A novel diffusion cell for in vitro transdermal

Chapter5

A novel diffusion cell for in vitro transdermal permeation,compatible with automated dynamic

sampling

5.0.Summary

In this chapter,we describe the development of a new diffusion cell for in vitro transdermal permeation.The so-called Kelder-cells were used in combination with the ASPEC-system(Automatic Sample Preparation with Extraction Columns)which is designed for the automation of solid-phase extractions(SPE).Instead of SPE-columns,20Kelder-cells were placed in the racks.This allowed automatic sampling of up to20cells for24hours in a dynamic mode.The cells consist of an inlet compartment,a donor compartment and a receptor compartment.The size and the depth of the inlet compartment was important to avoid entrapment of air bubbles in the receptor compartment.The Kelder-cells mimic blood flow beneath the skin in vivo by replacement of the permeating drug every two minutes.Hence,sink conditions are more easily maintained in comparison with the static Franz diffusion cell.

The performance of the cells was tested with permeation experiments using atropine as a model drug permeating through an artificial membrane(Silastic?).The use of this skin model minimized the variability in permeation of atropine compared to human skin.

66Chapter5 5.1.Introduction

Recently there has been an increased interest in drug administration via the skin for both local therapeutic effect on diseased skin(topical delivery)as well as for systemic delivery(transdermal delivery).The permeation of chemicals through the skin can be measured by in vivo and in vitro techniques.Frequently this has been done by in vitro techniques because of the simplicity of the experimental conditions. Although many variations exist,there are two basic approaches to measure skin permeation in vitro:the static,or nonflowing cell and the flow-through cell[1-3]. One of the most widely used static designs for studying in vitro permeation is the Franz diffusion cell.This cell has a static receptor solution reservoir with a side-arm sampling port.In previous experiments,we used these static cells to measure the in vitro permeation of anticholinergics through pig skin[4].Permeation was monitored by sampling the stirred receptor chamber solution.This manual procedure is labour intensive,and large variations were found between the experiments.

To enable automatic sampling of the receptor solution in a dynamic mode,we developed new diffusion cells(Kelder-cells)which can be used in combination with the ASPEC-system(Automatic Sample Preparation with Extraction Columns).This liquid handling system has been designed for the automation of solid-phase extractions(SPE)[5].Instead of SPE-columns,20Kelder-cells were placed in the racks and experiments can run for24hours automatically.These cells mimic blood flow beneath the skin since the receptor solution is replaced every two minutes (discontinuous flow-through).

In this paper,we describe the development of Kelder-cell designs for in vitro transdermal permeation.The emphasis of the designs were compact,small diffusion area,small dead volume,air bubble free,variable rinsing of the receptor compartment,flexible and reproducible collection of receptor solution,and adequate temperature control.Experiments were performed with atropine as a model drug permeating through an artificial membrane(Silastic?)which was used as a model skin.Atropine in the receptor solution was determined by radioreceptor assay.The results are discussed and compared with those obtained using the static Franz diffusion cell.

5.2.Materials and Methods

5.2.1.Materials

[N-methyl-3H]Scopolamine methyl chloride([3H]NMS,80.4Ci/mmol)was obtained from Du Pont NEN(Du Pont,Wilmington,DE,USA).1-Dodecylazacycloheptan-2-one(Azone?)was kindly supplied by Nelson Research(Irvine,CA,USA). Sigmacoat?was obtained from Sigma(St.Louis,MO,USA).Plexiglas?and Delrin?were obtained from Vink Kunststoffen BV(Didam,The Netherlands). Viton?O-rings were obtained from Eriks(Alkmaar,The Netherlands).Atropine base was obtained from Merck(Darmstadt,Germany).Propylene glycol was

A novel diffusion cell for in vitro transdermal permeation67 purchased from Brocacef(Maarssen,The Netherlands).All other chemicals and solvents were of analytical grade and obtained from Merck(Darmstadt,Germany). Polyethylene tubes(12ml)were obtained from Greiner(Alphen a/d Rijn,The Netherlands).The GF/

B glassfibre filters were from Whatman(Maidstone,UK). Rialuma was used as scintillation liquid,obtained from Lumac(Olen,Belgium),in combination with mini-scintillation counting vials from Packard(Groningen,The Netherlands).

5.2.2.Preparation of solutions

Isotonic phosphate buffered saline pH7.4(PBS-buffer)was prepared by dissolving

8.00g NaCl,0.20g KCl,0.20g KH

2PO

4

and1.44g Na

2

HPO

4

.2H

2

O in1l distilled

water.PBS-buffer was used as the receptor solution.

The50mM sodium phosphate buffer pH7.4(assay buffer)was prepared by

dissolving1.38g NaH

2PO

4

.H

2

O and7.12g Na

2

HPO

4

.2H

2

O in1l distilled water.

The drug solution consisted of15mg/ml atropine in ethanol/propylene glycol/ PBS-buffer/Azone?60:20:15:5v/v[6].

The atropine stock solution of1.23x10-3M was prepared in ethanol and stored at -20°C.

The tissue suspension was prepared by dissolving5mg of lyophilized receptors[7] in1ml assay buffer.

5.2.3.Preparation of human skin

Human skin was obtained from females having undergone breast surgery (University Hospital Groningen,Department of Pathology).The subcutaneous fat was removed and the whole skin membranes were frozen in liquid nitrogen and stored at-80°C,until used.Before experiments were performed,the membranes were thawed and epidermal membranes were prepared by soaking the whole skin membranes in water for2min at60°C,followed by blunt dissection[8,9].

5.2.4.Preparation of Silastic?membranes

Non-reinforced silicone membrane(Silastic?,polydimethyl siloxane,type500-1, Laboratoire Perouse Implant,Bornel,France)of0.125mm thickness was extensively rinsed in hot distilled water(60°C)until all sodium bicarbonate(present on the surface to facilitate handling)was removed.This was followed by a thorough rinse in distilled water(20°C)for one hour[10].

5.2.5.Experiments with Franz diffusion cells

Permeation experiments with Franz diffusion cells were performed using human epidermal membranes and Silastic?membranes.The Franz diffusion cells

68Chapter5

Figure5.1.T

he static Franz diffusion cell.

A=donor compartment;B=receptor compartment;C=membrane;

D=O-ring;E=water jacket;F=stirring bar;G=sampling port.

Figure5.2.The ASPEC-system.

A=Model401dilutor;B=sample processor;C1=solvent rack;C2;

sample rack;C3=SPE rack;D=reservoir;E=needle;F=

polypropylene tray;G=rinsing station.

A novel diffusion cell for in vitro transdermal permeation69 (Figure5.1)were made of glass with a contact area of1.35cm2(University Centre for Pharmacy,Groningen,The Netherlands)and pretreated with a silanizing agent (Sigmacoat?).The Franz diffusion cell consisted of a donor compartment(A)and a receptor compartment(B).The membrane(C)was mounted between the cell compartments and an O-ring(D)was used to position the membrane.The two cell compartments were held together with a clamp.The receptor compartment has a volume of4.3ml and was filled with PBS-buffer.It was kept at37°C by circulating water through an external water jacket(E).After30min of equilibration of the membrane with the receptor solution,200μl of the drug solution was applied in the donor compartment by means of a pipet.The donor compartment was then covered with parafilm to prevent evaporation of the solvent.The receptor solution was continuously stirred by means of a spinning bar magnet(F),at400rpm(Multipoint HP15,Variomag,München,Germany).Receptor solution samples,2.0ml aliquots, were withdrawn through the sampling port(G)of the receptor compartment at various time intervals.The cells were refilled with receptor solution to keep the volume of receptor solution constant during the experiment.The experiments were run for25hours.

5.2.

6.Experiments with Kelder-cells

ASPEC-system

The newly developed Kelder-cells were made compatible with the ASPEC-system (Gilson Medical Electronics,Villiers le Bel,France).As shown in Figure5.2,the ASPEC-system consists of three components:a Model401dilutor(A),a sample processor(B),and a set of racks and accessories to handle SPE-columns and solvents(C).The dilutor allows to transfer a specified volume of solvent from a reservoir(D)through the needle(E)into a container.The latter can be an SPE-column or one of the cell designs.It is also possible to aspirate air or liquid from a container into the needle.The needle of the ASPEC-system is able to move blocks with SPE-columns to a programmed position.These three features of the ASPEC-system were used to perform permeation experiments.

Five racks were placed in the polypropylene tray(Figure5.2,F)and instead of blocks with SPE-columns,five blocks each containing four cells,were placed in these racks(Figure5.3and5.4).Polyethylene tubes(12ml)were positioned below the cells,to collect the receptor solution buffer flowing through the cells.For each cell,12collection tubes were available.This means that for an experiment of24 hours,samples were collected every two hours.

Cell designs

Three designs of Kelder-cells were developed and permeation experiments were performed at room temperature using Silastic?membranes.All the designs consisted of an inlet compartment(A),a donor compartment(B)and a receptor compartment(C)(Figure5.5).The main difference between the three designs is the size and the depth of the inlet compartment.

70Chapter5

Figure5.3.The ASPEC-system.

A=Model401dilutor;C=rack with block of4cells;D=reservoir,

containing PBS-buffer;E=needle.

Figure5.4.Block containing4Kelder-cells.

A=inlet compartment;B=donor compartment;J=polypropylene cap.

A novel diffusion cell for in vitro transdermal permeation71 The cells were made from Plexiglas?to enable viewing of air bubbles inside the receptor compartment.A circular piece of membrane(D)with a diameter of12mm was cut and placed into the receptor compartment which was filled with PBS-buffer.A Viton?O-ring(E)was used to position the membrane between the donor and receptor compartment.The donor compartment was fabricated from Delrin?and screwed onto the receptor compartment.After30min of equilibration of the membrane with the PBS-buffer,75μl of the drug solution was applied in the donor compartment by means of a pipet.The donor compartment was covered with parafilm to prevent evaporation of the solvent.The receptor solution entered the cell via the inlet compartment(A),flowed through the inlet channel(F)to the receptor compartment(C)and left the cell via the outlet channel(G).An outlet tube(H) made of stainless steel was fixed at the end of the outlet channel to make the outlet hole smaller.The inlet compartment of the cell is sealed with a polypropylene cap (J)to force the buffer to flow through the cell when fresh buffer is injected.The receptor solution with permeated drug was collected in polyethylene tubes,placed below the cells.The entire cell is very compact with an contact area of0.51cm2, defined by the internal dimensions of the O-ring,and a receptor compartment volume of77μl.

Programming

The ASPEC-system was programmed to run permeation experiments automatically for24hours(ASPEC-system user’s guide).The inlet compartments of the cells were defined by their x-y-position in the polypropylene tray(Figure5.2,F).The needle was programmed to move to the first cell and inject PBS-buffer in the inlet compartment.Then,the needle moved to the second cell and injected again.After twenty cells,the needle moved back to the first cell and the procedure was repeated.

The interval time between subsequent rinsings,the dispensing volume and flow rate of the buffer can be varied.In these experiments,aliquots of150μl were injected into the cells sequentially with a flow rate of50μl/s and the interval time between subsequent rinsings of one cell was in the order of two minutes.The dilutor syringe capacity is10ml which means that after three times injecting150μl to20cells,the syringe had to be refilled from the reservoir which took about30seconds.Before the needle moved back to the first cell,250μl was dispensed into the rinsing station (Figure5.2,G).After two hours,the needle shifted the blocks of cells in the y-direction above the next row of polyethylene tubes.In two hours,9ml(60times 150μl)of receptor solution was collected in each polyethylene tube.In the program,wait commands were used to postpone the operating procedure until the desired time was elapsed.

Optimization of the ASPEC-procedure

First,the programming of the ASPEC-system was optimized to prevent the injection of air.Before buffer was injected into the cells,2μl buffer was dispensed at a flow rate of12μl/s above the inlet compartment to create a droplet on the

72Chapter5

Design1.

Design2.

Design3.

Figure5.5.Cross-sections of the three Kelder-cell designs.

A=inlet compartment;B=donor compartment;C=receptor

compartment;D=membrane;E=O-ring;F=inlet channel;G=outlet

channel;H=outlet tube;J=polypropylene cap.

A novel diffusion cell for in vitro transdermal permeation73 needle.Air bubble formation was further minimized by degassing the PBS-buffer and the use of a bubble trap in the dilutor.Other problems such as thickness of the needle,height of injecting into the inlet compartment,diameter of the O-ring,had to be resolved to avoid leakage or flooding of the cells.

5.2.7.Analytical Procedure

Atropine in the receptor solution was determined by means of a radioreceptor assay. Calibration curves were prepared from the atropine stock solution to provide concentrations ranging from1x10-9M to1x10-5M.These calibration samples, together with the receptor solution samples,were analysed as follows.Fiftyμl of receptor solution samples or50μl aliquots of calibration samples(in duplicate) were added to polyethylene tubes.Then50μl of radiolabelled[3H]NMS(4x10-9M) and400μl tissue suspension were added to polyethylene tubes.The tubes were vortexed and incubated for30min at37°C.After the addition of4ml ice-cold assay buffer,the samples were immediately filtered through Whatman GF/B glass fibre filters under vacuum using a filtration apparatus(48S,University Centre for Pharmacy,Groningen,The Netherlands).The tubes were rinsed twice with4ml icecold buffer,which was also filtered.The total filtration and rinsing process, taking place in approximately15s,was carried out on each tube in turn.The filters were transferred into mini-scintillation vials and dispersed in3.5ml scintillation cocktail by shaking for120min.The vials were counted for40,000counts or5min in a liquid scintillation counter(Minaxi,Packard,Groningen,The Netherlands), whatever came first.Fiftyμl of the used[3H]NMS solution were added to2mini-scintillation vials and counted as well to measure the total activity added. Calibration curves were fitted with the Ligand curve fitting program[11].The obtained binding values(Bq)of the receptor solution samples were introduced in the calibration curves and the unknown concentrations of atropine were calculated. When the final concentration of the receptor solution samples exceeded the upper limit of quantitation of the calibration curve,the receptor solution samples were diluted(10-10,000fold)and reanalysed.

5.3.Results and Discussion

5.3.1.Experiments with Franz diffusion cells

Silastic?membranes have been used in many drug permeation studies as a reasonable alternative to skin[3].In the development of new diffusion cells, synthetic membranes may be useful because they may minimize the variability associated with animal and human skin[10,12].To compare the variability in permeation between human epidermal membrane and Silastic?membrane,some initial experiments were carried out in Franz diffusion cells.

Table5.1.Variability in atropine permeation through human epidermal membranes (n=4)and Silastic?membranes(n=6)using Franz diffusion cells.

74Chapter5

Sampling time Coefficient of variation*1

(hours)Human epidermis Silastic?

1 3 5 17 19 21 23 25116.2

108.9*2

160.2*2

65.8*2

83.7*2

66.6*2

68.0*2

65.6*2

91.1

16.8

12.4

21.3

14.7

25.1

21.0

19.5

*1Coefficient of variation=sd/mean*100%.

*2Significantly higher than Silastic?:F-test,p<0.05.

The mean cumulative percentage of atropine permeating through the membranes,is shown in Figure5.6.After five hours,the permeation of atropine through human skin was significantly higher compared to Silastic?membranes(Mann-Whitney rank sum test,p<0.05).The higher permeability found for human skin may be due to the fact that the material had been subjected to freezing and thawing[13-15]. Degeneration of the skin during the experiment may have played a role as well.

To investigate the difference in variability between the two membranes,an F-test was performed on all measured time intervals(Table5.1).The tested variances, corrected for the mean percentage of atropine permeation,were significantly higher for human skin at every time interval,except at t=1h(p<0.05).Therefore, Silastic?membrane was used in further experiments for the development of new cells.

5.3.2.Kelder-cell designs

The first design consisted of a small inlet compartment(Figure5.5,design1).Yet, air could be trapped easily when the needle injected PBS-buffer.The trapped air was pumped through the cell together with the buffer but then stayed behind in the receptor compartment.These air bubbles under the surface of the membrane could reduce the permeation by reducing the diffusion area[16].Therefore,a second design was developed with an enlarged inlet compartment to prevent air bubbles (Figure5.5,design2).Although the problem with trapped air bubbles from the inlet compartment was solved,another problem became apparent.In some cases,the buffer was siphoned over to the outlet,resulting in empty cells.A third design was made with a large,but lowered inlet compartment to prevent emptying of cells (Figure5.5,design3).Also,the inlet channel,connecting the inlet and receptor compartment,was shifted from the centre of the bottom to the side corner.This circumvented the passage of air bubbles to the receptor compartment.

A novel diffusion cell for in vitro transdermal permeation75

5.3.3.Experiments with Kelder-cells

With the first two designs only one third of the cells could be analysed because the others contained air bubbles below the membrane.However,this problem was completely solved with the third design by changing the shape of the inlet compartment.In Figure5.7,the mean cumulative percentage of atropine permeating through Silastic?membrane is shown,using the third Kelder-cell design.At room temperature(22°C),a mean(±sem)of5.1%(±0.4)atropine permeated through the Silastic?membrane in24hours.With the Franz diffusion cells,the receptor solution was kept at37°C,and8.3%(±0.7)of atropine permeated through the Silastic?membrane in24hours(Figure5.6).The higher percentage of permeated atropine using the Franz diffusion cells can be explained by increased permeation rates at higher temperatures[10].

5.3.4.Advantages and disadvantages

The objective in designing the Kelder-cells was to develop an automated alternative for the Franz diffusion cells and run experiments unattended for at least24hours. The manual sampling of the Franz cells requires constant attention and is often limited to the normal laboratory hours which means a less accurate fitting of the curve.Also,air bubbles are easily formed in the receptor compartment while withdrawing samples[12,17].Initially,the entrapment of air bubbles was also a problem for the Kelder-cells but could be avoided by improving the design of the cells.

The replacement of the receptor solution in the Kelder-cell mimics the blood flow beneath the skin in vivo.This can be varied by changing the volume and flow rate through the receptor compartment.In order to maintain sink conditions,the volume of buffer pumped through a cell should be many times the volume of the receptor compartment[9,18].Therefore,the total volume of the cell was minimized to84μl (receptor compartment plus dead volume in the outlet channel and tube).Magnetic mixing of the receptor compartment was not necessary because the flow rate of buffer through the Kelder-cell was sufficient to completely remove the permeated drug[19].However,with Franz cells magnetic stirring is crucial to provide a homogeneous receptor solution[1].

The temperature of an in vitro system should be controlled to maintain a target temperature and minimize variation in experimental conditions.With Franz diffusion cells,temperature control of receptor solution is maintained with a water jacket.However,the temperature of the donor compartment is not thermostated and is therefore subject to variations in environmental temperature[2].Our present

76Chapter5

Figure5.6.Perm

eatio

n of

atrop

i n e

throu

gh human epidermal membranes(n=4,)and Silastic?membranes(n=6,

)using Franz diffusion cells;each point represents the mean and standard

error of the mean.

Figure5.7.Permeation of atropine through Silastic?membranes(n=5)the third Kelder-cell design;each point represents the mean and standard error of

the mean.

A novel diffusion cell for in vitro transdermal permeation77 experiments with Kelder-cells were performed to focus on designs and were not temperature controlled.In future experiments,the ASPEC-system will be placed in a temperature controlled cabinet,which provides a uniform temperature of the entire system.

Another advantage of Kelder-cells is the reduced skin surface area which allows smaller pieces of skin to be used.Also,the system can be linked to an HPLC system and automatically analyse the amount of drug present in the receptor solution samples.

5.4.Conclusions

The third design of the Kelder-cell is compact with a contact area of0.51cm2and a total volume of84μl(receptor compartment volume of77μl plus dead volume). The size and the depth of the inlet compartment was important to avoid entrapment of air bubbles in the receptor compartment.With the liquid handling system ASPEC,permeation experiments could easily be automated.

The Kelder-cells mimic blood flow beneath the skin in vivo by replacement of the permeating drug every two minutes.The volume and flow rate through the receptor compartment of the cell can be varied and the collection of receptor solution is reproducible and can be adapted to the needs.The ASPEC-system can be placed in

a temperature controlled cabinet for adequate temperature control.

5.5.Acknowledgement

Bert Schreuder and Chris Nienhuis are thanked for their technical assistance,and Renger Jellema is thanked for preparing Figure5.5.

5.6.References

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