Hydroxypropylmethylcellulose gel application delays Der p 1 diffusion in vitro

doi:10.4236/ns.2010.22012

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Vol.2, No.2, 79-84 (2010) Natural Science

http://dx.doi.org/10.4236/ns.2010.22012

Hydroxypropylmethylcellulose gel application delays

Der p 1 diffusion in vitro

B. Diethart1, J. C. Emberlin2, R. A. Lewis3

1School of Human and Health Sciences, Swansea University, Swansea, United Kingdom; [email protected]

2National Pollen and Aerobiology Research Unit, University of Worcester, Worcester, United Kingdom

3Worcestershire Royal Hospital, Worcester, United Kingdom

Received 16 November 2009; revised 10 December 2009; accepted 30 December 2009.

ABSTRACT

Background: A special hydroxypropylmethyl-

cellulose powder (Nasaleze®) has been used for

the alleviation of nasal symptoms of allergic

rhinitis since 1994. The efficacy of the product

has been recently proven but the mechanism of

action was still largely unknown. The aim of the

study was to investigate the hypothesis that the

gel formed after moisture absorption in the nose

might act as mechanical barrier that prevents

allergen diffusion towards the nasal epithelium.

Methods: The diffusion of Der p 1 through

HPMC and agar gels was measured in vitro after

15, 30, 60, 180 and 360 minutes using ELISA.

Agar blocks were used to simulate the nasal

mucosa. Control samples without gel layer were

obtained. Results: The control samples with no

applied gel barrier absorbed 72.2 % of the Der p

1 solution after 15 minutes and 100 % after 60

minutes. In comparison, the HPMC and agar gel

layers both significantly delayed Der p 1 diffu-

sion. After 15 minutes 0.76 % had diffused

through the HPMC gel layer compared to 28.1 %

which diffused through the agar layer. After 360

minutes, 14.1 % of the baseline Der p 1 crossed

the HPMC gel layer while 100 % had diffused

through the agar layer. Conclusions: HPMC gel

significantly reduces Der p 1 diffusion in vitro

compared to no barrier and an agar gel layer.

This is likely to be due to the small mesh size of

the polymer network of HPMC and could have

important implications for a preventative treat-

ment of allergic rhinitis.

Keywords: Allergic Rhinitis; Der p 1;

Diffusion Barrier; Hydroxypropylmethylcellulose

1. INTRODUCTION

Allergic rhinitis (AR) is a global health problem which

affects up to 25 % of the adult population in industrial-

ised countries and more than 40 % of children [1,2]. The

rising prevalence of allergic rhinitis imposes a huge

burden on the economy due to costs of treatment and

loss of work productivity. Recent estimates of annual

costs range from $2 to 5 billion in the U.S. alone [3-5].

The pathology of AR is associated with a severe im-

pairment of the quality of life for those who suffer from

it [6,7]. A reduction of quality-of-life impairment can be

achieved by appropriate treatment of allergic rhinitis

[7,8]. Modern medications such as antihistamines or

corticosteroids can do a lot to help to alleviate symptoms

and restore a normal lifestyle but many of them have

unwanted adverse effects or are limited in their applica-

tion [1,3,4]. Many people distrust these conventional

medicines and therefore prefer to use complementary

and alternative treatments. However, the therapeutic

efficacy of many of these treatments is not supported by

evidence and they might not be devoid of side effects

[3,9].

A recent approach is offered by the use of an inert hy-

droxypropylmethylcellulose (HPMC) powder (Nasaleze

®) for allergy prevention and alleviation in the nose.

Although the product has been registered as a class 1

medical device with the MHRA since 1991 and is sold

over the counter in more than 50 countries worldwide,

little work has been done on the effect of the powder on

nasal symptoms. However, the efficacy of HPMC in

decreasing symptoms of allergic rhinitis caused by grass

pollen and house dust mite allergens was recently proven

[10-12]. The investigators observed an improvement of

symptoms when using HPMC for treatment of SAR and

PAR. Nasal peak inspiratory flow (PIF) and peak expi-

ratory flow (PEF) increased compared to placebo and

some symptoms of allergic rhinitis including sneezing,

itching and runny nose were alleviated significantly.

Also the need to use rescue medication was found to be

reduced. Considerable variance was observed in the re-

sults and some participants did not show any improve-

ment. This was partly attributed to the application device

which is suspected not to deliver constant doses [12,13].

B. Diethart et al. / Natural Science 2 (2010) 79-84

The HPMC powder is applied to the nose using a spe-

cially designed dry powder dispenser bottle and forms a

gel on the nasal lining by absorbing moisture from the

nasal mucosa. It was hypothesised that this gel might act

as a mechanical barrier preventing allergens from enter-

ing the mucosa [11,12]. However, no investigations on

the mechanism of action of HPMC as an allergy treat-

ment have been published as yet leaving the question

how an inert cellulose derivative can offer relief to indi-

viduals affected by allergic rhinitis unanswered. Similar

HPMC powders which also form hydrogels upon contact

with liquids are widely used in controlled drug release

formulations where they restrict the release of drug

molecules through the tablet by serving as a barrier to

drug diffusion [14]. Also, high-viscosity HPMC gels

have been shown to limit glucose and cholesterol ab-

sorption in the gastrointestinal tract by creating a me-

chanical barrier [15,16]. Thus, it is assumed that HPMC

gel might impede the passage of allergens in a similar

manner.

The aim of this study was to investigate the possibility

that HPMC gel might constitute a mechanical barrier to

house dust mite allergen in vitro in order to gain infor-

mation about the mechanism of action of HPMC in the

alleviation of symptoms of allergic rhinitis.

2. METHODS

2.1. Materials

Hydroxypropylmethylcellulose powder was supplied by

Colorcon Limited, Kent. Der p 1 solution (in house ref-

erence, 7.5 μg Der p 1 per millilitre) was provided by

Alk-Abello, Madrid.

2.2. Sample Preparation

Preparation of the samples took place in a cleanroom to

minimise contamination by dust or allergens. All equip-

ment needed for preparation was washed in isopropyl

alcohol (70 %) for sterilisation and dried before each use.

Ten ml of agar (1.5 %, prepared with 0.9 % saline solu-

tion) were cast into a petri dish. After cooling, small

rectangles of equal dimensions (1 x 1 cm) were cut from

the agar and then transferred to cleaned slides. Two lines

of warm and therefore liquid Vaseline were drawn with a

brush from the two edges of one side of the agar block to

the edges of the slides to avoid diffusion of allergens

through the side of the block (Figure 1). The position of

the agar was marked on the bottom of the slide and the

agar block was covered by a cover slip that sealed the

upper surface of the agar. Allergen solution could there-

fore diffuse into the agar through only one free edge

(Figure 1).

To test the barrier function of HPMC, a thin layer of

HPMC gel was applied covering the edge of the agar

which was used for allergen application. For this, 50 mg

of HPMC powder were mixed with 1 ml physiological

saline solution (0.9 %) to form a 5 % gel. Immediately

after the mixing of the gel, 0.2 ml was applied to the

open edge of the agar block using a 1 ml sterile syringe.

The initial thickness of the gel layer was measured at 3

standard points. After covering with a cover slip, 20 µl

of the allergen solution were applied to the HPMC gel

covering the one side of the agar blocks limited by the

Vaseline lines.

The slides were incubated at 35°C and 90 % relative

humidity to simulate nasal conditions for 15, 30, 60, 180

and 360 minutes. After incubation the thickness of the

HPMC layer was again measured. The agar blocks were

then carefully removed from the slides and transferred to

labelled microtubes containing 0.5 ml PBS-T as elution

medium. Samples were shaken on an Autovortex for 20

seconds followed by shaking overnight on a lab shaker.

Samples were stored frozen at -20°C.

2.3. Reference and Control Samples

To investigate the difference of diffusion through HPMC

and agar, control samples were produced with an addi-

tional agar layer of 1.5 mm (average thickness of the

HPMC gel layer calculated from measurements of

HPMC samples using a digital caliper) to replace the

HPMC gel and treated in exactly the same way as the

HPMC samples.

Additionally, control samples with no allergen addi-

Figure 1. Photograph (A) and diagram (B) of experimental

setup for sample preparation for ELISA measurements of Der p

1 diffusion through HPMC gel.

B. Diethart et al. / Natural Science 2 (2010) 79-84

tion and no barrier addition, respectively were obtained.

Baseline measurements of the allergen amount in 20

µl of allergen solution were conducted by applying 20 μl

of allergen solution directly to a microtube containing

0.5 ml of PBS-T. The microtubes were then treated in

the same way as the microtubes containing the agar

blocks.

2.4. ELISA Measurements

The monoclonal antibodies (mAbs) and Der p 1 allergen

standards used in the assays were purchased from Indoor

Biotechnologies, and the assays were performed accord-

ing to the manufacturer’s instructions.

2.5. Statistical Analysis

One-way ANOVA was applied for statistical analysis of

the differences between Der p 1 diffusion in HPMC gel,

agar gel and control samples, respectively. No serious

violations of assumptions were observed. P values of

0.01 or less were considered to be statistically signifi-

cant.

3. RESULTS

The mean baseline allergen content in 20 µl of the stan-

dard solution used was found to be 151.0 ng/ml (SD =

4.0 ng/ml). This is in good agreement with the calculated

value of 150 ng/ml for the given dilution of a 7.5 µg/ml

stock solution. All control samples with no allergen ap-

plication were negative in the ELISA measurements.

The diffusion of Der p 1 molecules into the 1 x 1 cm

agar blocks eluted for measurements was delayed with

both gel barriers applied (Table 1 and Figure 2). The

amount of allergen diffused through 1.5 mm of 1.5 %

agar gel was significantly different from the baseline

values for the first 180 minutes (p < 0.005) but did not

reach statistical significance after 360 minutes (p =

0.628). After 15 minutes of incubation, 28.1 % of the

baseline allergen amount had diffused through the gel

into the agar block (Table 2, p < 0.0001). The amount of

allergen detected in the elutes of the agar blocks then

steadily increased until it reached baseline level after

360 minutes of incubation (Figure 2 and Table 2). The

thickness of the agar layer applied as a barrier did not

change during the measurement times from 15 to 360

minutes. In contrast, an initially 1.50 mm thick HPMC

gel layer swelled to an average 3.34 mm in 360 minutes

upon allergen solution application. Diffusion of Der p 1

molecules through 5 % HPMC gel showed a significant

reduction of diffused allergen for all test times (p <

0.001). After 15 minutes 0.76 % of the baseline amount

had diffused through the HPMC gel layer into the agar

block compared to 28.1 % which diffused through the

agar layer (Table 2). After 360 minutes, 14.1 % of the

baseline Der p 1 crossed the HPMC gel layer while 100

% had diffused through the agar layer (Table 2). How-

ever, the HPMC data include several outliers and the

standard deviation is high (Table 1). The mean coeffi-

cient of variation for all measurements for the HPMC

gel was found to be 201.9 % which is very high com-

pared to 37.8 % for agar.

Control samples with no barrier had absorbed 72.2 %

of the baseline allergen content after 15 minutes and

differences to baseline did not reach statistical signifi-

cance after 60 minutes using a 99 % confidence interval

(p60min=0.042, p360min=0.990).

4. DISCUSSION

Most of the commonly available treatments of allergic

rhinitis affect the inflammatory processes (e.g. by abat-

ing mediator release or blocking receptors) initiated after

100

120

140

160

180

050100 150 200 250 300 350 400

Time (in minutes)

Amount of Der p 1 (in ng/ml

)

HPMC

Agar

Base line

No barrier

Figure 2. Amount of Der p 1 diffused through a 1.5 mm thick

HPMC and agar gel layer, respectively compared to control (no

barrier) and baseline allergen amount.

Table 1. Amount of Der p 1 diffused through a 1.5 mm thick HPMC and agar gel layer, respectively, amount of allergen absorbed

without barrier (control) and baseline allergen amount in 20 µl of the applied solution.

Amount of Der p 1 measured in samples (in ng/ml)

Time (in min) 15 30 60 180 360

HPMC 1.15 1.57 8.98 13.17 21.34

Agar 42.46 78.98 93.92 116.46 163.59

No barrier 109.26 no value 126.62 no value 154.92

Baseline 151.04 151.04 151.04 151.04 151.04

B. Diethart et al. / Natural Science 2 (2010) 79-84

Table 2. Fractions of allergen amount diffused through a 1.5 mm thick HPMC and agar gel layer, respectively and with no barrier

compared to the baseline value of 151.04 ng/ml.

Diffused fraction of Der p 1 (in % of baseline)

Time (in min) 15 30 60 180 360

HPMC 0.76 1.04 5.94 8.72 14.13

Agar 28.11 52.29 62.18 77.11 108.31

No barrier 72.34 no value 83.83 no value 102.57

Baseline 100.00 100.00 100.00 100.00 100.00

allergen penetration into the mucosa and binding to IgE

[1,17,18] and therefore represent symptomatic treatment.

This means that inflammation and the associated damage

of the mucosa are already established and the medication

decreases signs of this inflammation while it is still on

going. An ideal allergy treatment would inhibit the es-

tablishment of an allergic reaction altogether. Anti-IgE

prevents binding of allergen to IgE antibodies and so

inhibits a reaction while the allergens are already inside

the epithelium [19]. HPMC might work at an earlier

stage by preventing allergens from entering the mucosa

in the first place by the generation of a mechanical gel

barrier.

The present study aimed to investigate this possible

barrier function of HPMC to allergens. The results ob-

tained by ELISA-measurements show that HPMC sig-

nificantly delays Der p 1 diffusion and that the amount

of allergen diffused through the gel is even lower than

indicated by preliminary tests [20]. This retardation

might allow the mucosa to recover its physical integrity

and the allergic reaction to decline. However, a complete

barrier to Der p 1 diffusion could not be confirmed.

The retarded diffusion of solutes in hydrogels like

HPMC gel or agar gel is well known and widely used for

biotechnological separation methods such as electro-

phoresis or gel chromatography and in controlled release

formulations [21,22]. The most comprehensible model

developed to explain the diffusion delay of solutes in

gels is the obstruction theory which assumes that the

impenetrable polymer chains are obstacles that cause an

increase in diffusional path length and additionally act as

a sieve [21,24]. Therefore the mesh or pore size of the

polymer network is a crucial parameter in the reduction

of diffusion in hydrogels [25]. Hydrogels consist of high

molecular weight molecules forming a threedimensional

network which is dispersed in a continuous liquid me-

dium [22,25]. Due to cross-links and entanglements of

these molecules hydrogels can be described as a mesh

with solvent filled spaces between the individual poly-

mer chains which act as a filter for molecules larger than

the spaces available [26,27]. Controlled release studies

with FITC-dextran molecules of different molecular

weights revealed that the critical molecular weight for

diffusion in HPMC gels, which are characterised by a

mesh size of 12 nm, lies between 65 and 66.5 kDa de-

pending on the molecular weight of the polymer and the

concentration of the gel [28]. Allergenic proteins usually

have a molecular weight between 5 and 80 kDa [29,30].

This means that a great proportion of allergens theoreti-

cally are small enough to diffuse through the HPMC

mesh spaces. Although Der p 1 (24 kDa) lies well below

the mesh size of HPMC gels, a substantial delay in dif-

fusion has been observed. Even though molecules larger

than 65 kDa are stopped from diffusing through HPMC

almost completely, all other smaller molecules will still

be delayed by the longer diffusional path due to obstruc-

tions by the macromolecular chains and the slower water

movement due to binding of water to the polymer. Fur-

thermore, the mesh size and therefore the size of the

spaces available for diffusion in weakly cross-linked

homogenous gels is not stable but time-dependent and

the size and location of the spaces change due to

Brownian motion of the molecule chains [22,31].

In comparison to HPMC, the mesh size of a 1.5 %

agar gel as used in this study has been observed to be

between 70 and 800 nm [21,26]. Even the lowest of

these values is almost six times larger than the mesh size

of HPMC which explains the higher allergen diffusivity

within agar gel.

The values obtained in the present study are valid for

Der p 1 and allergens of the same or very similar mo-

lecular weight. It has been shown that the diffusion coef-

ficient for globular proteins in agar decreases with in-

creasing molecular weight and therefore radius of the

proteins [21]. This leads to the assumption that allergens

smaller than Der p 1 like Bet v 1 (17 kDa) or grass group

2/3 allergens (10-12 kDa) might be expected to diffuse

faster whereas larger allergens like Amb a 1 (38-50 kDa)

or Art v 1 (28-60 kDa) might exhibit slower diffusion

velocities through the HPMC gel network.

The variability of the results of the measurements of

Der p 1 diffusion through HPMC gel was high with a

coefficient of variation (CV) of just over 200 %. In

comparison, the CV of Der p 1 diffusion in agar gel was

only about 37 %. For this reason the variation in the

amount of allergen diffusing through the HPMC gel

layer cannot solely be attributed to limitations in the

methods that were applied. Similarly high variability of

diffusion coefficients was obtained for mucus gels [32].

This was attributed to the heterogeneous nature of the

B. Diethart et al. / Natural Science 2 (2010) 79-84

mucous gel producing uneven penetration profiles. Re-

lease from HPMC matrices for controlled drug release

was found to be sensitive to alterations in the chemical

composition and the polymer gel conformation and sub-

stantial batch-to-batch variations in release and swelling

could be observed for a single type of HPMC [33,34].

The authors suspect that this might be due to aggregate

formation in the gel causing transient cross-linking that

could perturb diffusion in some places throughout the

gel which cannot be predicted.

Due to its importance in controlled drug release, the

effect of HPMC as a diffusion barrier for drugs has been

studied extensively. However, no investigations of aller-

gen diffusion in HPMC have been found in the accessi-

ble literature. It was confirmed in this study that HPMC

gel delays Der p 1 diffusion in vitro. Other allergens

need to be tested to extend the evidence for the efficacy

of the product. Also many other factors will influence

the efficiency of the product in vivo. For practicality

reasons, the gel layer used in the experiments is thicker

than the gel layer that can be expected to be established

within the nasal cavity. Diffusion velocity is a crucial

parameter needed to make assumption for in vivo condi-

tions and should therefore be addressed in future re-

search. A complete diffusion barrier is essential for the

retardation of drug release [14] and similarly optimal

coverage of the nasal mucosa is important since uncov-

ered areas may allow free allergen entry and the provo-

cation of an allergic response. Sub-optimum coverage is

likely to reduce the efficiency of the product. The provi-

sion of a suitable powder delivery device therefore poses

an important challenge for the maximisation of the effi-

cacy of HPMC in the alleviation of allergic rhinitis.

In conclusion, a diffusion delay of Der p 1 in HPMC

gel has been confirmed in vitro. This means that even

though HPMC gel does not constitute an impermeable

barrier to allergens, the significant delay of allergen en-

try into the mucosa could be beneficial to hay fever suf-

ferers through the reduction of allergen exposure. This

fairly novel way of treatment reduces the allergen load

itself and not the symptoms caused after allergen entry

into the mucosa. Thus, with the appropriate delivery

device, HPMC could be a valuable, drug-free alternative

for the treatment of allergic rhinitis. The efficacy of

HPMC in hay fever treatment has been recently proven

[10-12]. However, the research presented in this paper is

the first to address the mechanism of action of HPMC in

the alleviation of allergic rhinitis. This knowledge will

allow improvements on the product to be made in order

to increase its benefit to hay fever sufferers.

5. ACKNOWLEDGEMENTS

This study was sponsored by the University of Worcester, UK and

Kisska International Ltd.

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