diffusion testing fundamentals_definisi

8/11/2019 Diffusion Testing Fundamentals_definisi

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INTRODUCTION

When deciding which type of di usion cell system you need,or before running an experiment with your new di usioncell or system, it is important to not only be familiar with theequipment but have a well planned experimental protocol

with clear objec ves. What do you want to know about apar cular compound or membrane? Does it ma er which typeof membrane you use? What membranes are available that willt your needs? How will you interpret and present the results?There are many parameters to consider, and working out thedetails beforehand will reduce the number of failed a emptsand produce consistency between experiments that is crucialwhen comparing results. This paper will address basic conceptsof di usion, describe various materials from which to choose foryour di usion study, and describe the components of di usioncells and di usion systems.

BACKGROUND/TERMINOLOGY

Difusion is, by de ni on, the random movement ofmolecules through a domain driven by a concentra ongradient, from high concentra on to low concentra on.In vitro di usion is generally passive di usion of apermeant from a vehicle in the donor chamber, throughan ar cial or biological membrane into a receptor uidin a receptor chamber, disregarding delivery systemssuch as iontophoresis and microneedles. The permeant

is the molecular species moving through or into thessue/membrane. Permea on is the movement of thepermeant through the membrane that encompassesrst par oning the membrane and then di usionthrough the membrane. Penetra on can occur into themembrane without necessarily di using, or passingthrough, the membrane.

Flux is the amount of permeant crossing a membraneper unit area into the circula ng system per unit me,and for in vitro permea on this system is the receptorchamber, expressed in units of mass/area/ me.Similarly, accumula on is the amount of permeantcrossing a membrane within a certain me, expressedin units of mass/area. Difusivity is a property of thepermeant and is a measure of how easily it penetratesa speci c membrane expressed in units of area/me. The permeability coe cient (Kp) describes therate of permeant penetra on per unit concentra onexpressed in distance/ me.

DIFFUSION TESTING FUNDAMENTALS

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Any compound applied to either ssue or anar cial membrane will have a lag me , the meit takes to permeate through the membrane anddi use into the receptor uid and then nally reacha steady state of di usion. The lag me is the periodduring which the rate of permea on across themembrane is increasing. Steady state is reachedwhen there is a consistent, unchanging movement

of the permeant through the membrane. Theamount of me it takes to achieve steady state willdepend on several factors, the permeability of thessue or membrane being studied, the proper esof the compound itself, and the ow rate of thereceptor uid if ow-thru type di usion cells arebeing used.

The permeability barrier is a lipid barrier dependenton the amounts, types, and organiza ons of lipidspresent in a ssue. Permeability of a par cularssue does not directly correlate to the thicknessof that ssue. For example, skin structure varies tosome degree over the human body and di erentpermeants exhibit di erent rank orders for variousskin sites.


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Sta c cell (ver cal or Side-Bi-Side):Descrip on: Franz type cell or Side-Bi-Side Cell, xed volumereceptor chamber, controlled temperature, port to samplereceptor uid, s rred receptor uid (Side-Bi-Side Cells allows rring of both the donor and receptor chambers.)

Uses: Evalua ng compound uptake into a membrane, nitedose permea on, steady state ux of compounds (eitheralone or in formula ons.)

Permeability of the compound&

Permeability of the ssue

Determines rate across membrane ( ux)

Determines concentra on in receptor chamber

Considera ons:1. If you are using a highly permeable compound with alarge volume receptor chamber, the large volume reduces

the gradient, so build up of the compound in the receptorpart is not a problem (sink condi ons are maintained).

2. If you are using a highly permeable compound with asmall receptor chamber, the buildup of compound reducesthe concentra on gradient and therefore slows the ux ofthe compound (non-sink condi ons).

3. If you are using a low permeability compound, thendetec on of the compound in a large volume receptorchamber can be a problem.

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Con nuous ow cell or ow-thru cell:Descrip on: Franz type cell or In-Line Cell, xed volumereceptor chamber, controlled temperature - Franz type cellsare s rred, In-Line Cells have con nuous ow which causesturbulence in the receptor chamber and simulates s rring,

ow rate is adjustable, permits automated sampling.

Uses: Mimics in-vivo ( ow equates blood ow), evalua ngcompound uptake into membrane, nite or in nite dosepermea on, steady state ux and K p of compounds (eitheralone or in formula ons.)

Permeability of the compound&

Permeability of the ssue

Determines rate across membrane ( ux)

Determines concentra on in receptor chamber

Determines rate of clearance ( ow rate)

Considera ons:

1. If you are using a highly permeable compound, the highow rate necessary to clear the receptor chamber to maintaingradient can result in a large volume of permeant to analyze.

2. If you are using a low permeability compound, the ow ratenecessary to clear the receptor chamber can result in a largevolume of permeant for which detec on can be a problem.

3. A smaller receptor chamber requires less permeant to clearthe receptor chamber which results in less permeant volumesand be er detec on.

IN-LINE CELL

SIDE-BI-SIDE CELL

I. EXPERIMENTAL DESIGN/EQUIPMENT/PRINCIPLESChoice of di usion cells: sta c or con nuous ow

FRANZ CELL


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II. MEMBRANE TYPES

1. Human ssue ex vivo: It may be di cult to obtain su cient quan es ofnormal, healthy human ssue to perform permeability experiments with largeenough sample sizes for sta s cal analysis. Many laboratories use human cadaverskin obtained from accredited U.S. human ssue banks. There might be ethical andlegal considera ons in obtaining human ssue biopsies and surgical specimens.

2. Small animals: Models such as rats, mice, and rabbits have tradi onally been usedin research for human ssue permeability as they are usually inexpensive to purchaseand maintain. Drawbacks are the ssue areas are usually thinner than human skinand have a di erent morphology resul ng in higher compound permeabili es.

3. Large animals: Monkeys, dogs, pigs, and other large animals have also been usedextensively but may be expensive to purchase, especially in the cases of monkeysand dogs. Pig and monkey so ssue is very similar to human so ssue in terms ofmorphology and func on, therefore is widely used as a surrogate for human skin.

4. Polymeric membranes: These kinds of membranes are usually used for in vitro release tes ng (IVRT). According to the FDA SUPAC-SS (May 1997), any appropriateinert and commercially available synthe c membranes such as polysulfone, cellulose

acetate/nitrate mixed ester, ... of appropriate size to t the di usion cell diameter

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TISSUE PREPARATION/STORAGE

Once you have selected themembrane you will use foryour study, it is impera ve thatyou know the speci cs for thatmembrane such as the source,age, integrity, and shelf life. If itis human or animal ssue, youwill need to know the healthand age of the animal, me ofexcision, race and gender of thehuman, site of excision (for skin)and storage condi ons beforeu liza on. Most skin can befrozen and used at a later datewithout a ec ng permea on.Certain ssues degrade quicklyand must be used immediatelyor kept moist and refrigeratedun l use. It is also importantto note if any chemicals wereapplied to the ssue such as analcohol wipe that could alterthe lipid permeability barrier orif there was possible damagefrom instruments or mechanical

damage. Any damage to ssueor altera on of the permeabilitybarrier can bias your results.Consistency in ssue retrieval,handling and storage canminimize erroneous data, andprecise informa on about thessue can help to determine ifthe results are valid. You cannd informa on per nent tothe ssue type you plan to use

and appropriate ssue handlingtechnique by referencingpublished literature.

Polymeric membrane

Photo courtesy of MatTek Corp.h p://www.ma ek.com/ pages/products/epiderm

can be used. Usually hydrophilic polymericmembranes with a pore size of 0.45 mare used. Once set during the IVRT methoddeveloping phase, the same membraneshould be used for the en re dura on ofthe project.

5. Human skin equivalents (HSEs):These are ssues engineered 3D skin

constructs (such as MatTek products)that u lize a combina on of culturedhuman skin cells and extracellularmatrix components under controlledculture condi ons. The advantage ofthese ssues is that viability can bemaintained. The main disadvantage isthat the permeability of the simulatedssue is generally higher than that of in

vivo .

Ar cial membranes and animal modelscan be valuable predictors for compoundsand formula ons for in vivo use. You canaddress metabolism, penetra on, anddistribu on of the compound of interest,but be careful about making direct claimsfor that compound in a speci c model tohuman in vivo use.

Human skin equivalent


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III. DONOR FORMULATIONS

The compound of interest can be prepared in a variety offormula ons - liquid solu ons, suspensions, creams, gels,ointments, lo ons, pastes, powders, or adhesive patches.The formula on chosen should mimic the real worldapplica on as much as possible. Varying the formula on,concentra on, and adding permeability enhancers can also

help you gain valuable informa on about the permeantcharacteris cs and behavior.

1. Applica on - in nite dose/ nite dose: The concentra onof the permeant will depend on the study aims.


2. Vehicle formula on: Also named blank or controlformula on, vehicle formula on is the applica on dosewithout the ac ve pharmaceu cal ingredient (API) or drugagent. The vehicle in which you choose to incorporate thepermeant of interest, whether liquid, semi-solid or paste,will again depend on your study aims. There are several

things to consider:

An in nite dose, one in which the permeant will notbe depleted from the donor formula on over thecourse of the experiment, will produce fundamentalpermea on behavior and is used when tes ng thatbehavior in the presence of permeability enhancers.

A nite dose mimics the amount of permeant thatwould be prac cal in actual use; for example, aspeci c amount of drug to be administered to apa ent. The aim would be to determine what amountof the permeant needs to be incorporated in a speci cvehicle that will deliver the required amount of drugto the pa ent in order to be e ec ve.

An alterna ve applica on would be to mimicapplica on of a compound that would in actualitytemporarily remain on the ssue; for example, asalve applied to the skin or a drug to the inside of the

mouth. The permeant would be applied to the ssuefor a short period of me, it would then be removed,and samples would be collected for a length of mefollowing permea on.

a)

b)

c)

Aqueous solu ons and phosphate bu ers are goodop ons for ge ng basic permea on data.

Lipophilic compounds have a low aqueous solubilityand may require alterna ve vehicles such as water/alcohol mixtures or propylene glycol. It must be notedthat these addi ves may interact with the structure andpermeability barrier of the ssue and thus change theux of the permeant.

Inert materials can a ect the ux of the permeantthrough both ar cial and animal membranes.

The nature of the vehicle can alter the potency ofcertain enhancers. That is, the same enhancer cana ect the penetra on of a speci c permeant di erentlydepending on the vehicle it is incorporated in.

Selec on of bu er is important to control the degreeof permeant ioniza on. Adjus ng pH can also fullydissociate the permeant into charged ions.

The thermodynamic ac vity (the driving force for

permea on) of the same concentra on of permeantcan change dras cally by modifying the vehiclecomponents.

a)

b)

c)

d)

e)

f)

3. Permeant detec on: Considera on must be given tothe method of permeant detec on. This can be performedby chromatographic or spectroscopic methodologies,or the use of radio-labeled permeants. A discussion ofadvantages and problems associated with these methodscan be found under Sec on V. SAMPLING.

4. Other considera ons: For each formula on tested, aminimum of three parallel cells should be used for everyrun. Due to biological variability in skin, in many cases5-6 replicates should be run per group. Evapora onof compounds, solvents, or formula ons from donorchambers may occur, especially if the cells are maintainedat high temperatures.


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IV. RECEPTOR MEDIA

Selec on of a receptor solu on depends on the nature ofthe permeant and the type of di usion cell used. An idealreceptor medium for an in vitro permeability experimentshould mimic the in vivo situa on. Flow-thru systemsminimize accumula on in the receptor chamber, thusaqueous receptor uid is usually adequate. This could

be a concern for sta c cells where the permeant is notcon nually cleared.

Solubility of a compound should be taken into considera onso that the compound is at its desired form in the donorformula on as well as being delivered at a certain amountin the receptor medium; which would a ect the di usiongradient and could slow di usion.

Aqueous receptors are common for hydrophilic andmoderately lipophilic permeants. Phosphate bu er sali ne(PBS, pH 7.4) should be used if the permeant is ionizable. For

un-ionizable permeants, a solubilizer added to the receptormedium should be su cient. More lipophilic permeants orpermeants with low aqueous solubility require addi onalsolubilizing agents such as surfactants, protein (bovine serumalbumin) or organic solvents (ethanol/water systems).The e ect of these solubilizers on the penetra on of yourcompound of interest needs to be considered as thesesolubilizers may cause damage to the barrier or changesin the ssue which can alter the permeability results. Theyshould, therefore, be used at the minimum concentra onnecessary to solubilize the permeant .

V. SAMPLING

The best method of sampling in terms of me intervals,frequency, and volume, and the best method of repor ngthe permea on of a compound of interest will depend onyour research ques on. Do you want to know how muchcompound crosses a membrane at short me intervals, thetotal amount of compound that crosses the membrane overa long sampling period, or the total amount of compoundfound within the membrane a er a given exposure period?Do you want to calculate ux, accumula on or permeabilityconstant (K p)? The following addresses factors that willhelp you determine these parameters.

Flux and permeability can be determined using radio-labeled or unlabeled compounds. The calcula on of eachparameter can be found in the next sec on. The ux of acompound can be converted to an amount of compound (inng or g) per me interval. Regardless of how your analysiswill be performed, you need to know the me points of thespeci c samples to report the results as either of two types


of accumula on: 1 - The total quan ty of the compoundin the collected sample per me interval, regardless ofvolume, and 2 - As the concentra on of the compound, i.e.the amount of compound per standard volume of sample.

Radio-labeled compounds can be detected in very smallquan es and in a large volume of receptor medium.Analysis is usually quite rapid and accurate. The en recollected volume can be easily counted using a scin lla oncounter, and then the ux or K p calculated from the countsper minute (cpm) obtained can be converted to the amountof compound in any given sample at any given me intervalby knowing the speci c ac vity of the labeled compound.Many compounds are readily available and inexpensive topurchase, but other compounds, such as pharmaceu cals,are very expensive to synthesize and may have handling andstorage issues. For example, a radio-label could be unstable inthe donor formula on.

Non-radio-labeled compounds can also be used in

permeability experiments and detected with methodsincluding high-performance liquid chromatography(HPLC), enzyme-linked immunosorbent assay (ELISA), anduorescence. These methods are versa le; they can beadapted to many permeants, and you can o en conductmul ple analyses. It is possible to determine if the samespecies of compound that permeated the membrane wasthat which was applied. The disadvantage to these detec onmethods is that very small quan es of compound cango undetected or under-detected because of the largevolume in the receptor chambers of sta c cells or large

quan es collected from ow-thru cells. As small samplesare required for quan ca on by HPLC or ELISA, you needto take aliquots of the sample and then calculate the totalcompound in a given sample over a given interval. You canalso decrease the volume of the sample by drying it downto a total quan ty of sample that can then be analyzed.

As the results of your experiment will be reported as theamount of compound penetra ng a membrane over me(see Sec on VI.), the volume of aliquots taken for analysisand the me intervals between samples needs to be precise

to obtain reliable data. When using a sta c system, thereceptor uid volume will be the same for all the di usioncells, therefore, the amount of compound determined in analiquot can be easily calculated for that speci c volume. Thesamples should be taken quickly and consecu vely from allreceptor chambers so that the me interval is consistentfor all samples. If there is a delay of minutes betweentaking samples instead of seconds, this should be takeninto account when performing calcula ons. When usinga frac on collector with automated sampling, the me


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intervals for the samples from all the cells will be exactlythe same. There may, however, be slight varia ons in thevolume of receptor uid pumped through each di usioncell into the collec on vials. This is due to slight varia onsof the tubing element diameters or clamping pressuresin the pump casse es but is not a concern if the totalvolume of sample is being analyzed. If aliquots are takenfor analysis, then the volume of each collected sample

must be measured so that the total amount of compoundin the sample can be calculated precisely. For example, ifa 100 l aliquot of sample is determined to contain 2 pgof compound, then there would be 20 pg total in a 1 mLsample but 24 pg in a 1.2 mL sample.

It may be necessary to obtain a small volume of receptoruid over a long period of me with your con nuous owsystem. This would occur when you want to reduce theamount of sample for analysis to avoid pipe ng or todetect a compound that you know would only penetratein very small quan es over a period of me. In essence,you are conver ng to a sta c system. In this instanceyou would turn the pump o for a period of me andthen quickly purge the receptor chamber by running aspeci c volume through it to obtain your sample. This isa valid method if you report the me interval correctly.In this case, the me interval would be from the me thepump was turned o through the end of the purging ofthe receptor chamber. Keep in mind with this methodthat the driving force for di usion is the concentra ongradient. If the membrane is very permeable, thecompound could accumulate quite rapidly in the receptormedium, consequently increasing the concentra on in thereceptor chamber and thus slowing di usion. You mustalso be sure to adequately purge all the compound thathas accumulated in the receptor chamber.

A buildup of compound in the ssue followed by a slowrelease into the receptor medium can occur. This hasbeen demonstrated in experiments where a compoundof interest formulated in a liquid, gel or paste is appliedto a membrane and then removed a er a short periodof me but samples are collected a er the removal. The

compound may con nue to di use for some me up toseveral hours a er removal of the compound from themembrane surface. This can be a ributed to a reservoire ect. The upper layers of the ssue absorb the compoundfairly rapidly, and once saturated, the compound di usesthrough the deeper layers of the ssue and into thereceptor medium at a fairly constant rate. Again, as longas the concentra on gradient remains, the di usion willcon nue.

The frequency of samples collected will be determinedby the goals of your experiment. If you have no priorconcept of the permeability of a membrane to a par cularcompound, it is ini ally prudent to take small samplesover a long period of me to determine the onset ofsteady state ux and the overall permeability. If you knowthe membrane is highly permeable to the compound,you will want to take very short and frequent samples. If

you only want to know the total amount of compound tocross the membrane in a given me period, you can takefewer samples with longer me intervals. It is best to doseveral replicates for a given compound and condi on,and then average the amount of compound collectedat each me interval for those replicates. You can thenplot these results over the en re experimental period,which will provide you with of reliable data. For example,one scenario would be to collect for very short intervalsin the beginning of an experiment, such as 15 minuteintervals for one hour, 30 minute intervals for 2 hours

and then 60 minute intervals for several hours. This willallow you to determine how quickly a drug permeates,when it reaches steady state ux, and nally the totalquan ty of compound permea ng the ssue over theen re sampling period. Using this you can calculateux or Kp for whatever me period is of interest for thiscompound. You can determine the amount of radio-labeled compound s ll in the ssue by autoradiographyor by solubilizing the ssue and coun ng in a scin lla oncounter. You can also iden fy compound in the ssue bygrinding and/or homogenizing it in liquid and centrifuging

o the liquid to be analyzed with HPLC, ELISA, Luminex orpyrosequencing.



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VI. CALCULATING FLUX ANDPERMEABILITY COEFFICIENT (K P)

Flux (J) is the amount of permeant crossing themembrane per me. It is given in units of mass/area/me or in units of radioac vity/area/ me. Note: If thepermeant was applied in a nite dose, then you can onlycalculate a ux by the formula: J = Q/(At) where Q is the

quan ty of compound traversing the membrane in me t,and A is the area of exposed membrane in cm 2 . Units ofux are quan ty/cm 2 /min.

Examples: (g/cm 2/h) or (pg/cm 2/min) (cpm/cm 2/min)

Steady state ux (J ss ) is the amount of permeant crossingthe membrane at a constant rate; this occurs a er the lagphase when the amount con nues to increase. When theamounts measured at successive sampling intervals are

not signi cantly di erent, this is considered steady state.Jss = Q/(At) where Q is the quan ty of compound

transported through the membrane in me t, and A is thearea of exposed membrane in cm 2. The unit of steady stateux is quan ty/(cm 2 hr).

If the amount of permeant applied to the membrane wasan in nite dose, then the permeability constant (K p) can becalculated from the rela onship K p = Q/[At(C o-Ci)] whereQ is the quan ty of compound transported through themembrane in me t (min), C o and C i are the concentra onsof the compound on the outer side (donor side) and theinner side (receptor side) of the membrane respec vely,and A is the area of exposed membrane in cm 2. Usually C o can be simpli ed as the donor concentra on and C i as 0.The units of K p are cm/min or cm/hr.


VII. METHODOLOGY

However you design your experiments, you should beconsistent and precise in your methods. It is impera ve tobecome familiar with your equipment, to set some baselinevalues for the membranes or ssues you plan to use, andto run several compounds if possible to determine if yourdetec on method will be adequate. Ini ally run some

simple experiments with as few variables as possible. Onceyou have set baseline values, it will be easier to ascertainthat your experiment is running smoothly or if there is aproblem with the methodology.

Be consistent with prepara on of the ssue and membranes.Mount the ssue carefully to avoid overstretching ordamaging it by perfora on. Specimens must be largeenough that there is an edge of ssue which can be securelyclamped in the di usion cell around the ori ce. Care mustbe taken to ensure that ssue does not protrude into thereceptor chamber which could block the ow of receptoruid.

Use ssue as soon as possible a er excision or freeze itat -80C un l you do use it. Keep notes on the me fromharvest to use when excising ssue from live animals.

Be precise in compound prepara on, receptor mediumprepara on, pipe ng samples, and sampling intervals.Changing the pH of or adding solubilizers to your solu onscan a ect the penetra on of some compounds. You shouldbe aware of this and be able to jus fy your methods.

Temperature can a ect the results of your experimentso you must be consistent in using room temperature,skin temperature, or body temperature when comparingcompounds. Report temperature in your methodologies.Always a empt to reproduce the in vivo situa on as closelyas possible.


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diffusion testing fundamentals_definisi

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