pub47 rhodes

29

In Vitro Systems for Assessment of DrugRelease from Topical Formulationsand Transmembrane Permeation

ERIC W. SMITH and JOHN M. HAIGHSchool of PharmaceUlical Sciences, Rhodes University, Grahamstown,South Africa

~

BASIC PRINCIPLESOF DESIGN

Numerous experimental methods have been developed to investigate drug releasefrom vehicles and the percutaneous absorption of topically applied chemicals.The objective of this reSearchis often to find correlation between laboratoryresults and the transdermal absorption experienced by living subjects so that invivo experimentation may be curtailed. In many instances, the diverse experi-mental techniques tend to obscure absorption-controlling factors and compli-cate interstudy comparisons, rather than clarify the complex transdermal absorp-tion process. Moreover, lack of agreement between results may occasionally beascribed to shortcomings in the in vitro methodology employed. The benefitsof using an in vitro cell system for the preliminary testing of drug permeationin the laboratory are obvious. The environmental and diffusion variables maybe controlled in an attempt to elucidate specific factors affecting the kineticprocesses and drug bioavailability. Investigations are complex because of themultiple, interrelated events underlying the processes of drug partitioning fromthe applied vehicle and diffusion through the portals of the stratum corneumto the myriad of metabolic, binding, and clearance activities in the lower epi-dermal and dermal strata.

In any investigation there are severalcontrolling variables that may be con-sidered and the permutations of these variables that are selected for study willdict~te the research protocol. Experiments are usually designed (and cell sys-tems adopted) to formulate answers to specific investigational questions. Forexample, experimental protocol may differ when investigatinghow molecularstructure affects intrinsic diffusivity through a membrane, in comparison withthe investigation of formulation factors affecting the release of drug from a

465

466 Smith and Haigh

dosage form. Experimental design that allows the greatest number of variablesand variable permutations to be monitored is a sensibleobjective at the pre-liminary planning stage. Mter critical review, the beneficial features of severalexperimental designsmay be incorporated into one composite, which shouldthen provide optimal methodology for the specificproject at hand. Factors thatrequire consideration when selectingan in vitro system include:

1. The rate-limiting process: drug solubilization or diffusion in the vehicle,partitioning from the vehicle, diffusion through the test membrane or par-titioning and removal by the receptor phase.

2. The intrinsic diffusivity of the permeant (structure-activity relationship)and apparent diffusivity.

3. The predominating route of diffusion during the experiment and the rela-tive extents of drug binding and metabolism occurring in the membrane,delivery, and receptor phases.

4. The intrinsic barrier potential of the membrane (if one is used) and theeffects that vehicle components may have on its retardive properties. Hy-dration of the membrane and the presence of penetration enhancers maybe important here. Interspecimen variability between membranes of thesame type (especially biological material) may markedly influence experi-mental results.

A diffusion cell system cannot duplicate exactly the events that occur in vivo:the clearance of dif~usant by the vasculature and enzymatic metabolism areespecially difficult to simulate in vitro, as are the pharmacodynamic eventssuch as corticosteroid-induced vasoconstriction. However, researchers have pro-posed that it should be possible to correlate in vitro diffusion results with in vivodata, provided diffusant clearance from the distal surface of the membrane isnot the rate-limiting step to diffusion (Franz, 1975, 1978; Marzulli et al., 1969).

- Investigatio~f drug release from topical dosage forms have been performedin systems without rate-limiting membranes (the classicimmersed ointment jarexperiments). Drug release from formulation vehicleshas also been investigatedby systems incorporating porous, retaining membranes that negligibly affect therate of drug diffusion. Experimentation involvingrate-limiting membranes(more applicable to percutaneous absorption) generally requires an agitated bi-chamber or flow-through apparatus. In each case the appearance of the drug ina liquid receptor phase, or diminution from the donor phase, is analyticallymonitored as a function of time, and this data is used to generate release or per-meation profUes.

DIFFUSIONSYSTEMSWITHOUT A MEMBRANE

. Diffusion systems have been used that have no membranous barrier to the pas-sage of permeant (Table 1). Typically the formulation is immersed in an im-

In Vitro Chamber Design 467

Table 1 In Vitro Diffusion SystemsWithout a Separating Membrane

miscible, agitated receptor liquid maintained at constant temperature. Thesesystems have limited applicability because they lack similarity to in vivo permea-tion, or even to diffusion through barrier media in vitro. At most, they can bedescribed as drug-partitioning systems between two or more immisciblephases;however, they may provide information on the release of drug from the donorvehicle. No parallel is implied between this drug diffusion and that occurringwhen the vehicle is applied to the skin, although this may exist.

Busse et a!. (1969) describean immiscible triphasic solvent system used torepresent the topical absorption of corticosteroid esters. The system comprisedan upper liquid paraff'm/hydrogenated lanolin layer containing betamethasone17-valerate (donor), a hydroalcoholic layer (representing the skin), and a lowerchloroform layer (sink). A triple-blade paddle stirred the system gently overseveral days of experimentation, avoidingmixing of the phases, and the chloro-form layer was assayed for drug content by thin-layer chromatography (TLC).Although basic in design the authors demonstrated that drug partitioning wasgreater from the vehicle containing lanolin than from the donor phase withoutthis solubilizer.

Researchers have packed topical formulations into jars or dishes that havebeen inverted or immersed into liquid receptor phases. Generally, the immisci.bility of the phases enables these experiments to be performed without a mem-

Author Description Receptor Diffusant

Busse (1969) Agitated triphasic system Chloroform Betamethasone

Di Colo (1986) Immersed jar for gels Water (acidified) Benzocaine

Chowan (1975) Immersed jars (floated) Water Corticoids

Ong (1988) Immersed jars Propylene carbonate Lonapalene

Poulson (1968,1970) Petri dishesfPG gels Isopropyl myristate Fluocinolone

Haleblian (1977) Petri dishes/PG gels Isopropyl myristate Fluocinolone

Katz (1972) Petri dishes/PG gels Isopropyl myristate Fluocinolone

Rosvold (1982) Petri dishes/PG gels Isopropyl myristate FluocinolOne

Amundsen (1981) Petri dish/gels (stirred) Isopropyl myristate Betamethasone

Dempski (1969) Petri dish/gelled IPM Water Dexamethasone

Behme (1982) Immersed sieve/cream Water Estradiol

Won Jun (1986) Agar plate with recess Agar Sulfonamides

Mazzo (1986) Transdermal device Water Scopolamine

PG, polyethylene glycol; IPM,isopropylmyristate.

468 Smith and Haigh

brane sequestering the donor formulation. The rate of appearance of diffusantin the receptor phase can, therefore, be directly attributed to the physico-chemical release characteristics of the vehicle.

Di Colo and co-workers (1986) have used this technique to investigate theeffect of surfactants on the release of benzocaine from gel systems. Chowan andPritchard (1975) applied ointment bases, containing suspended cortico~eroids,to the bases of Teflon dishes that were floated on aqueous receptor fluid to in-vestigate drug release from the formulations. Ong and Manoukian (1988) haverecently used a similar system to evaluate lonapalene release from ointments topropylene carbonate receptor.

Poulsen and co-workers (Haleblian et al., 1977; Katz and Poulson, 1972;Poulson, 1970; Poulson et al., 1968) and Rosvoldet al. (1982) have used petridishes containing formulation, submerged in isopropyl myristate, to investigatethe release of radiolabeled fluocinolone acetonide from propylene glycol gels.Similarly, Amundsen et al. (1981) have used tl1}.ssystem for determining beta-methasone 17-valerate release from gels. The researchers used a large stirringblade positioned above the surface of the gel to nUxthe bulk fluid and, com-mendably, calibrated the drug release rate to stirring speed so that their ex-periments could be conducted at an optimal agitation rate.

Filled petri dishes were also used by Dempski and co-workers (1969) to in-vestigate dexamethasone release from gelled isopropyl myristate into an aqueousreceptor phase. They concluded that drug release, as measured by these in vitrosystems, is a function of the relative drug solubility in the base and in the recep-tor medium.

Behme et al. (1982) report that their attempts to measure estradiol releasefrom a cream yielded higher valueswhen the formulation was contained bycellulose or fdter membranes, than when immersed in the receptor solventwithout a covering medium. However, they note that in the latter situation

-the cream swelledappreciably and sloughed into the fluid. These problemsprompted the development of a novel method to contain the cream: spreadinto the interstices of an 80-mesh stainless-steelsieve. The screen, containinga weighed amount of formulation, was immersed into gently stirred water,thereby exposing a large surface area of cream to the receptor phase.

A novel method is reported by WonJun and Bayoumi (1986), who punchedcylindrical holes into gelled agar plates and filled these with the dosage formsunder study. The extent of drug diffusion into the medium was assessedby dis-solving the agar in water and analyzing the solution by high-performance liquidchromatography (HPLC); however, no mention is made of the treatment usedto remove residual formulation from the donor hole. They report a linear cor-relation between the amount of drug released and logarithmic time and concludethat the drug solubility in the base and the partition coefficient between baseand medium are the major determinants of the release profdes.


Generally, these systems have been employed only when the donor vehicleand receptor phase are immiscible, thereby preventing phase homogenization.The lack of retaining membrane would preclude the use of this apparatus forexamining drug release from solution or lotion dosage forms. Although theseexp'eriments may provide data on drug partitioning from the delivery vehicle,these systems do not parallel the partitioning that occurs from a vehicle to theskin or the subsequent percutaneous diffusion and, thus, their usefulness islimited.

DIFFUSION CELLS WITHOUT A RATE-LIMITING MEMBRANE

Diffusion cells without a rate-limiting membrane are generally designed to inves-tigate the characteristics of drug releasefrom topical formulations, rather than -transmembrane diffusion. Typically the donor vehicleis retained within an openglassjar or petri dish by cellulose or similarporous membrane that will preventdispersion of the formulation into the receptor phase but will negligibly influ-ence the movement of drug molecules into the liquid (Table 2). In this situa-tion, the mass of drug released from the formulation is proportional to thesquare root of the time, provided the membrane is in no way rate limiting andless than 60% of the total amount of drug initially present in the vehicle hasbeen released (Beastall et aI., 1986; Hadgraft, 1979; Higuchi, 1967). As mole-cules partition from the formulation into the receptor, the remaining drug in thematrix must reequilibrate into the new volume, thereby decreasing the concen-tration in the vehicle. Drug release, therefore, commences on contact of thevehicle with the receptor phase, and the rate decreases continuously with time.The absence of observed lag times in the resultant permeation profile impliesthat there are no significant diffusion-controlling steps mediated by the sepa-rating medium; release characteristics in these studies are governed almost en-tirely by the donor formulation. These tests may be valuable in elucidatingoccurrences such as drug solubilization within the formulation and the potentialof the molecules to partition into the receptor solvent, but they have limitedapplicability to estimating the complex process of percutaneous absorption.

Modified ointment jars have been used extensively as donor formualtioncontainers (Ashton and Hadgraft, 1986; Billups and Patel, 1970; Gilbert et aI.,1986). The jars, filled with the test formulation, have membranes secured overtheir mouths and are inverted or submerged in the receptor fluid. Althoughseveral liquids have been used as receptor media, aqueous systems may benonideal for relatively water-insoluble drugs (Turakka et aI., 1984). Ayresand Laskar (1974) used such a system to investigate the release of benzocainefrom ointment bases. The formulations were packed into glasscontainers,covered with hydrated cellulose membrane, and inverted in aqueous receptorphase but, remarkably, the latter was not agitated. This experiment demon-

Author

Table2 In Vitro Diffusion SystemsWithout a Rate-Limiting Membrane

Description

~.....0

Receptor Diffusant

Busse (1969)

Billips (1970)

Ashton (1986)

Gilbert (1986)

Turakka (1984,1985)

Ayres (1974)

Kazmi (1984)

Parikh (1986)

Muktadir (1986)

Mazzo (I 986)

Malone (1974)

Polyethylene cups/filter paper

Immersed jar/cellophane

Immersed jar/cellophane

Immersed jar/Cellgard-IPM

Immersed jar/polycarbonate

Immersed jar/cellulose



Plastic jar/semipermeable membrane

Transdermal aevice/cellulose

Teflon chamber/filter paper

IPM

Water

Water

Water

Propylene glycolWater

Water

Water

Betamethasone

Salicylic acid

Nicotinates

Benzoates

HydrocortisoneSorbic acid

Indomethacin

Testosterone

Ibuprofen

ScopolamineFlucloronide

Buffer

Water

WaterVI3g:~Co:J:11/~


strated that drug release rates varied greatly, dependent on the character of theointment and drug concentration, but it also served to exemplify the followinglimitations of this apparatus. The containing membrane is nondiscriminatoryin its barrier properties, and a bidirectional diffusion of the components of bothchambers was experienced. Severalformulation constituents, such as polyethy-lene glycol, were detected in the receptor fluid, whereas the movement of waterinto the donor chamber, and its interaction on the donor side of the membrane,resulted in the precipitation of the drug because of its low water solubility.

A similar, agitated system has been reported by Ezzedeen et al. (1986) forassessingthe release of sorbic acid from ointment bases. They, too, report leach-ing of formulation constituents into the receptor phase and dissolution of thevehicle on the donor side of the membrane. The research limitations of nondis-criminating membranes in this type of diffusion cell are evident. From theirstudies Turakka et al. (1984,1985) concluded that the water content of the -donor vehicle appears to be the most important factor affecting hydrocortisonerelease into propylene glycol receptor phase. In their in vitro system this corti-costeroid was not released from anhydrous dosageforms, and the release ofhydrocortisone acetate was less than that of the parent alcohol.

Immersed ointment jars and cellulose membranes have also been used to in-vestigate the release of indomethacin (Kazmi et aI., 1984) and testosterone(Parikh et al., 1986) from ointment bases to aqueous receptor phases. Muktadiret al. (1986) used plastic jars covered with semipermeablemembrane and in-verted in stirred phosphate buffer to determine the release of ibuprofen fromointment bases. The shortcomings of this methodology are apparent in theirresults obtained from the addition of known in vivo penetration enhancers tothe ointment bases. This addition generated little or no improvement in drugrelease in vitro. This is probably because the ointment released the drug, in theabsence of a rate-limiting barrier, as fast as intraformulation diffusion would al-low replenishment of drug at the receptor interface. Ointment additives would'not necessarily improve this diffusion, unless they modified viscosity, for ex-ample, and would not, therefore, show any enhancement in the in vitro releaseprofile. On the other hand, it is feasible that improved drug permeation in thepresence of enhancers may be demonstrated by incorporating a rate-limiting bar-rier to diffusion, such as excised skin.

Mazzo et al. (1986) compared three methods for assessingdrug release from atransdermal device. Two of the methods require modified solid dosageformdissolution/disintegration apparatus, and the third uses a diffusion cell withouta membrane. Cellulose membrane was used to anchor the deviceto the disin-tegration apparatus and separate the former from the receptor phase. The diffu-sion cell used was of cylindrical design,mounted horizontally with a verticalsampling port, and the device was attached directly to the open end of thecylinder. Agitation of the receptor cell contents, of dubious efficacy, was

472 Smith and Haigh

effected by a magnetic stirrer bar positioned midway between device and sampl-ing port. The steady-state flux rates obtained from the diffusion cell were ap-proximately 25%lower than those from the two sets of dissolution apparatus,and the authors commented that this may be due to unstirred diffusion boundarylayers at the device surface. These methods pertain mainly to the assessment ofdrug release and quality control of marketed transdermal devices.

An interesting variation is presented by Busseet al. (1969), who soaked f:tlterpaper in isopropyl myristate (representing the skin) onto which ointment-filledpolythene cups were inverted. Mter severaldays incubation, a concentric sec-tion of the f:tlterpaper surrounding the cup was cut, the solvent extracted, andthe corticosteroid content assessedby TLC. Malone and co-workers (1974) havealso used f:tlterpaper to contain ointment formulations within Teflon chambersthat were inverted in aqueous receptor phase.

These methods without a rate-limiting membrane are, therefore, useful asscreening tools for drug release or for detecting ~rug-vehicle interactions, butthe limitations resulting from intimate contact of donor and receptor vehiclesand the aselectivity of the membrane makes extrapolation of results to in vivodrug absorption unreliable. The limitations of the methodology are also exemp-lified by the fact that most of the publications concern the release of moleculesfrom viscous, lipophilic, ointment bases that are essentially immiscible withaqueous receptor phases; little work has been attempted using cream, lotion,or solution donor vehicles. Furthermore, Hadgraft (1985) has warned that spu-rious results may be obtained using this methodology if aqueous formulations(such as gels) are tested, due to water absorption by the dosage form. Thismethodology does not, therefore, appear versatile in its spectrum of applica-bility.

DIFFUSION CELLS INCORPORATINGA RATE-LIMITINGMEMBRANE

To obtain a closer approximation of the complexities of transdermal drug ab-sorption, some form of rate-limiting barrier to diffusion should be included in thecell apparatus (Table 3). The movement of molecules from the delivery vehiclewill then be governed by the vehicle-membranepartition coefficient, the rateat which partitioned molecules diffuse from the interface through the mem-brane matrix, and the rate of molecule removal from the distal membrane sur-face.

Several diffusion cell designshave been described, each having features that,reportedly, overcome certain experimental limitations of inferior designs.Certain features are common to many of the designs: two chambers, one con-taining the drug donor vehicle and the other containing agitated receptor sol-vent, separated by a diffusion rate-limitingmembrane. In many instances hori-zontally mounted cylinders have been used for the half-cellswith the membrane

Table 3 Steady-State Diffusion Systems with a Rate-Limiting Membrane

Author Description Receptor Diffusant ::S<

Stehle (1972) Flasks/filter disks Buffer Pyridines;:;'...0

Valia (1984a,b) r;'bichambers/skin Water Estradiol Cj::s-1\1

Chien (1984) L-bicham bers/ skin Water Estradiol 3cr

Southwell (1984) Bichamber/skin Water Various ...

Broberg (1982) Recessed plate/silicone Isopropyl myristate Lidocaine1;1<II

Julian (1986) Immersed jar/cellulose silastic Agitated water Benzoates/oa'::s

hydrocortisone

Bottari (1974) Recessed plate/silicone Water Salicylic acidBarry (1976;1977) Bichamber cell/cellulose Water Corticoids

Garrett (1968) T cell + gas/ silastic Buffer Phenones

Lovering (1974) T cell (agitated)/silicone Buffer Chlordiazepoxide

Foreman (1976) Flow-through/cadaver skin Ethanolic solution Nandrolone

Astley (1976) Flow-through (agitated)/skin Buffer Tritium

Wurster (1979) Flasks (shaken)/skin Heptane Sarin

Durrheim (1980) Bichamber cell/mouse skin Saline Alkanols

Harper-Ballantone (1986) Bichamber cell/mouse skin Water Benzoates

Nakano (1970) Bi- and trichamber/silicone Water Salicylic acid

Touitou (1 985a,b ;1986) Bicylinders/cellulose, silicone Water Benzoic acid

Galey (1976) Bichamber/skin Buffer Various

Flynn (1971) Bichamber (stirred)/silicone Water Phenone

Tojo (1985a,c,d) L-bicham ber / silicone Water Corticoids

Ghannam (1986) L-bicham ber /silicone Water \ Corticoids.j::o."-JUJ

Liu (1986) L-bichamber/silicone Water Nortestosterone

474 Sm ith and Haigh

clamped between these chambers. Generally, homogeneous mixing of the fluidwithin these cylinders is difficult to achieve. Bar magnets will cause fluid mixingin the immediate vicinity of the stirrer bar but will not generate extensive lateralfluid movement within the cylinders. The problem of adequate thermal controlhas been addressed either by total immersion of the assembled cell or by indivi-dual water jackets enclQsingeach chamber. In many, the membrane flange is notheated and this may cause temperature gradients between the membrane surfaceand the bulk chamber fluid, especiallyin the presence of poor fluid agitation.

On reviewof the diffusion cell designs,one emphasis is clear: the entire con-tents of each fluid-filled cell must be adequately agitated to prevent the develop-ment of localized concentrations of drug and the agitation must also be suffi-ciently vigorous to minimize the diffusion boundary layers at the membraneinterface (thereby maintaining sink conditions, as far as possible). The existenceof relatively large, static diffusion boundary layers at the membrane surfacesprovide additional resistance to the passageof the'permeating drug. Occasion-ally, the magnitude of this resistance may approach that of the membranethrough which the diffusant has passed. The importance of adequate agitationon the results obtained from in vitro diffusion experiments has been shown byLovering and Black (1974) and by other workers (Stehle and Higuchi, 1972).They concluded that as receptor phase agitation increases, the thickness of theunstirred boundary diffusion layer decreases,and drug permeation rate increases.Moreover, if stirring increases sufficiently, the rate of permeation is insignifi-cantly influenced by the relatively thin diffusion layers, providing optimal per-meation monitoring conditions. It is assumed that the diffusion boundary layersin the dermal capillary vesselsare negligiblysmall and, therefore, do not makea substantial contribution to the permeation resistance in vivo. Occasionally,the existence of the diffusion boundary layer is acknowledged by researchers butis ignored in comparative work because of its uniformity throughout the experi-

-mentation (Ackermann and Flynn, 1987; Ackermann et aI., 1985,1987; Satoand WanKim, 1984), or is assumed to be negligible(Bottari et aI., 1977). Thisis acceptable provided it is remembered that calculation of the diffusion coeffi-cient will include the contribution by the boundary layer barrier and, thereby,yield only an apparent diffusion coefficient. In other cases, failure to calibratethe diffusion cells for diffusion layer thickness will produce unreliable results.

Several solvents have been employed for the donor and receptor vehicles ofin vitro permeation systems. Most use aqueous media, presumably becausethe assay of drugs in aqueous solution is facile; however, diffusants of low watersolubility may not partition readily from delivery vehicles or from the receptorsurface of the membrane into the aqueous phase, thereby retarding further dif-fusion. Bronaugh and Stewart (1984) have cited this as a reason for the mis-leading results observed in some standard diffusion cell techniques and for dis-crepancies between in vivo and in vitro results. Diffusants that may not parti-

InVitro Chamber Design 475

tion into aqueous receptor phases in vitro may be sufficiently soluble in biologi-cal fluids in vivo to maintain adequate vascular clearance and, thereby, sink dif-fusion conditions. These authors caution that compounds having an aqueoussolubility of 10 mg' L-1 or less may demonstrate limited in vitro partitioninginto hydrous receptor environments.

Several instances are reported in which limited solubility of the diffusant inthe receptor phase has produced anomalous permeation results, and enhance-ment of solubility or increased lipophilicity of the receptor phase has improvedpartitioning of diffusant from the membrane (Riley and KemppaInen, 1985).Scott et al. (1986) reported that estradiol and testosterone were insufficientlysoluble in saline solution to maintain adequate sink conditions, and they used50% ethanol solution as a receptor medium. Valia and Chien (1984a) haveshown that the measured skin permeation of estradiol increases as the percentageof polyethylene glycol in the aqueous receptor phase increases. Franz (1975)reported that he omitted highly lipophilic compounds from his aqueous in vitroexperiments to avoid insolubility problems. Bronaughand Stewart (1984) sug-gest that simply using a receptor solvent that will solubilizethe diffusant andnot damage the membrane is inadequate; they maintain that the establishmentof greater overall lipid solubility in the receptor phase is necessary to competewith the lipophilic properties of the skin. These researcherssubstituted rabbitserum, dilutions of serum albumin, organic solvents, and surfactant solutionsfor saline receptor phase in an attempt to more accurately measure the permea-tion of sparingly water-soluble compounds that demonstrated minimal diffusioninto the aqueous solvent.

In contrast, the prospective diffusant may be so soluble in the donor solventthat it has little aff'1nityfor the membrane and reluctantly partitions into it. Theflux rates measured by in vitro techniques are, therefore, dependent on the solu-bility of the diffusant in the liquid phasesemployed and, thereby, the partitioncoeff'1cientsthat are in effect. In addition, the susceptibility of aqueous mediato microbial contamination and the potential for aqueous degradation of thediffusant may make the choice of a nonaqueous solvent for donor and receptorvehicle fairly appropriate. The question of hydration should alsobe addressedwhen membranes are immersed for prolonged periods in aqueous vehicles. Thefully hydrated stratum corneum is, generally, more permeable to many diffusingspecies, and this may yield anomalous results if aqueous donor and receptorphases are used. Sloan and co-workers (1986) reported that contact of full-thickness hairless mouse skin with phosphate buffer for 120 h did not signifi-cantly affect the permeation rates of theophylline. Southwell and co-workers(1984) reported insignillcant increases in the penetration of caffeine throughhydrated human skin compared with dry skin maintained under controlled con-ditions, although more recently (Southwell and Barry: 1984), they reported thatthe diffusion coefficients of aspirin and caffeine increasedwith skin hydration.

476 Smith and Haigh

Thus, the effect that hydration may have on permeation appears to vary, depen-dent on the drug and membrane under investigation.

On the other hand, the use of cosolubilizersmust in no way affect the bio-chemical composition of the barrier membrane. Surfactants added to aqueousreceptor phases may have independent penetration-enhancing effects on biologi-cal media. Bronaugh and Stewart (1984) demonstrated that polyethylene glycol20 oleyl ether has negligibleeffect on certain biological membranes, although en-b,ancingpermeant solubility in the receptor phase. Presently, this surfactantappears to be the most innocuous solubility modifier for use in laboratory per,meation systems;however, its usefulnessmay be surpassed as further investiga-tions are conducted.

Similarly,alcoholic or nonpolar organic solvents must not denature the mem-brane barrier. Sloan et al. (1986) report that a 3-min wash of hairless mouseskin with methanol had only a marginal effect on the flux of theophylline. Incontrast, Bond and Barry (1988) report a 16-fold increase in the permeabilityof hairlessmouse skin and no increasein the permeability of human skin afteracetone treatment. Bronaugh and Stewart (1984) observed that methanolic andethanolic receptor solvents damaged full-thickness rat skin in vitro as assessedbythe increases in cortisone flux rates.

Bearingthese points in mind, a nonviscous, lipophilic fluid may appear usefulas a choice for receptor medium. The bipolar character of isopropyl myristatemakes it a good solvent for most of the permeants investigated in transdermaldiffusion studies, and its nonaqueous nature will not support hydrolysis-medi-ated molecular rearrangement or cleavagereactions. In addition, the combina-tion of hydrophilic and lipophilic characteristics tend to simulate the polar andnonpolar characteristics of the skin, thereby more closely simulating the in vivodiffusion environment (Broberg et aI., 1982; Poulson et aI., 1968). However,the possible deleterious effects of a lipophilic solvent on the barrier properties

- of biological membranes must not be ignored. Lipid receptor phases must inter-act with the biochemical constitutents of the membrane, by acting as a solventfor the biogenic lipids, for example, and this will alter the permeability charac-teristics of the skin. As the lipophilicity of the receptor phase increases (i.e.,the closer its biochemical composition to that of the skin), the possibility forextraction of lipophilic components from the membrane increases (Bronaughand Stewart, 1984). Sloan et al. (1986) and Waraniset al. (1987) propose thatpretreatment of hairless mouse skin with isopropyl myristate increases the fluxof subsequently applied theophylline in propylene glycol. Notwithstandingthese observations, isopropyl myristate presents a highly favorable medium ofbiphasic nature for permeant partitioning and has, consequently, been used inlaboratory permeation experiments.

In attempting to minimize the experimental variables as much as possible,it may be prudent to avoid use of cosolubilizersor organic compounds, when-


ever possible. Generally, deviation from an isotonic receptor medium will mod-ify the barrier properties of biological membranes, and this modification must beborne in mind. Alternatively, the alteration of the barrier may pose little con-cern, dependent on the specific goals of the research.

CELLS FOR DETERMINING STEADY.STATEFLUX

Cell designs for determining steady-state flux usually comprise two similarchambers, containing permeant in solution and receptor solvent, respectively,separated by the test membrane (Ackermann et al., 1985; Langguth et al., 1986a;Michaels et aI., 1975; Morimoto et aI., 1986; Washitakeet al., 1980). The con.tents of both cells are adequately agitated to ensure thorough dispersion of drugmolecules. The donor vehicle is assumed to expose the membrane to a constant-concentration of the drug, and the receptor phase is assumed to maintain sinkclearance conditions for the permeant. Barry (1983) has reported that a deple-tion of donor phase or increase in receptor phase concentration not exceeding10%does not significantly violate zero-order flux conditions or deviate fromthe initial thermodynamic driving force for diffusion; this restriction is not vio-lated by most cell systems. These cell configurations, measuring solution to sol-vent permeation, are useful for assessingthe intrinsic diffusivity of a moleculethrough the membrane or the effect of partition coefficient, pH, or boundarylayers on diffusion. If donor and receptor solventsare identical, these experi-ments should indicate the tendency of the drug to diffuse through the mediumin the absence of any factors that may enhance permeation, and they may formthe initial "control" basis for the study of possible enhancing factors.

In many in-stances,the method of fluid agitation has dictated the variationsin cell design. An early bichamber system (Bettley, 1961) rotated about an axisin the plane of the membrane, forcing an air bubble in each chamber to flowthrough the fluid, generating mixing of dubious efficacy. The mechanics ofthis design seem overly complex in the light of more recently developed systems.Julian and Zentner (1986) used a modified designof the immersed ointment jarincorporating two magnetic stirrer bars, one in the bulk receptor solution andone resting on the ,donor surface of the horizontal membrane. This method ofagitation may be possible with relatively strong membranes, but it probablywould not suit a delicate biological medium. This design also allowed immersionof an ultrasonic probe into the donor chamber for investigation of ultrasound.mediated effects on permeation.

A sophisticated donor chamber is described by Bottari and co-workers(1974)for the study of concentration influences on the diffusion of salicylicacid fromointment bases. They used a recessed, stainless-steelplate, containing the for-mulation, covered by hydrated silicone membrane held in place by a steel ringand bolt assembly. The filled, assembled cell was immersed into stirred receptor

478 Smith and Haigh

medium and the release of drug monitored spectrophotometric ally. Commend-ably, the authors alkalinized the receptor medium so that the fluid presented afavorable environment for diffusion of the acid and, together with frequent re-placement of the medium, sink conditions were maintained. Furthermore; theauthors conducted blank diffusion runs with bases containing no diffusant toensure that permeating formulation constituents would not interfere with theassay procedure. They found that the stainless steel cell released chemicals intothe acidic receptor fluid and an improved design (Bottari et aI., 1977) was con-structed of polymethyl methacrylate. These are safeguards that other research-ers have overlooked. The siliconemembrane used here influences the rate ofdrug diffusion, to some extent, and the results indicated a definite lag time be-fore the appearance of drug in the receptor fluid. Lag times were also observedby Broberg et ai. (1982) when using silicone membrane in a similar apparatusbut were not observed when release of medicament was monitored in the ab-sence of the membrane.

Comparatively largevolume, large diffusion area, cubic cells have beenmolded from Perspex blocks (Barry and Brace, 1977; Barry and EI Eini, 1976;Patel and Foss, 1964) or Lucite (Corrigan et aI., 1980). The cell halves secur-ing the membrane were clamped together, and agitation was generated by Tef-lon-coated bar magnets or immersiblestirring units. Stoppered sampling portsallowed aliquots of receptor solution to be removed at suitable intervals withsolvent replacement. Although these cells of large diffusion area allow rela-tively high permeant concentrations to develop in the receptor phase, they areuseful only for testing membranes that are in copious supply; obtaining intactsheets of excised biologicalmembrane severalcentimeters in diameter is a fairlydifficult procedure.

Severalresearchershave used an inverted T-shaped cell with the membranecoveringboth horizontal ends (Washitakiet aI., 1980) or have used identical

-L-shaped chambers clamped together to resemble a V-tube (Dyer et al., 1979).Donor solution is filled into the T-cell,and the whole system is immersed in thereceptor medium. Adequate fluid mixing in the arms of the cell would appearto pose a problem for this design: recycling the fluid from a reservoir is onemethod of agitation that has been used, and mixing may further be enhancedby bubbling an inert gas through the solution (Garrett and Chemburker, 1968).However,variability among repetitive experiments was reported to be greaterwhen nitrogen purgingof the fluid was employed. If it is assumed that gaspurging would only improve turbulence and mixing within the cell, then itmust be concluded that recyclingof the T-shaped cell contents alone does notgenerate adequate mixing and does not maintain a constant donor concentra-tion at the membrane surfaces. Evaporation of the receptor solvent from thewide-mouthed cellsmust also be prevented with this apparatus.

In Vitro Chamber Design .479

Noting these shortcomings, Lovering and co-workers(1974) incorporated aninternal means of agitation within the T-cell. These authors omit factors of dif-fusion boundary layer thickness and drug partition coefficient from their calcu-lations of the drug permeability coefficient on the basis that the arithmeticproduct of these parameters is negligiblewhen the permeation rate is not limitedby diffusion boundary layers. However, they assumedfrom their data that theboundary layers were negligibly small and no validatingexperiments were con-ducted. The dangers of making assumptions concerningboundary layer influ-ence without adequate calibration data for the cell system is obvious. This isespecially important when, as in this case, the cellsare of elaborate designwithsevera1linkagesand include agitation of unproven efficacy.

Flow-through cells in which the contents of both donor and receptor cham-bers are continuously replaced with fresh solvent have been effectively employedin this research (Foreman and Kelly, 1976). This technique obviouslyrequiresa large reservoir of receptor fluid with which the membrane may be bathed overthe course of the experiment and, therefore, the method of analysisfor thedilute eluent solution must be appropriately sensitive. In many instances thefluid inlet in each chamber is directed on to the surface of the membrane; how-ever, this may generate some strain on the medium. Delicate tissuespreclude theuse of large flow rates because of the extensivehydrodynamic pressure that maybe generated and, thus, it is doubtful that regions of the cell distant from theinlet or outlet apertures are thoroughly mixed on fluid perfusion. A combina-tion of magnetically stirred donor chamber and flow-throughreceptor chamberhas been used by Astley and Levine (1976). The receptor fluid inlet o(the cellwas positioned at the center of the membrane with eluent collected from itsperiphery. Thereby, the authors claim, efficient elution of the penetrant fromthe whole surface of the membrane was ensured.

Bichamber Steady-State Cells

Most cellsJor determining steady-state flux reported in the literature consistof two chambers, usually constructed of glass, separatedby a membrane-fasten-ing device (Langguth, 1986b). These chambers may simplybe two Erlenmeyerflasks that have been modified to support a membrane between connecting portson each vessel(Shenouda and Mattocks, 1967; Goldbergand Higuchi, 1968). Theflasks may be immersed in a constant temperature water bath and independentlyagitated with a magnetic stirrer; however, the fluid-mixingefficiency at themembrane interface appears to be questionable in most of these modified flaskdesigns. Stehle and Higuchi (1972) used modified conical flaskswith ground-glassprojections that fit into a machined, Teflon membrane holder, the halvesbeing held together by nut-and-bolt fasteners through brass collars that preventdeformation of the Teflon (Fig. lc). The cells were independently heated by a

480 Smith and Haigh

b@X(Ed *(a) (b) (c)

Figure 1 Diffusion cell designs, adapted from Wurster et aI., 1979; HarperBellantone et aI., 1986; Flynn and Smith, 1971, respectively.

water ja:cket,leavingthe membrane holder exposed to ambient conditions.This appears to present a temperature gradient problem between the bulk cellcontents and the fluid juxtaposed to the membr~e, as it is not certain howefficiently the bar stirring within the bulk chambers will replace fluid at themembrane interface through the horizontal projection tubes.

A similarcell design,with presumably inferior hydrodynamics, is that pro-posed by Wursteret al. (1979) that has no internal means of agitation but isshaken gently in the place of the membrane (see Fig. la). Here, also, the mem-brane is offset from the cell chambers by horizontal tubes, and the membranearea for diffusion is relatively small in comparison with the diameter of thesetubes. The whole assembly is immersed in a water bath and, thus, tempraturegradients are not expected to pose a problem, but boundary diffusion layersin this system are assumedto be extensive. The membrane is held in positionby a complex slide mechanism that is inserted into the connector chassis. Thereplacement of one chamber with a specially designed aperture allowed the ap-plication of gel or gas to the donor side of the membrane. For rapidly diffusing

-compounds (sarin in this case), the small diffusion area and limited mixing ofthe receptor fluid are offset by the rapidly increasing concentration in theheptane receptor solvent. This cell system, therefore, appears adequate forcompounds that diffuse rapidly through the membrane; however, it is postu-lated that the system would not operate as efficiently for drugs that are in-herently slowerdiffusers.

Durrheim and co-workers(1980) used a bichamber glass or polycarbonatecell in the form of two cylindrIcalhalf-cells. Each chamber had two verticalports for samplingthe receptor solution and to accommodate the shaft of themotorized stirrer. The propellers attached to these shafts were positioned closeto the membrane surface within each cell. It is reported that this agitationwas gentle, but adequate, and distributed a dye homogeneously throughoutthe aqueous receptor fluid in less than 2 min. Whether or not this rate of


agitation would be sufficient to minimize diffusion boundary layers at the mem-brane interface is uncertain; the time for dye dispersionquoted here seem un-duly long for such a small chamber volume. However, this designgenerated agood diffusion area/volume ratio that would augment the detection of very smallpermeant concentrations.

Harper Bellantone and colleagues (1986) reported the use of a commercialsteady-state cell for investigating iontophoretically enhanced diffusion (see Fig.1b). This cell consisted of two cylindrical chambers of 5-mlvolume,with themembrane clamped between the connecting flange surfaces,and with electrodes,for iontophoresis, inserted through the vertical samplingports. Mixingin thebulk cell was generated by Teflon bar magnets; however, it is dubious that hy-drodynamics would be optimized by this means of agitation in horizontallymounted cylinders. From the reference diagrams, there appears to be a con-striction in each chamber at the flange that, presumably, would hinder mixing-of the fluid at the membrane surface. Furthermore, the narrowingof the cham-ber diameter near the membrane surface appears to have posed someproblemswith air bubble formation. Total immersion of the system is possible, therebypreventing temperature gradients.

Nakano and Patel (1970) used bi- and trichamber cells constructed of poly-methyl methacrylate (susceptible to adsorption of nonelectrolytes from solu-tion) to investigate salicylic acid diffusion from various ointment basestoaqueous solution through a silicone membrane. The chamberswere not inter-nally agitated, but the whole cell system was shaken in a horizontal plane.Touitou and Abed (1985a,b, 1986) employed a similarshakingtechniqueto agitate their cylindrical cells constructed of Perspex. The cellhalves, sepa-rated by membrane or drug delivery device, were clamped together on a modi-fied shaker plate. They stated that no stagnant diffusion layerswere detectedat shaking speeds between 100 and 200 rpm; however, they did not elaborateon the methodology used to validate this statement. It does not seemprobable,in the absence of a void volume or large air bubbles within the cylindricalcells,that horizontal shaking of the closed system would induce agitation of any sig-nificance. It is postulated that diffusant movement through the phaseswouldbe governed by kinetic motion, in contrast to hydrodynamic mixing,and sub-stantial diffusion layers would form at the membrane interface. Furthermore,Perspex, as a construction material, requires close attention becauseof thepotential for adsorptive interaction with permeants.

A glasscell having a good surface area/volume ratio (0.60) has been used byGaley et aI. (1976). Agitation here was effected by motor-drivenTeflon pro-pellers attached to vertical stirring shafts. A complex bichamber design(Fig.lc), requiring specialized mechanical manufacturing techniques, has beenevaluated by Flynn and Smith (1971). The chambers were machined frombrass and were held together by a large nut-and-thread assembly,the membrane

482 Smith and Haigh

being uniformly clamped between O-ringsseated within the flange surfaces.Agitation was generated in each chamber by relatively large, speciallyformed,Teflon stirrers that were mounted in the vertical plane of the membrane. Thisnecessitated horjzontal stirrer shafts to protrude from each chamber and gasketseals to prevent any leakageof chamber fluid through the shaft housing sleeves.Gears, attached to the protruding stirrer shafts, were interlocked with the gearsof a synchronous motor that would drive the stirrers in each chamber at thesame rate. Samplingof chamber fluid was effected through vertical samplingports and a perforated screen could be used to support the membrane whennecessary. This designappears ideal in many respects: good membrane arealvolume ratio (0.67), total temperature control by complete immersion of theassembled unit, even sealingpressure on the membrane, and optimal agitationat the membrane surface. As anticipated, there was a plateau stirring speed abovewhich no increase in the drug permeation rate was observed. However, thecomplexity of this designdictates that it be manufactured by an engineeringconcern, and it requires mountings for motors, Shafts, and cogs. The cost is,therefore, assumed to be appreciable, whereas all the advantages of this designmay possibly be duplicated by a well-planned, simply-constructed, glasscell andmodified, common laboratory apparatus. The large diffusion area of this model,although useful from an analytical viewpoint, limits the applicability of thesystem to membranes in abundant supply.

The most useful designappears to be one proposed by Southwell and Barry(1981,1984). Two glasschambers with a relatively small diffusion area (morein keeping with the sizeof readily available biological membranes) are immersedhorjzontally in a water bath (Fig. 2a). Agitation is effected by Teflon-coatedbar magnets; a singlelaboratory magnetic stirrer positioned below the cell sys-tem effectively induces synchronous stirring of the bars in each cell. Stainless-steel mesh has been used to support delicate membranes without, apparently,

(a) (b) (c)

Figure 2 Diffusion cell designs, adapted from Southwell et at, 1984; Tojoet at, 1985b,a, respectively.

484 Smith and Haigh

of the stirred receptor phase is attained approximately 1 min after the intro-duction of a drug aliquot through the sampling port. This value comparedfavorably with the 10 min required for equilibration in the Franz cell (describedlater). Furthermore, calculation of the theoretical diffusion boundary layerthickness, using data from the aqueous dissolution profile of benzoic acid disks,yielded an approximate value of 0.054 mm for the Valia-Chien cell, which wasfive times smaller than that calculated for the Franz cell. This cell system hasbeen theoretically characterized in terms of Sherwood-Reynolds-Schmidt rela-tionships by Tojo et al. (1985a). They propose that this characterization allowsthe estimation of aqueous diffusion layer thickness and calculation of the intrin-sic permeation rate of any compound under ideal hydrodynamic conditions fromdiffusion measurements taken under nonideal conditions. This exercise seems tobe of only academic interest, as it may be possible to design a diffusion cell con-figuration that has near-ideal fluid-mixing hydrodynamics, and measurementsunder these near-idealconditions may be made qirectly without the necessityfor resorting to theoretical interpretations.

A similar,commercial,diffusion cell (see Fig. 2c) has been developed andevaluated by Chien and co-workers (Ghannam et al., 1986; Liu et aI., 1986;Tojo et al., 1985a,c,d) and has been used by severalresearchers (Lee et al.,1986a,b; Liu et al., 1985; Sun et al., 1986). The cell comprises two L-shapedchambers ofvolume 140-250 ml which, when fastened together, provide anarea of 13. 9 cm2 for drug diffusion. The base of the vertical arm of each cham-ber has a recessedstirring platform that accommodates a magnetic stirrer bar,and both chambers of the assembled cell are placed over a synchronous stirrerunit. The vertical arms of each chamber are water jacketed for temperaturecontrol, and each has a samplingport with cap to prevent evaporation of thefluid. Again,stirring the fluid in the horizontal arms of the chambers may benonideal, and the absence of temperature control along these arms may generate

gradients between the membrane surface and bulk fluid. However, as with theprevious design,the temperature of each chamber may'be individually controlledwith this apparatus, a distinct advantage over simple immersion of the entiresystem in a water bath.

Tojo et al. (1985a,c) evaluated this system along similar empirical principlesas described before, using data from the dissolution rate of a benzoic acid disk.Valuesfor the theoretical hydrodynamic constants and their interrelationshipare proposed for correcting the effect of diffusion boundary layers on the drugpermeation rates obtained under nonideal flow conditions'. Cell shape here de-viates markedly from the ideal; the authors again proposed that this may validlybe counteracted by the application of correction factors based on theoreticalmathematical hydrodynamic models. Furthermore, certain of their assumptionsmade in applyingthe theoretical model (initially proposed for longitudinal fluidflow in cylinders) to static hydrodynamics in diffusion cells may be debatable.


In essence, this practice appears to complicate the permeation monitoring pro-cess: a computational correction is required for a nonideal cell designin prefer-ence to the adoption of a design with more optimal hydrodynamics.

These zero-order, steady-state diffusion cells are useful in that a number ofkinetic events may be monitored simultaneously. The massof drug entering themembrane may be assessedby monitoring its diminution in the donor chamberwhile the mass of drug passing through the membrane is represented by its ap-pearance in the receptor fluid. The fraction of drug bound within the membranemay then be assessedat any time by the difference in massof the drug enteringand leavingthe membrane (Scheuplein, 1978). This ability to estimate thedegree of binding or reservoir formation within the membrane is useful in manyinstances., Moreover, the cells are useful for determining intrinsic diffusivityofa molecule through a specific membrane in the controlled absenceof factors -that may enhance or retard the permeation process.

CELLS FOR SIMULATINGIN VIVO CONDITIONS

The steady-state diffusion techniques described in the precedingsectionsgen-erate nonphysiological solvation of the membrane throughout the experiment,and this may, in part, account for the discrepancies between reported permea-tion results. A system that parallels in vivo conditions more closelywould requirehydration and temperature gradients to be established acrossthe membrane,with only the receptor surface of the medium exposed to the solvent(Akhterand Barry, 1983a). This has been achieved by single,fluid-filledchamberscon-taining agitated or recycled receptor phase (simulating the sink clearancecondi-tions of the vasculature in vivo; Table 4). The donor chambersof these cellsneed not resemble the receptor chamber in volume or shapeand may comprisea simple cylinder to contain the vehicle applied to the horizontally mountedmembrane. The donor vehicle is usually not agitated in the simulatedconfigura-tion, rep~~sentingthe clinical situation in which a topical product would be ap-plied to the skin. In this situation the drug and vehicle interact with the mem-brane and atmosphere in a manner (evaporation, permeation, or depletion) simi-lar to that occurring in clinical use of the delivery system. Absorption of com-pounds may thus be studied under experimental conditions most applicabletotheir in vivo usage.

Coldman et al. (1969) have made use of this technique for examiningtheeffects of supersaturated drug solutions, with concomitant thermodynamicpotential, prepared by a combination vehicle of volatile and nonvolatilefrac-tions. On application, the volatile component evaporates, leavingthe drug inlipophilic solution of high concentration; an experimental procedure that isimpossibleusing the steady-state apparatus described previously.

- In this "in vivo-mimic"situation the donor vehicle character may vary widelyfrom the solutions used in steady-state experiments. A film of drug depositedby solvent evaporation, one of the numerous types of topical formulations ofdiversematrix nature, or a transdermal delivery device may be used as the sourceof permeant. Other benefits of this cell configuration include the ability tocontrol variablesof the donor compartment, such as humidity or exposure toatmospheric conditions, thereby simulating occluded or nonoccluded clinicalconditions. In this manner, the influence of hydration on the permeability ofthe membranemay be assessed. Additionally, the membrane may be treatedwith chemicals(e.g.,penetration enhancers) before or during exposure to thepermeant, to ascertainwhat effect they have on permeability. It is feasible toapply sequential treatments to the same membrane, the latter may thus act asits own control. Cooper and co-workers (1985) investigated the use of fattyacids and alcoholsas penetration enhancers for acyclovir. Barry (1983) and

486 Smith and Haigh

Table 4 In Vivo-Mimic Diffusion Systems with a Rate-Limiting Membrane

Author Description Receptor Diffusant

Frimz (1975) Dumbbell (stirred)/skin Water Various

Bronaugh (1984) Flow-through/skin Various Lipophilic drugs

Washitake (1980) T-cell/egg shell membrane Water Betamethasone

Coldman (1969) Vertical cylinder/skin Water Fluocinolone

Akhter (1982;1984; Biconcave glass/skin Buffer Profens1985)

Ainsworth (1960) Flow-through-stirred/skin Water Tributyl phos

Foreman (1978) Flow-through/ skin Water Nandrolone

Michniak (1983) Plexiglass mouse skin Buffer Corticoids

Ogiso (1986) Franz cell/rat skin Buffer Indomethacin

Chien (1983) Vertical cylinder/mouse skin"

Saline Nitroglycerin

Keshary (1984) Vertical cylinder/mouse skin Saline Nitroglycerin

Kneczke (1986) Vertical cylinder/cellulose Water Salicylic acid

Crutcher (1969) Flow-through/guinea pig skin Saline Testosterone

Blank (1964) Flow-through-stirred/ skin Water Alcohols

Guy (1986) Flow-through/mouse skin Buffer Nicotinates

Okamoto (1986) Flow-through-stirred/ skin Saline Parabens

Hawkins (1986) Penetration-evaporation/pig skin Aqueous Various

Bronaugh (1985a) Flow-through-collected/rat skin Saline Cortisone


Akhter et al. (Akhter and Barry, 1983a,1984,1985; Akhter et al., 1982) re-ported good experimental results when investigating the sequential effects ofocclusion and of adding penetration enhancers, such asN-methylpyrrolidone,on the permeation of nonsteroidal anti-inflammatory agents from acetone-de-posited fIlms through human skin. Therefore, a number of experimental vari-ablesmay be investigated using the same in vivo-mimicapparatus; the advantagesof using these systems are apparent in comparison with the steady-stateappara-tus described previously.

In most investigations it is assumed that the drug concentration in the donorphase does not diminish significantly during the time course of the experiment("infmite-dose experiments"). Mter an initial transient period of membranesaturation (lag time), and provided sink conditions are maintained in the recep-tor chamber, the concentration gradient across the skin (and thus the drug per--meation rate) should remain constant. This may provide valuableinformationabout the steady-state drug diffusion and lag times when semisolidtopical form-ulations are applied to the membrane, in comparison with simpledrug solutions.

However, this infmite dose/steady-state situation does not parallel the formu-lation use in vivo, in which a fInite amount of product is usually spread on theskin in a thin layer. The concentration or solubility of the drug in this layer maychange as water evaporates from the dosage form, or formulation constituentsare absorbed by the skin, thereby changing the thermodynamic potential of thediffusant in the vehicle (Tanaka et al., 1985). Furthermore, a relativelythicklayer of vehicle applied in vitro may maintain the membrane in a hydrated state,swellingthe tissue and decreasing its barrier properties. Therefore, if in vivo useis to be simulated as closely as possible, severalfactors favor a semifmite-dosetechnique as described by Ainsworth (1960), or a fInite-dose technique as pro-posed by Foreman et al. (1977,1978) and Franz (1978), that has been used byother researchers (Akhter and Barry, 1983a,1984,1985; Akhter et al., 1982;Southwell and Barry, 1984; Southwell et at, 1984). This method requires thata fInite vol~me of drug solution, in volatile solvent, be applied to the membraneand evaporated to dryness to form a fIlm of drug on the medium. Initially, aperiod of rapid permeation may be experienced because of enhancedpartition-ing of the drug from the deposition solvent, prior to the total evaporation of thelatter (Akhter and Barry, 1985). Maximum partitioning usually occurswhen thedrug solution is saturated, representing the condition of maximal thermodynam-ic potential. The permeant flux rate decreases as drug is precipitated from thesupersaturated solution, and permeation from the deposited ftlm supersedesthatinduced by initial partitioning from the solvent. Permeation of drug from thisfilm passes through a maximum value with time, and then declinesas the per-meant is exhausted from the deposited layer (Scheuplein and Ross, 1974).

However, although it may be facile to produce an even ftlm of drug on themembrane by solvent evaporation, it is postulated that spreadinga fmite amount

488 Smith and Haigh

of ointment, for example, on to fragile biological tissue in a thin, uniform layerwould be difficult, although this has been accomplished with full-thickness ratskin and petrolatum vehicle (Bronaugh and Stewart, 1986a). In those situationsin which a relativelyviscous semisolid is to be treated, the use of an infinite-dosetechnique is the most feasible method of experimentation.

It must be assumed that dissolution of the deposited drug is not the rate-limiting step to absorption and that the solvent does not alter the biochemicalproperties of the membrane appreciably; both important assumptions that mayrequire validation. Akhter and Barry (1985) observed that permeation of certainnonsteroidal anti-inflammatory agents from deposited films was governed by therate of drug solubilization at the skin surface, demonstrated by the fact that in-creasesin the concentration of drug in the deposited film did not increase thepermeation rate, whereas more concentrated solutions produced greater fluxvalues. They found that occlusion of the donor chamber did not increase therate of solubilization from this deposited film, but it did enhance the flux ofdrug already present in the stratum corneum. Additional care, therefore, is re-quired if this methodology is to be adopted; it must be shown that solubiliza-tion of the drug is not the rate-limiting step to diffusion. One remedy has beenimplemented by Michniak and co-workers (Michniak, 1983; Michniak-Mikolaj-czak and Bodor, 1985) who applied drugs in acetone or methanol/isopropylmyristate solvent combinations: the evaporation of the volatile componentleavesthe drug in lipophilic solution spread thinly over the membrane surfaces,thereby alleviatingdissolution rate problems.

The evaporation of small solvent volumes does not, presumably, adverselyaffect the diffusion medium. Akhter and co-workers (Akhter and Barry, 1985;Akhter et al., 1984) report that contact of the skin with acetone, used to de-posit drug films, for periods from 2 min to 2 h did not significantly alter thepermeability of the tissue, confirming earlier reports by Scheuplein and Ross(1974). In addition, Bennett and Barry (1983) observed unchanged tritiatedwater permeability through human skin after sequential treatment with a num-ber of alkanolsand conclude that the skin had not been damaged by this treat-ment. In contrast, Waraniset al. (1987) reported that a 3-min methanol washof hairlessmouse skin nearly doubled the flux of theophylline in propyleneglycolvehiclethrough this tissue. It would, therefore, appear that each finite-dose technique must be individually evaluated to determine the effect of thedeposition solventon the barrier properties of the membrane.

Static InVivo-MimicDiffusion Cells

Most of the cellsemployed in this experimentation are similar in design: a lowerreceptor chamber incorporating some means of agitation (usually a Teflon-coated bar magnet) with an inclined, stoppered side-arm attached to the cham-


ber through which the receptor solution may be sampled. The membrane ismounted horizontally between the flanged edges of the lower chamber and theupper donor container and may be supported by a stainless-steelsieveor parallelrods spanningthe aperture. Teflon washers may be used to sandwichthe mem-brane and, thereby, provide a better seal. The flange edgesare mechanicallyheldtogether making the joint watertight, and the donor container may have someform of closure, dependent on the experimental conditions. Fluid is filled intothe cell to the levelof the membrane so that no hydrostatic strain is imposed onthe medium, and any air bubbles that form may be expelled through the in-clined samplingport by tipping the cell. The entire unit is usually immersedin awater bath to the level of the flange so that the donor vehicle is not heated. Anumber of these cells may be arranged around a singlemagnetic stirrer for repli-cate runs. One feature found in several reported cell designs is a narrowing of -the chamber diameter in the flange region, so that the membrane is offset slight-ly from the cell body by a narrow cylinder. It is envisagedthat fluid flow wouldbe hindered into this cylindrical connection, thereby generating inferiormixinghydrodynamics and extensive diffusion boundary layers at the membraneinter-face.

An early cell design (Fig. 3a) proposed by Coldman et al. (1969), and subse-quently used by severalresearchers (Chowhan and Pritchard, 1978;Haleblianet al., 1977;Ostrenga et al., 1971; Young-Harveyet al., 1986), consistsof avertical cylindrical chamber with an inclined sampling side-arm,of slightlysmaller diameter, attached to the chamber body near its base. The membranewas mechanically clamped between two Teflon disks to the upper surfaceof thecell body. Fluid agitation was generated by a Teflon bar magnet to which apolyethylene sail, slightly shorter than the cell body, was attached. The authorsreported that this provided efficient mixing, but they did not present validatingdata. It is assumed that a large portion of the 1D-mlreceptor fluid would becontained within th~ sampling side-arm and would not mix adequately with the

tV(a) (b) (c)

Figure 3 Diffusion cell designs, adapted from Coldman et aI., 1969; Southwellet aI., 1984; Franz, 1978, respectively.

490 Smith and Haigh

bulk fluid of the cell, generating a possible source of sampling errors. The diam-eter of the fluid-filled cylinder connecting the under surface of the membraneto the bulk cell fluid is approximately half the diameter of the cell body. Fluidmixingandmasstransferwithin this constrictionmaybe further impaired..

A useful design is that of Southwell and co-workers (Southwell and Barry,1984; Southwell et al., 1984) and Akhter et al. (Akhter and Barry, 1985; Akhteret al., 1982), who used a biconcave cell, agitated by a bar magnet, with an in-clined, stoppered side-arm (see Fig. 3b). The membrane is supported by a stain-less steel screen and the upper, donor compartment has an innovative well thatprevents spillageof the donor vehicle when the cell is tipped to expel air bubblesfrom under the membrane. One postulated drawback of this design is the con-nection of the inclined samplingport near the base of the cell. This may notsupport efficient mixing of the fluid in the sampling tube with that in the bulkcell. The connection near the base dictates that the tube be especially long sothat its orifice is positioned above the level of the membrane and, hence, a sub-stantial proportion of fluid is contained within tllis sampling port. It is impera-tive that efficient mixing of fluid in this port with that of the cell body be in-duced for accurate sampling. A similar design constructed of Plexiglashas beenused by Nacht et aI. (1981) for measuring benzoyl peroxide permeation.

Franz (1978) designed and tested a cell system along these lines (see Fig.3c) that has subsequently been commercially marketed and has been widelyused (Chow et aI., 1984; Iyer and Vasavada, 1979; Mirejovsky and Takruri,1986;Touitou, 1986, Waraniset al., 1987). This cell has a small, upper, donorcompartment open to the atmosphere and a dumbbell-shaped receptor com-partment (of 12-mlvolume) comprising a lower ellipsoid bulb (containing themagnetic stirrer bar); an intermediate, narrower cylindrical tube; and a wideupper chamber in contact with the membrane. An unstoppered samplingportis connected to the upper segment of the cell. The compartments are held to-gether by a springclamp and, surprisingly, only the central, cylindrical, portionof the receptor chamber is surrounded by a thermostated water jacket. Severalof these cells may be mounted in a specially designed housing block containingthe manifold connections for the heated water jacket and the magnetic stirrers.

Ogisoet aI. (1986) used the Franz cell with an a-ring flange to measurethe diffusion of indomethacin from various ointments and gels, and they re-ported good correlation with in vivo drug absorption studies. These workersconcluded that it is reasonable to assume the results from the in vitro experi-ments adequately reflect the in vivo transderrnal absorption of the drug. Thisreport exemplifiesthe fact that even though mixing hydrodynamics in a diffu-sion cellmay be inadequate the system may still be useful for screeningpur-poses. However,the variance between experiments is expected to be relativelylargebecause smalldifferences in agitation speed/may influence diffusion bound-ary layer thicknessextensively. Sheth et al. (1986) used the Franz diffusion cell


for penetration-enhancing investigations, but they incorporated a speciallymanufactured elongated stirrer bar, presumably to improve mixing hydrody-namics.

Although the Franz cell has been used extensively for skin permeationstudies, it has severalobvious nonideal features. Keshary and co-workers(Chienet al., 1983; Keshary and Chien, 1984) summarized these shortcomingsafterevaluatingthe cell for the release of nitroglycerin from severalcontrolled-releasetransdermal therapeutic systems, and Chien and Valia (1984) compared the hy-drodynamics of the Franz cell to that of their own design. They reported thearchitecture of the Franz cell did not provide adequate solution hydrodynamics,mixing efficiency, or temperature control required for quantitative permea-tion evaluations. Closeex.amination of the Franz cell designoffers immediateexplanation for some of these inadequacies: it is improbable that adequate -agitation of the entire receptor cell contents may be transmitted from thestirred lower bulb, through the narrow cylindrical connection to the widerupper compartment. The resistance to laminar fluid flow generated by thisinternal cell topography is simply too great to induce adequate verticaifluidmovement and, therefore, the existence of large boundary diffusion layersatthe membrane interface is anticipated. These relatively static, diffusion layersare reported to be fIvefold greater in the Franz than in the Valia-Chiencell(Chien and Valia, 1984), and may greatly increase the overall resistanceto drugpermeation.

A permeant concentration gradient would also be established through thecell, being greatest at the membrane and most dilute in the lower bulb. Equili-bration times of 30 min are reported by Chien and Valia 1984) for the Franzcell after introduction of a drug aliquot into the upper portion of the chamberand subsequent sampling of the drug solution from the lower bulb. Samplingwould, therefore, take place from a solution hetergeneous with the drug con-centration. When this is coupled to the poor vertical fluid movement, therewould probably be inadequate thermal transmission from the centrallyheated portion of the compartment to the membrane surface and lowerbulb:a 4°C discrepancy has been measured (Chien and Valia, 1984). Thiswouldintroduce further variables into the system if ambient conditions vary duringan experiment. Obviously, these problems are amplifIed if solventsmore vis-cous than water are used; a factor that has received little investig~tion.

These design shortcomings have been experimentally verified by Gummeret al. (1987), who evaluated various Franz cell designs for uniformity of stirring.By measuring the time required for homogeneous dye dispersion, they ob-served that the fluid in the sampling side-arms and in the upper portions of thesedumbbell-shapedcells was not adequately agitated by magnetic bar stirringinthe lower portion. In some caseshomogeneity was not achieved, evenafter 30min of stirring.

492 Smith and Haigh

On the basisof these shortcomings Keshary et aI. (Chien et aI., 1983;Kesharyand Chien, 1984) proposed a number of modifications to the basic Franz designthat they believedwould greatly improve the hydrodynamics of the cell. The re-ceptor compartment was widened into a simple cylinder of diameter 20 mm, andthe height of the cell was reduced to 50 mm, producing a volume of approxi-mately 15.7 mI. The dimensions of the heated water jacket were increased sothat the lower 38 mm of the cell was completely enclosed and a star-head mag-net was incorporated in place of the simple bar magnet to generate a more effi-cient fluid mixing pattern. A glassstopper was introduced into the samplingport to minimize evaporation of the receptor solvent; an important factor whenusingvolatile solvents (including water) at 37°C for severalhours or days. Theperformance of the improved design was compared with that of the commercialFranz cell for a number of physical variables. Equilibrium temperature main-tenance in the bulk phase and at the membrane interface was more easily attain-able, with lessvariation than in the Franz design., A temperature difference ofS.SoCwasobserved between the two cells, as measured at the membrane surface,that produced a 12.4%difference in the measured release rate of nitroglycerin.Solution mixing was substantially improved by a fourfold factor, as assessedbythe rate of attainment of solution homogeneity after introduction of an aliquotof diffusant. The calculated thickness of the boundary layer is reported to bethree times smaller in the Keshary-Chien cell, thereby proportionately reducingthe contribution of this layer to the overall diffusional resistance. It can be seen,therefore, that simple modifications to cell shape may yield greatly improvedpenetration profiles of greater validity, simply by improving the hydrodynamicfluid flow characteristics. It is unfortunate that the authors did not reportpermeation results at agitation speeds other than 600 rpm or the use of receptorsolventsother than water so that a full evaluation of the performance of theirimprov~dcell could be assessed.- - This Keshary-Chiencell design has been used successfully by Kneczke et al.(1986) to investigate the effects of white petrolatum crystallinity and viscosityon the releaseof salicylicacid from ointment formulations through a cellu-lose membrane. Their experiments were performed at 400 rpm agitation; againno data for alternative agitation speeds or temperatures were presented.

A number of alternative designs for simulating in vivo conditions have beenreported. Washitakeet al. (1980) used a modification of the T-shapedcell toinvestigatethe diffusion ofbetamethasone 17-valerate from ointment bases.Eggshellmembrane was placed over both open ends of the inverted T-cellandointment applied to the external surfaces of these membranes. The cell wasfilled with aqueous receptor fluid, and the unit shaken horizontally to induceagitation. Severalshortcomings are immediately obvious in this design. Thequestion of agitation efficacy derived from simple shaking of the cell has beenaddressed previously. Furthermore, the authors noted that the receptor fluid


diffused through the membrane, presumably augmented by the hydrostaticfluid pressurewithin the cell, and interacted with the donor foI'mulations.

Flow-Through InVivo-MimicCell Designs

As an alternative, cells having a flow-through receptor compartment instead ofa samplingport have been used. Here the contents of the receptor cell are reoplaced entirely with fresh solvent at specific intervals or continuously by pumpmechanism. The receptor chamber is connected to a peristaltic pump that re-places the solvent several times each hour at a rate sufficient to maintain sinkconditions for diffusion (typically 70 times an hour for a small cell). The flow-through receptor chamber is generally much smaller than the static cell type,typically having a volume of 30-40 Jll. This is mainly because severalcellvol- -urnes of receptor fluid must be pumped through the chamber during the courseof an experiment and, thus, to maintain the total amount of fluid at manageablelevels,the cell sizemust be relatively small. Furthermore, most cellshave a rela-tively small diffusion area of approximately 0.1 cm2, making these systemseconomical as far as membrane requirements are concerned.

Sink conditions are maintained with relative ease in flow-through systemsbecause permeant is immediately swept away by the fluid. Crutcher and Mai-bach (1969) suggestedthat the increasing concentration of penetrant in thereceptor solution may limit further permeation (especially of relativelywater-insoluble permeants), a situation that is not experienced in vivo. They found thatmeasured permeation rates increased in proportion to an increase in the receptorfluid perfusion rate. They conclude that in vitro work should strive to duplicatethe true sink conditions experienced in vivo by either frequ~nt perfusate re-placements or continuous flow of the receptor medium. Wester and Maibach(1985) reported that flow.through receptor cells are more useful than staticcells for this, because sink conditions can be maintained more adequately inthe flow-through cell for sparingly soluble diffusants.

Although continuous flow of solvent through the receptor cell may inducesome agitation, it is assumed that a periodic replacement of the entire cell solu-tion, without further means of agitation, would create considerable diffusantboundary layers at the membrane interface. Solvent infusion pressure cannotusuiuIybe used as an agitation aid, for undue stress may be placed on the memobrane. The cell designer must, therefore, infuse and effuse liquid in such a waythat permeant is completely swept away from the undersurface of the memobran.e,minimizing diffusion layer formation. Foreman and co-workers(1977,1978) have used such a cell for fmite-dose diffusion studies. The inlet tube oftheir flow-through design directed the fresh solvent against the center of themembrane undersurface while effusate was removed through a port at the baseof the cell. Obviously, appropriate receptor cell shape would enhance mixing

494 Smith and Haigh

~(a) (b)

Figure 4 Diffusion cell designs, adapted from Marzulli, 1962; Okamoto et aI.,1986, respectively.

hydrodynamics and it is assumed that basic cylindrical chambers (Aguiar andWeiner,1969; Marzulli, 1962) are not ideal in this regard (Fig. 4a).

Noting this, Blank (1964) included a stirrer in,the receptor chamber togetherwith the flow-through fluid arrangement, similar to the designproposed earlierby Ainsworth (1960). This inclusion improves mixing hydrodynamics and, sub-sequently, has been adopted by severalworkers, including Guy et al. (1986) andOkamoto et al. (1986), who have used this combination stirrer/flow-throughdesignin a relative large-volumereceptor chamber (see Fig. 4b). In this instancethe fluid agitation was totally dependent on the magnetic stirrer and no relianceis placed on the infusion of fluid to generate mixing. The hydrodynamics ofthis systemare, therefore, assumed to be superior to the solitary flow-througharrangement. A penetration-evaporation cell designed along similarprincipleshas been used by Hawkins and Reifenrath (1986). The receptor chamber intheir designconsisted of a vertically mounted, thermostatically jacketed, cylin-der with flow-through fluid attachments, to which the membrane was fixed by

3D O-ring,and agitation was affected by a magnetic stirrer bar. The elaboratedonor chamber was designed to enhance evaporation from the membrane sur-face by forced, heated, air ventilation. Gummer et al. (1987) have designed twoagitated/flow-throughpermeation cells that have been validated for stirringefficiency. They have completely enclosed their conical/cylindrical chamberswith a waterjacket and have maintained the cell volume as smallas possiblewhile optimizingthe diffusion surface area for their particular membrane re-quirements.

Severalmodifications of this basic designhave been promulgated in anattempt to relieve the researcher of the repetitive samplingand solvent replace-ment duties during each experimental run. A continuous flow-through stainlesssteel cell connected to an automatic sample fraction collector has effectivelybeen employed for this by Akhter and co-workers (Akhter and Barry, 1983b;Akhter et al., 1984). The conical donor compartment of their cell facilitateddeposition of donor vehicle on to the membrane. Smallmetal rods may be


placed under the membrane to further reduce the volume of the cell and to gen-erate additional turbulence as the fluid flows through the cell. The formationof air bubbles is reported to be a problem in this apparatus, even smallbubblestrapped beneath the membrane will reduce the diffusion area substantially. Theuse of in-line bubble traps and the degasing of solvents, especiallyhydroalcoholicfluids, usually eliminates this drawback. The chambers are distally connectedto vials in a rotating fraction collector that will sample the perfused solution atregular intervals. Severalcells may be assembled radially on a heated, coppermounting block, over a circular turntable that will sequentially move the col-lection vialsunder the effusion ports of the cells. Although this cell designisespecially useful for scintillation counting of radiolabeled permeant, a directconnection.of the fraction collector to spectrophotometric detection instru-ments is also possible. -

A similar,sophisticated design is described by Bronaugh and Stewart(1985a,b; 1986a,b) and Fisher (1985). Their two-piece flow-through cellsweremachined from Teflon so that the top section could be screwed into the base,securing the membrane fmnly over the receptor chamber (O.4-mlvolume). Theinlet tube diameter is approximately equal to the depth of the chamber and, tocreate a slight backpressure and facilitate mixing, the chamber has a smalleroutlet tube diameter. This generally ensures intimate contact of fluid with themembrane. As expected, observed permeation rates increased with perfusionrate of the receptor celt up to a limiting value. This plateau value would obvi-ously be dictated by the partitioning of the diffusant into the perfusingmedium.A smallglassplug inserted into the base of the cell allowed inspection of mem-brane undersurface for air bubbles. Several cells may be fitted into a heatedaluminum holding block for temperature maintenance. Again, a fraction collec-tor is employed to sample the effusate at specific intervals.

The performance of this cell configuration was compared with results fromstatic diffusion cell systems for the permeation of tritiated water, cortisone,and benzoil;acid through rat skin. The results demonstrate insignificantdif-ferences in the absorption profiles obtained from the two cell designsand goodcorrelation between these data and in vivo studies. For relatively water-insol.uble compounds, the flow-through system demonstrated greater overallper-meation than the static designusing saline receptor fluid. Semisaturation ofthe static fluid is suspected here, with lowered partitioning potential. Maxi-mal permeation of these compounds in either system was effected by the useof surfactant solutions, as receptor media, and resulted in flux values ofapproximtely equal magnitude for the two designs. These results are impor-tant from a developmental viewpoint because they indicate that if the recep-tor medium is optimized for the specific permeant and membrane systemundertest, in conditions of optimal agitation, the flow-through diffusion cell designoffers few obvious advantagesover the static fluid design relative to the kinetic

496 Smith and Haigh

data generated, the only advantage being that sample collecion is automated.Bronaughand Stewart concluded that the flow-through system, using nonopti-mized receptor fluid, could not outperform the use of cosolubilizers in eithercell system. Bearingthe cost and complexity of flow-through cell fabrication inmind, it is postulated that optimization of a static designmay be the best entry-levelposition for researchers in this field.

In summary, the advantages of a flow-through system are multifold: smallmembrane samples are required and, therefore, several, replicate experimentsmay be initiated using a typically sized autopsy skin specimen. The flow-through system may be employed with both fmite and infinite doses of diffu-sant from a variety of donor vehicles for the simulation of in vivo or steady-stateconditions. Sequential treatment of the skin or deposited donor ftlm is possible.The perpetual infusion of solvent maintains sink conditions for the permeant,and the sample collector eliminates the tedium of repetitive sample acquisition.However,the design is complex from a manufacturing viewpoint, and the sys-tem is totally dependent on the synchronization of pump and fraction collec-tor. The expense of establishing such a system is, therefore, fairly large. More-over, overt performance superiority of the flow-through system compared withoptimized static fluid cells has not been clearly established.

SUMMARY

Howeverideal the in vitro cell design may be, the discrepancy between thekinetic events occurring in the laboratory and those that take place in humanskin is extensive. Invitro methods measure steady-state or finite-dose permea-tion from infinitely concentrated donor vehicles that may contact the mem-brane for severaldays. Clinical use of the drug may involve inunction of a small

_amount of formulation into a large area of skin, with removal of residual formu-lation by washingsome hours later. Correlation of data from laboratory investi-gationswith in vivo trials has also proved difficult because tests in humans oftenmeasurea physiological response to the permeant (such as vasoconstriction) oruse urinary excretion assays (with their complications of systemic metabolism,binding, and compartmentalization). Especially obvious is the difference in massof permeant required to elicit a measurable response in the two modes of investi-gation. Severalmicrograms of diffusant generally mark the limit of detectionin an in vitro cell system, whereas a number of molecules of drug reaching thevasculaturemay induce a vasoconstrictive response and be assessedby humanblanchingtrial methodology. Furthermore, laboratory techniques generallymeasure steady-state diffusion across the entire stratum corneum barrier, where-as in vivoresponsesmay be elicited during the transient phase by rapid drugdiffusion through shunt routes-this initial, rapid drug absorption (before steady-state achievement) is undetected in many in vitro protocols. Notwithstanding


these restrictions, the in vitro experimental techniques do have an importantpart to play in developmental dermatopharmacy. Well-designedinvestigationswill, in many instances, provide useful information about the diffusivecharac-ter of a molecule or the release characteristics of a topical formulation, theacquisition of this knowledge being essential before human trials are initiated.

HavingconfIrmed the necessity for laboratory studies, the foregoingaccountof in vitro diffusion systems exemplifies the formidable task in the choice of arepresentative model for permeation studies. The multitude of cellvariables,such as temperature, degree and mechanism of agitation, samplingprocedure,permeant solubility, and the delivery vehicle, must be controlled precisely,along with the area, thickness, integrity, and biological properties of the mem-brane (if one is included in the system). A design equilibrium must be achievedbetween the variables and controls, so that accuracy and precision are maxi-mized. Care exercised in the design and evaluation of a new in vitro diffusion'cell system will obviously enhance the credibility of the results obtained. Anynew system, therefore, initially requires a complete analysis of its performancewith respect to the common in vitro variables. Only once this has been ac-complished, and satisfactory results obtained, may experimental permeationdata be reported with any degree of confidence.

The major problem envisagedwith most of the cell designs is the absence,inadequacy, or nonuniformity of fluid agitation within the chambers. Otherproblems that may be experienced, but are seldom mentioned, include airbubble formation on the underside of the membrane (that may be diffIcult todislodgeonce the diffusion run has been initiated), the presence of a hydro-static fluid pressure that may cause the membrane to' distend, and obtaininga uniform seal around the membrane flange. The following, summarizedfea-tures may be proposed as those of an ideal in vitro diffusion cell system:

1. An optimal diffusion area/volume ratio that will allow sensitivepermeantanalysisin the receptor medium, especially at early samplingtimes whenpermeant concentrations are low. The proviso here is that the diffusionorifice, and the surrounding membrane-securing flange, should accommo-date the smallest area of biological tissue usually obtained by the particu-lar sampling technique employed. Large sheets of synthetic media are inample supply and will not dictate the diffusion area of the cell.

2. Homogeneous fluid mixing must be generated throughout the chambers(at the membrane surface, in the bulk fluid, and within the samplingarm)and should be sufficiently vigorous to minimize diffusion boundary layers

. at the membrane interface. This would require an internal, mechanicalmixing procedure in contrast to simple shaking of the cell.

3. The cell design must enhance rapid temperature equilibration within thechambers so that gradients are not established between the bulk cell con-

498 Smith and Haigh

tents and the membrane interface. Adequate thermal control would prob-ably require total thermostatic envelopment of the cell chambers andmembrane region, either by complete immersion of the cell in a water bathor total water jacketing of the chambers and flange.

4. The conformation of the diffusion cell chambers should be as uniformas possible,with a minimum of appendages and no physical constrictionsbetween the agitated bulk cell fluid and the membrane surface.

S. The samplingport. should form part of the cell body and should be at-tached in such a fashion as to facilitate adequate mixing of its contentswith the bulk chamber fluid. The port should not contain a relativelylargeproportion of the chamber fluid and should have some stopper sys-tem to prevent evaporation of chamber contents. A hydrostatic fluidpressure should not be generated on the membrane, and the port should,ideally, allow the facile expulsion of any air bubbles that may fo~ andbecome trapped on the undersurface of the membrane.

6. The cell design should be easily constructed with basic laboratory materi-als. Glassis the ideal construction material because it is inexpensive,it iseasilyworked into any idealized conformation, it is fairly inert to thenormal laboratory chemicals, and it supports rapid thermal conduction.A totally inert material is not yet available, hence, the need for appro-priate performance validation. Careful design and planning may incorpor-ate other basic laboratory equipment into the diffusion cell system, suchas thermostated water baths 01'magnetic stirrers, thereby further simpli-fying construction and reducing cost without sacrificingsensitivity in themonitoring of permeation.

7. Optimal designwould also produce a cell that is versatile in performance:one that may be used for both steady-state and in vivo-mimic,ftnite- andinfmite-dose diffusion experiments.

8. A rate-limiting, discriminating membrane appears essential for demon-strating subtle differences between the drug release characteriSticsofsimilartopical formulations and for estimating drug absorption rates thatmay be expected in vivo. Equally, the receptor phase should comprisea relatively innocuous solvent, but one in which the permeant is sufft-ciently soluble to facilitate partitioning from the membrane. This mayrequire surfactant or lipophilic addition to aqueous media to enhanceits biochemical similarity to the physiological environment of the skin.

Although these features may represent the ideal diffusion cell design, the in-corporation of all these facets into a single system may be impractical;however,as many as possible should be included into any cell system proposed for study.In addition, it has been established (Bronaugh and Stewart, 1985a,b; 1986b) thatflow-through sell designsexhibit little advantage over static designsrelative topermeation kinetics obtained, if the conformation, receptor medium, and agita-


tion of the static systems have been optimized. Bearingin mind the additionalcost of construction and ancillary equipment required for the flow-through cells,the choice of a static system may be appropriate if sufficient attention is appliedto the foregoing details.

The ongoing research in this field will, undoubtedly, produce laboratory sys-tems that are more precisely and accurately able to simulate the percutaneousabsorption experienced in vivo. It appears as though the diversity in designsseento date will grow as more researchers embark on this important field of study,and this diversity is important for progressive evolution. However, the impor-tance of extensive designvalidation in each case cannot be overstressed;only inthis manner may meaningful comparisons of results be made. The FDA inter-laboratory in vitro permeation study that is currently being undertaken underthe direction of the Maibach group will certainly improve our understandingof the variance experienced between different study groups and, furthermore,may suggestspecific factors to beneficially narrow the diversity of future celldesign.

ACKNOWLEDGMENTS

E.W.S.acknowledgespostdoctoral research grants from the Council for Scien-tific and Industrial Research, The Pharmaceutical Manufacturers Foundation,ScheragPharmaceuticals, and ICI Pharmaceuticals, with sinceregratitude.

Wethank Dr. H. I. Maibach for his helpful comments and suggestionsin thepreparation of this chapter.

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