflect both rate and extent of drugabsorption from the respective application site andcan, therefore, be used for BA and BE assessment. Infact, MD has been recommended by the FDA as asampling tool for BE evaluations of topical dermato-logical products.2,14,66,95-97

To date, most of the current BA studies are per-formed in healthy volunteers. However, physiologicalprocesses can be altered in patient populations com-pared to healthy volunteers. Consequently, the assess-ment of BA in patients can be expected to becomemore important in the future. Initial progress hasalready been made in this area by determining free,active drug concentrations in diseased tissue (egtumors, diabetic foot, psoriasis, etc) using MD. Oncefree, active drug concentrations have been deter-mined, they then can be further correlated with appro-priate biomarkers in order to predict clinical efficacy.

The authors are thankful to Dr Sabarinath Sreedharan Nair forhis valuable comments and suggestions.

Financial disclosure: None declared.

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clinical microdialysis in skin and soft tissues - an update.full

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