Dev el op me nt

of a s ui ta bl e pr ot oco l to meas ur e ad he si ve fo rce o f a mu co ad hes ive

tab le t

I. ABOU-RABII1, J-M. CARDOT1, J-M. AIACHE1

1
Biopharmaceutics Department, Faculty of Pharmacy, Auvergne University, 28 place Henry Dunant 63000 Clermont-
Ferrand, France.

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Summary:

The utilisation of a buccal mucoadhesive tablets may be one of the most successful way to treat buccal

local pathologies. The sustained release of drugs in the buccal cavity helps to overcome constant

salivary secretion in the mouth. The aim of this study was to develop a protocol to measure the

adhesive forces of mucoadhesive tablet, by the mean of a texture analyser.

The different parameters of the protocol (time of moistening and volume of hydration), were determined

by using placebo tablets (20% metolose, 80% milk protein). The ideal time of adhesion was found to be

12 minutes divided into two parts, 6 minutes for the initial moistening in which the tablet was completely

immersed in 4 ml of deionised water, and six other minutes of contact between tablet and aluminium

adhesion surface.

In second series of analysis, several series of tablets with variable concentrations of adhesive

substance (milk protein) were prepared, and their adhesion forces measured using previous

parameters. The linear relationship between the milk protein concentration and the adhesion forces

proved the validity of the method. The results were homogeneous, and the force of adhesion

proportional to the amount of milk protein used in the tablet which proved that this excipient possesses

a high adhesive power. The calculation allows the determination of the smallest concentration of

excipient necessary to produce an adhesion.

This method is able to measure the adhesivity, and can be used as quality and qualification control for

new excipients.

Key words : bioadhesion, mucoadhesion, milk protein, adhesion, trexture analyser.

Introduction:
In order to treat buccal pathologies, conventional lozenge, mouthwash, or gel would be the simplest

dosage forms for the delivery of drugs in the buccal cavity, but these conventional dosage forms had the

disadvantage of an initial burst of salivary concentration followed by a rapid decrease. A lozenge

produced effective salivary drug levels for more than one hour but repeated administration was

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restricted due to systemic toxicity coming from the large quantity of ingested drug. The action of

mouthwashes was even more transient than that of lozenges, and gels/pastes were difficult to retain in

the mouth [1]. So buccal mucosal sustained release devices might prove to be a viable alternative to the

conventional local oral medications.

In case of oral Candida infections, a prolonged therapy with antifungal agent was required, and some

papers documented prolonged release of antifungal agent from buccal devices [2] in the form of an

adhesive tablets. This adhesion was obtained by the use of special excipients like Carbopol 974P,

sodium carboxymethylcellulose, polyethylene oxide, polymethylvinylether/maleic, tragacanth, etc.

Besides these excipients, J. M. Aiache et al [11] described the use of “milk protein concentrate” as a

new and convenient excipient in oral drug dosage forms due to its adhesive properties. “Milk protein

concentrates” are naturally-occurring substances obtained directly from pasteurised raw cow milk by

physical method (skimming and ultra filtration) without denaturing or any marked modification of their

nutritional or technological characteristics. The product obtained is a more or less yellow-white powder,

with a milky smelling, and contain beside milk proteins, lactose, fat and mineral salts in varying amounts

[3, 4, and 5].

According to the proportion and nature of the protein fractions, and the proportion and nature of

associated substances, these powders displayed various properties directly linked to the functional

properties of the proteins they contain. These properties were abundantly studied in food science [6, 7,

8, and 9].

The functional properties of a protein was defined as those non-nutritional properties that influence how

the protein could be used in a food, i.e., the physical and chemical properties that help achieve the

desired characteristics of the food.

These properties could be classified into three main groups:

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 (i) Hydration properties (protein-water interaction): these included adsorption, swelling,

adhesion, dispersibility, solubility and viscosity [10].

(ii) Protein-protein interactions properties : these were involved in processes such as

precipitation, gelling and the formation of structures.

(iii) Surface properties: these included surfactant, emulsifying and foaming properties.

Last but not the least property, “milk protein” had the advantage to be well tolerated, without toxicity due

to its natural origin [11].

During the first steps of the development of an adhesive formulation, it is really difficult to realize early in

vivo study. However the data published by Khanna and al [12] proved that the in vitro measurement of

adhesion was proportional to the in vivo adhesion time. So at this step, it may be sufficient to develop

an in-vitro method to estimate the adhesive power of a tablet, before in vivo testing to confirm the in vitro

data.

Several methods in vitro were described in order to estimate the adhesive power with adequate

materials:

1. Simple test of traction (Chang et al [13], Robinson [14], Marvola [15], Forget [16], and Ishida

[17]), in which the necessary force to tear away a tablet stuck on a standard surface of

adhesion was measured.

2. Traction test with establishment of curve constraint/deformation (Gurny [14], Duchene and

Ponchel [18]): in this method the sample was stuck between two disks, which moved apart with

a constant speed until the sample was pulled out, and then a constraint/ deformation curve was

established.

3. Method using a molecular probe described by Park and Robinson [15]: it consisted in

measuring by an adequate probe the change in the membrane viscosity of cells hanged in

cellular culture when this membrane was in contact with the polymer to be studied.

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 4. Piccerelle and al (1999), described the use of a texture analyser in order to measure the

adhesive power of a polyvinlpyrrolidonne film [19].

The aim of this paper is to present a new and easy method, using a texture analyser, to measure the

adhesion power of bio adhesive tablets containing milk protein, in order to compare several formulations

and select the most appropriate composition.

Materials and method

Tablets

The placebo bio adhesive tablets were made by direct compression after mixing of the different

excipients used for the formulation.

• Milk protein concentrate was the Prosobel LR 85(supplied by Armor Protein., Bretagne, France).

• Metolose SH 95000 (Hyroxypropyl Methylcellulose) (supplied by Shin-Etsu Chemical Co., Tokyo,

Japan.)

The table 1 shows the tablets’ composition used for the experimental procedures

Texture analyser:

The texture analyser or texturometer (Texturometer TEC 025 with software TEC v 6.0, supplied by ETIA

SA., Compiègne, France. (Figure 1), was specially designed for the analysis of mechanical texture

properties of both solid and gel paste products.

It consisted essentially of a displacement unit with a sensitive probe, the movement of this unit being

ordered by two displacement control keys (ascending, and descending) connected to a computer.

The original method for a substance’s texture analysis is based on the real time analysis of the stress

progression in a material. The stress is provoked by the descending probe at a controlled speed, which

makes a compression on the system tested. During this operation, the work, in joule, starting at the

beginning of the compression until the displacement of the unit is automatically stopped, is measured.

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This value called “positive work” represents the resistance to modification under pressure provoked by

the descending probe. Then the probe gets up and the force is again measured, describing some of the

system properties (viscosity, adhesion, etc.): “negative work”

However the direct use of the Texturometer was not possible in the case of bio adhesive tablets in

function of the different requirements (and steps of use) of this type of formulation:

- adhesion of the tablet on a standardised predefined surface,

- evaluation of the “positive work”,

- evaluation of the force needed to extract out the system,”negative work”.

So the tablets have to be fixed on the probe, then wetted so that the adhesive excipient can develop

its properties and stuck on a standardised predefined surface by compression (positive work).After

a certain time, the extraction force is evaluated (negative work).

The experimental procedure was the following:

The upper area of the tablet to be studied was fixed by means of cyanoacrylate glue on a poly

methacrylate disposable device which was directly screwed on the special 20 Newton probe. The tablet

was immersed in a determined volume of deionised water, introduced in a plastic container, the bottom

of which was made of a smooth aluminium surface, figure (2).

The descent of the displacement unit pressed the tablet on the aluminium flat support. The tablet was

then maintained during a given period before ascending the displacement unit to measure the

necessary force to extract of the tablet.

The protocol adopted was as follow:

1. The upper area of a tablet was fixed by means of cyanoacrylate glue on a poly methacrylate

disposable device which was screwed directly on the special 20 Newton probe.

2. Using the manual switch, the displacement unit got moved down until the tablet was completely

immersed into deionised water avoiding any contact with the aluminium surface. This phase

allowed the moistening of the lower surface (initial moistening).

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 3. Then under computing command, the displacement unit got down at a constant rate (0,25

mm/s) until reaching the aluminium plate( figures 2) so that the tablet got in touch with it (figure

3, point a), the probe exerted some pressure on the tablet before it was automatically stopped

(figure 3, point b).

4. After some minutes (figure 3, point c), the displacement unit was moved up at the same speed

ordered by the computer until reaching its initial level. The adhesion force of the tablet was then

measured by recording the negative work using the software TEC v6.0 (E.T.I.A).

The software TEC v6.0 allowed having a graphical display of all the forces, positive and negative work

(figure 3).

The experiments have been conducted in two phases:

First phase: determination of the parameters
Several parameters of this protocol were to be determined:

1. the total amount of water in which the tablet might be immersed

2. the total time necessary for the two phases of the experiment (hydration in the deionised water,

and contact with the adhesion surface).

The experiment was accepted if the tablets:

• stayed stuck on the disposable plastic device and can be completely separated from the

aluminium surface without leaving any tracks.

• remained intact without any broken part at the end of the test.

The parameters have been determined in the following order:

a) Smallest immersio n duratio n.

This determination was necessary in order to obtain a correct and continuous adhesion layer due to the

hydration of the hydrophilic components.

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15 tablets (Formulation CP, table 1) were immersed into deionised water for 1 to 15 minutes; one of

each was controlled every 2 minutes by broking in 2 parts the tablets and evaluated by visual

examination. The time for a good adhesion gel layer is determined.

b) Dete rminatio n of water moisteni ng volume

This parameter was necessary to obtain the best volume of water to fill just the plastic container in

function of the different size of tablets.

Forty tablets (CP formulation, table 1) were tested with several water volumes (1, 2, 4, 5, and 6 ml), 8

tablets were used in each volume.

c) Determi nation of moistening time an d comp ression du ration: total exp erime ntal

time

With the same protocol, and with a volume of water fixed to 4 ml, forty two tablets (CP formulation, table

1) were tested, six at each run; the total time of experiment was variable from 2 to 20 minutes.

In these two last experiments, the percentage of success was calculated, and the value of related

parameter was retained.

Second phase: application to the study of different bio adhesive

formulation
With the parameters found in the preliminary phase, four groups of tablets were tested, each group

containing various concentrations of milk protein (20%, 40%, 60%, and 80%). The adhesion force

calculated and the statistical analysis described below was carried out in order to find a possible

relationship between the excipient concentration and the adhesion force which would validate the

system and the possibility to study adhesion by the mean of a texturometer.

Statistical analysis

A chi square test (Proc Freq of SAS 8.02) was applied on the volume and moistening time results, if the

probability was lower than 0.05 then the difference could be was considered as statically significant.

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A one way ANOVA (pro GLM of SAS 8.02) was applied to the negative work values obtained in order to

study the influence of the variation of milk protein concentration and to determine which concentration

were different together (multiple analysis).

In addition a linear relationship was studied in order to establish the potential relation between milk

protein concentration and negative work (proc GLM of SAS 8.02).

Results and Discussion

First phase
a) Water moistening volume

When the tablets were immersed in the water lower than 10 minutes, the adhesion gel layer was not

continues or very thin. With more than 10 minutes of hydration, a continuous and enough thick layer

was obtained.

So, to determine the water volume, an initial total moistening time of 10 minutes was chosen (5 minutes

for initial moistening phase, and other five minutes for final moistening phase).

Thus, with a total moistening time of 10 min, the following results were obtained (figure 4)

The Chi square test applied on volume results demonstrated a difference between the volume (p=

0.0047).

This figure shows that two values of water (4 and 6) gave the most accepted results; the lower value

was chosen which allowed studying tablets without any loss of water, and excessive moistening.

b) Determination of moistening time and compression duration: total experimental time

The results obtained are presented in the Figure 5

The chi square values applied on the moistening time results demonstrated a difference between them

p= 0.0012.

The results demonstrated the optimal immersion time in the water was 12 or 14 minutes. With lower

time, the tablet was broken into two fragments: the upper part remains stuck on the mobile head of the

texture analyser, the lower part remains stuck on the surface of adhesion, because the moistening was

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not homogeneous (insufficient time of moistening). When the moistening time was relatively long (about

20 min), few accepted results was obtained, certainly due to the excessive moistening of the tablet and

the disintegration phenomena.

Thus, final results of the preliminary phase was that the water volume of 4 ml and the moistening time of

12 minutes divided into two parts, six minutes for the initial moistening phase, and six other minutes for

final moistening phase where the tablet was in contact with the aluminium surface.

Second step
The adhesion force (represented by the negative work) was measured and the mean was calculated for

all the tablets of each series. The results are presented in table 2.

The symbol NA indicates that the results were not accepted (broken tablet or tablet separated from

disposable device). This effect was certainly due to an important power of adhesion, so important that

the tablet was broken or detached from the disposable device before been extracted from the aluminium

surface..

The mean of negative work was calculated for every group of tablets (20%, 40%, 60%, and 100%); the

figure 6 demonstrated the variation of adhesive forces vs. the milk protein percentage.

Even when the tablets had no milk protein a negative work values were recorded, this is certainly due to

an adhesive proprieties of other excipient (metolose). The regression analysis demonstrated a linear

relationship between the milk protein concentration and the negative work with a slope of

0.0000111±0.0000035 (p=0.0053) and an intercept of 0.000478 (p=0.0260), the coefficient of correlation

was of 0, 61 (p=0.0053).

Theses values stayed indifferent when the milk protein concentration rose, but from milk concentration

of around 38%, negative work values started statistically to rise, this might help to find optimal milk

protein concentration. That being confirmed by the ANOVA that showed a difference between the

various milk protein concentration (p=0.0467), the difference being located between 80% and 20%-40%

of protein, 20 and 40 being not different.

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Concl usi on

• The described method allows an objective and accurate measurement of the adhesion power or

adhesion force of a dosage form containing one or more specific adhesive excipients.

it “mimic” quite the use of these dosage form in vivo, i.e the moisture of the dosage form ,and

the initial pression applied to obtain the adhesivity.

The results are reproducible with the parameters determined, and the force of adhesion was

found to be proportional to the quantities of adhesive excipients contained in the formulation. So

this method could be used to determine the optimal concentration of excipients necessary to

obtain a convenient adhesivity.

This method has been used for other mixtures containing different adhesives excipients and the

results, published in an international meeting shown the confirmation of the first data [20].

• However other studies are required to insure that a relationship exists between in vitro and in

vivo adhesion measurements

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Refe rence s:

[1] A. E. COLLINS and P. B. DEASY, J. Pharm. Sci., 79-116 (1990).

[2] S. SHADOMY and M. A. PFALLER, in Manual of Clinical Microbiology, 5th ed., A. BALOWS et al.

(eds.), American Society for Microbiology, Washington, DC, 1991, p. 1173.

[3] B. MIRABEL, A nn. Nutr. Alim., 32, 243-253 (1978).

[4] J.N. WIT and R. BOER, Neth. Milk Dairy J., 29, 198-211 (1975).

[5] G. HUMBERT and C. ALAIS, La technique laitière., n° 952, 73-74 (1981).

[6] C.V. MORR, J. Dairy Sci., 58, n°7, 977-984 (1975).

[7] J.P. CHERRY, Washington D.C., Am. Chem. Soc., (1981).

[8] J.E. KINSELLA, C.R.C. Crit. Rev. Food Sci. Nutr., 7, 219-280 (1976).

[9] J.C. CHEFTEL et al., in Protéine alimentaire, Paris, Tec & Doc Lavoisier (1985).

[10] D.H. CHOU and C.V. MORR, J. Am. Oil Chem. Soc., 56, 53A-52A (1979).
[11] J. M. AIACHE et al., 19: 401-405 (1998).
[12] R. KHANNA.et al., Drug Development and Industrial Pharmacy, 23 (8), 831-837 (1997).

[13] MERIOT. F, Ph.D thesis. University of Tours, France (1997).

[14] N. A. PEPPAS and A. G. MIKOS, S. T. P Pharma 5 (3) 187-191 (1989).

[15] M. MARVOLA et al., J. Phar. Sci, 71, 975-977 (1982).

[16] P. FORGET et al ., S. T. P Pharma 4 (3) 176-181 (1988).

[17] G. PONCHEL et al., S. T. P Pharma 4 (8) 688-697 (1988).

[18] D. DUCHENE and G. PONCHEL, European journal of Pharmaceutics and biopharmaceutics 44 15-

23 (1997).

[19] B.BERY, Ph.D thesis. University of Aix-Marseille, France (1999).

[20] J. M. AIACHE et al., Proc. 4th World Meeting ADRITELF/APGI/AP, Florence, (2002).

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Se ries Com Se ries Aver age wei ght Averag e hard ness

position (mg) (m g) (Newton )

CP 20% milk protein 250 60

80% Metolose
2C 20% milk protein 265 70

80% Metolose
4C 40% milk protein 248 65

60% Metolose
6C 60% milk protein 258 63

40% Metolose 60
100C 100% milk protein 240 60
Table 1: tablets’ composition

Milk protein Number of mean Standard

concentration experimentation deviation
s
20% 5 0.00074 0.00023
40% 5 0.00092 0.00012

60% 4 0.00102 0.00022

80% 6 0.00143 0.00062

Table 2: negative work measurement (NA : broken tablets)

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Figure 1: The Texture analyser

Figure 2: Photo of aluminium adhesion support with its base, and plastic container

Figure 3: graphical display of the adhesion force (negative work)

a: the contact between inferior surface of the tablet and the adhesion surface
a to b: initial pressure applied by the probe on the tablet
a to c: the tablet is maintained on the aluminium surface (six minutes in this example)
c to d: the displacement unit is going up and the negative work presenting adhesion force is measured
d: detachment of the tablet

Figure 4: Water volume results (n= negative results, p= positive results)

FREQ: Frequency of occurrence
CUM FREQ: cumulative frequency
PCT: Percent of the total positive results
CUM PCT: cumulative percent of the total positive results

Figure 5: moistening time results (n= negative results, p= positive results)

Figure 6: variation of adhesive forces when the milk protein percentage changed

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Figure 1

Figure 2

15
Figure 3

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Figure 4

Figure 5

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Figure 6

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