AU2010294008B2 - Use of guaifenesin for inhibiting mucin secretion - Google Patents

Abstract

A method of inhibiting mucus secretion in an individual that includes administering an effective amount of a composition which comprises guaifenesin.

Description

USE OF GUAIFENESIN FOR INHIBITING MUCIN SECRETION BACKGROUND OF THE INVENTION IL Field of the Invention The present invention relates to the use of a pharmaceutical compound for the inhibition 5 of mucus secretion in an individual, In particular, the present invention relates to the use of guaifenesin for the inhibition of mucus secretion. 2. Description of:Related Art Guaifenesin, whose chemical name is 3-(2-methoxyphenoxy) ,2-propanedioi, is an expectorant. An expectorant is a drug that helps bring up mucus and other material from the 0 lungs, bronchi, and trachea. Guaifenesin is thought to act by thinning the mucus, loosening phlegm and bronchial secretions, and also by lubricating the irritated respiratory tract. By thinning the mucus, guaifenesin reduces the viscosity of the mucal secretions, and as a result increases the efficiency of the cough reflex and of ciliary action in removing accumulated secretions from trachea and bronchi. The effect felt by an individual is that a nonproductive 5 cough becomes more productive and less frequent. In the prior art there are disclosed methods of inhibiting mucin. However, these methods are directed to the treatment of chronic conditions, such as asthma. WO 2004/043392 discloses a method of modulating mucin synthesis and the therapeutic application of compounds in controlling mucin over-production associated with diseases such as chronic obstructive 20 pulmonary diseases (COPD), including chronic bronchitis, and, inflannatory lung diseases, asthma, cystic fibrosis and acute or chronic respiratory infectious diseases using compounds of a defined formula having at least two aromatic rings. BRIEF SUMMARY OF THE INVENTION The applicant has developed a method of inhibiting the secretion of mucus in an 25 individual which comprises administering an effective amount of a composition which comprises guaifenesin. According to a first aspect of the present invention there is provided a method of inhibiting mucus secretion of bronchi and trachea in an individual which comprises 1 administering an effective amount of a composition which comprises guaifenesin wherein the composition comprises an inu-nediate release portion and a sustained release portion, such that the inhibition of mucus secretion is therapeutically achieved for a period of approximately 12 hours and further wherein the composition comprises from 600mg-1200mg of gaifenesin ai 5 further wherein the composition inhibits mucus secretion of bronchi and trachea. The guaifenesin can be administered in many suitable forms such as a tablet., powder, capsule, liquid or liquigel, The guaifenesin can be administered orally. The mucin can be produced in the upper respiratory tract of an individual. The composition can contain one or more additional active agents selected -from the 0 group including, but not limited to, an antitussive such as dextromethorphan hydrobromide, a decongestant such as phenylephrine hydrochloride, pseudoephedrine hydrochloride or ephedrine, an antihistamine such as chlorpheniramine maleate, brompheniramine maleate, phenindamnine tartrate, pyrilamine maleate, doxylarnine succinate, phenyitoloxamine citrate, diphenhydramine hydrochloride, promethazine, and clemnastine fumerate, fexofenadine or a combination thereof. 5 The daily dose of guaifenesin can he 2 400ing. According to a second aspect of the present invention there is provided a method of treating an individual having a disease or condition characterized by increased mucin secretion of bronchi and trachea with an effective amount of a composition which comprises guaienesin. wherein the composition comprises an immediate release portion and a sustained release portion, 20 such that the inhibition of mucus secretion is therapeutically achieved for a period of approximately 12 hours and further wherein the composition comprises frorn approximately 600mg- 1200mg of guaifenesin and further wherein the composition inhibits mucus secretion of bronchi and trachea. The disease or condition characterized by increased mucin secretion infectious cart be 25 selected from inflammatory conditions of the airways. BRIEF DESCRIPTION OF THE FIGURES Example embodiments of the present invention will now be described in more detail with reference to the accompanying figures. 2 Fig. I illustrates the treatment protocol. Fig. 2 is a graph showing the effect of guaifenesin on MU~C5AC inucin secretion: 30 mm Figs. 3a and 3h are graphs showing the effect of guaifrriesin on MUCSAC inucin secretion: 6 hours / / / / / / / / / / / / / / / / / // / / / / / / / / / / / / / / / / / / / / / / / / / 2A WO 2011/030163 PCT/GB2010/051525 figs. 4a and 4b are graphs showing the effect of guaifenesin on MUC5AC mucin secretion: 24 hours Figs. 5a and 5b are graphs showing the effect of guaifenesin on MUC5AC mucin secretion: 48 hours S Fig. 6 is a graph showing the effect of guaifenesin on mucociliary clearance. Figs. 7a and 7b are graphs showing metabolic activity. Figs. 8a, 8b and 8c are graphs showing mucus rheology. Figs. 9a and 9b are graphs showing the vector sum of viscosity and elasticity against time and dose, 10 DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS MATERIALS AND METHODS Cens: EpiAirway cultures (normal human bronchial epithelial) cells grown on Millipore Transwels, I or 4.2 cm 2 surface area. The cells were purchased from MatTek_ and were cultured. 15 at air-liquid interface for two (mucus synthesis and secretion) or three (mucociliary transport and mucus theology) weeks prior to use. Guaifenesin (GGE) Treatment: For mucociliary clearance, a stock guaifenesin solution of 2 m gmL in culture medium was prepared in the coming of each experiment and kept cold until diluaion into warmed 20 medium to the target concentrations of 02, 2, 20 or 200 pig/mL The medium in the hasolateral compartment of each culture was replaced with the GGEcontaining medium, and the cultures were returned to the 37C. 5% CO 2 incubator for as the times indicated, The experiments were repeated three times on independent cultures. The concentrations used in the in vitro experiments range from 0.2 pg/mL to 20 mg/mi 25 and thus bracket the clinical doses used in hmnans. Measurement of Mucin Secretion: WO 2011/030163 PCT/GB2010/051525 GUAIFENESLN solutions were prepared by dissolving in PBS (phosphate buffered saline) immediately before treatment of the cells. MUCSAC mucins were quantified by ELISA using 45M1 antibody (Labvision, Fremont. CA). Confluent I cnN grown on an air/liquid interface were washed from the apical surface with 200 pL PBS and incubated with 5 fresh complete growth medium added to the basal chamber, Cultures were incubated 24 hours to collect the apical fluid (pretreatment sample or PT) by adding 100 pL PBS to the apical surface of the cultures. PBS was added to dilute the highly viscous, thin mucus layer on the surface. Because of the small size of the insert, it was not feasible to collect a sufficient amount of mucus for both phannacology and rheology without the addition of PBS, After collecting 100 pL of the 1.0 diluted mucus samples (PT), cultures were divided into three groups (6 hr 24 hr and 48 hr), 16 inserts per group, and treated with varying concentrations of guaifenesin (0, 0 2,2 20 pg/miL) for each tine group, 4 inserts per each dose Thus, a total of 48 inserts were used for this study (4 inserts/dose x 4 doses/time point x 3 time points), The apical fluid was collected at 30 minutes following drug treatment from all the cultures to see whether guaifenesin affects the 15 "secretion" of mucins. The apical mucus sample was collected in two steps -first by adding 100 p4 PBS to the apical surface (I" wash) and then by adding 100 pL PBS containing 5 mM dithiothreitol (DTT) (2" wash). Samples from each wash were assayed for MUC5AC content arid the sum of the two values (the It land 2"n wash) was expressed as the "released MUCSAC" of the culture. At the three different tine points (i.e, 6 24 and 48 hr cultures were washed to 20 collect the apical fluid as described above ("released mucin") and lysed using a lysis buffer (PBS pH1 7.2, mMnt Triton X-100, 2 mM EDTA, I mM PSMF and 5 mM DITT) ("cellular mucin"). The amount of mucin in each sample eitherr secreted, released or ceH lysate) was divided by the amount of mucin in the PT sample collected from the same well in order to obtain a "secretory index" to compensate for the variations among the cultures. The treatment protocol is depicted 25 in Fig. 1, Measurement of Mucociliary Clearance: Cultures (4.2 cn 2 ) were exposed to basolateral guaifenesin for I or 6 hr. The cultures were removed from the incubator and placed. on the stage of digital imaging microscopy system. Video data were collected for 10 seconds using a 25x objective. The rate of movement of 30 endogenous cell debris was analyzed on the video images using a transparent template overlay 4 WO 2011/030163 PCT/GB2010/051525 on the video images and a stopwatch to measure at least 5 particles on each culture -fr a total of between 30 and 45 measurements per condition. Collection of MNeus: Following the analysis of clearance, mucus was harvested from the apical surface of the 5 cultures, without dilution. Viability: The apical surfaces of the cultures were then washed. with PBS and the metabolic activity, an indicator of viability, was measured using the Water Soluble Tetrazolium (WST) assay (Boehringer). 10 Rheologic Measurements: The ecological properties of apical mucus secretions (20 pL) were measured using an ARI000 controlled stress rheometer (TA Instruments, New Castle DE) using a parallel plate geometry. The dynamic linear viscoelastic behavior was determined f1om the strain response to an oscillating stress and reported as a storage or elastic modulus (C. and loss or viscous (' ) 15 modulus. as a function of frequency to such that viscosity, r- Go, Rheologic data can also be presented using vectorial notation as tangent 6 which is the ratio of viscosity to elasticity and G*; the vector sum of viscosiy and elasticity (mechanical impedance.). When stress in the linear range is used to evaluate the materials, the material properties are independent of stress. II order to conduct a frequency sweep from 0. 1 to 1000 rad/,s, we evaluated 20 viscoelastcity using a creep test at 0.5 Pa for 2 minutes. The strain response was fitted to a discrete relaxation spectrum, tran.sfbrmed to the retardation spectrum, and then to the storage and loss moduli, as a function of frequency, using methods developed by the PL We evaluated the linear viscoelastcity at I and 100 rad/s and we used an oscillatory stress sweep and. steady shear flow experiments to evaluate the behavior in the non-linear ranges. fe oscillatory sweep data 25 were analyzed by observing the stress where G' and G" crossed, This point indicates where the material shows more viscous behavior (irreversible deformation and f1ow) than. recoil behavior. All rheologic measurements were made by technicians who were blinded to the treatment group origin 5 WO 2011/030163 PCT/GB2010/051525 Statistics: For mucin secretion, differences between control and guaifenesin treatment groups were assessed by conparing the means using Student's t-test for unpaired samples and p<0.05 was considered significant. All the values in the figures represent means ± SEM of 4 cultures unless 5 otherwise stated. * p<005 **p<0.01 Por mucociliary clearance, differences between control and guaifenesin treatment groups were assessed by comparing the means using ANOVA, with a Bonferroni post-test to assess differences from the controls tested at the same time after treatment. A p value of < 0.05 was considered statistically significant; 10 For rheology experiments, data were analyzed using the StatView"ht 5 statistics package. Raw data were visually confirmed to be normally distributed about the mean. ANOVA was used to compare results of treating sputum with different concentrations of guaifenesin. Fisher's protected least significant diffLrence test was done to determine significance with multiple comparisons, Data are presented as group means + I standard error unless otherwise indicated 1.5 By convention p < 0.05 is considered statistically significant; RESULTS In Fig. 2. EpiAirway cultures were treated with the indicated concentrations of guaifenesin for 30 min, Secreted MUC5AC was compared with the pre-treatment values. During the 30 minute treatment period, there was no significant difference (p<:0.5) 20 between control and guaifenesin treatment groups. In Fig. 3a. the white boxes represent the amount. of mucin associated with the cell. whereas the black boxes represent the amount of mucin released during the given period of treatment, Therefore, the addition of the white box and the black box represents the total amount of nucin produced during the given period. The total amounts of MUC5AC were compared for 25 statistical differences between control (no guaifenesin) and guaifenesin groups, Treatment of NHFIBE cells with guaifenesin for 6 hours did not affect the amounts of mucins released (Fig. 3b). However, the total amounts of mucins produced during the 6 hour treatment period were significantly ()<0,0 1.) suppressed by the presence of guaifenesin (both 2 pg/ml and 20 pgml ). 6 WO 2011/030163 PCT/GB2010/051525 Twenty-four hour treatment with either 2 pg/mil or 20 gg/mL of guaifenesin signifiCantly suppressed mucin release (Fig. 4b) as well as mucin production (Fig. 4a). Treatment with guaifenesin (g pg/ml and 20 pgnL) for 48 hours significantly (p<0%'O) :uppressed the production of mucins (Fig 5a). However, the amount of nucin released during 5 this period did not seem to be significantly afkcted. Effect of Guaifenesin on Mucociliary Clearance: As shomn in Fig. 6, guaifenesin appeared to increase the mobility of the cellular debris on the surface of cultures treated for 1 hr, but there was little evidence of a dose-response and in fact, only the eflect of 2 pg/ml was statistically significant, Hlowcvs at the 6 hr time point, 10 there was a strong trend to a dose response and movement of the surface material for all three concentrations tested was significantly faster than the control as illustrated in Fig. 6, EpiAirway cultures were treated with the indicated concentrations of guaifenesin for 1 or 6 hrs. Mucociliary clearance was assessed. by the rate of movement of endogenous debris on. the surfaces. ** indicates significantly different from the control cultures at the same time p < 15 0.005. Viability: There was no adverse effect on the viability of the cells as indicated by the WST assay. In fit. there appeared to be a trend to increased metabolic activity in the cells treated with guaifenesn, however this did not reach statistical significance. Data from one of the three 20 replicate experiments is shown below. As shown in Figs. 7a and 7b, EpiAirway cultures were treated with the indicated concentrations of guaifenesin for I or 6 hr. Metabolic activity was assessed using the WST assay, separately added to the apical or basal surfaces of the cultures. Rheology: 23 A total of 96 specimens from 5 sets of experiments were analyzed. The mucus from the first fbur experiments was received at arbient temperatures and analysis of rheology Of these samples showed extreme heterogeneity and the rheologic sweep curves obtained were consistent with degradation. The results shown in figures 7 and 8 are therefore derived from the 22 specimens received from batch. five. All specinens were non-Newtonian, viscoelastic gels, WO 2011/030163 PCT/GB2010/051525 The results demonstrate a significant guaifenesin dose-dependent decrease in viscosity; elasticity, and complex modulus (G*) of specimens at 1 hour (p 0,05) and especially at 6 hours (p< 0.01) when measured at I rad/s or roughly ciliary frequency but not significantly at 100 rad/s corresponding to cough. 5 Mucus Rheology. Fig. Sa: G" viscosity, Fig. 8b: G' elasticity, Fig. 8c G nechanical impudence (vector sum of viscosity and elasticity). Data shown are the mean and standard error of data from the I and 6 hr time points combined, Gs* vector sum of viscosity and elasticity, at I rad/s (Fig. 9a) and 100 rad/sec (Fig. 9b), segregated by time as well as dose. 10 In all three treatment time periods (6 24 and 48 hours), guaifenesin at both 2 pghnL and 20 g/niL suppressed the production of inucins by NI4BE cells grown on. an air/liquid interface. Likewise, treatment with both 2 pg/iL and 20 pg/mL of guaifenesin for 24 hours showed a significant (p<0.05) decrease in mucin release. To address the effects of guaifenesin on mucociliary clearance. mucociliary transport 1 5 rates were measured. The purpose of these experiments was to investigate potential. alteratio-ns in mucoci -iry clearance induced by exposure of differentiated primary human tracheo-bronchial epithelial cells to Guaifenesin. The original plan was to deposit aerosolized I pm diameter fhorescent microspheres on the surface of the cultures using a nebulizer However. for reasons that are unclear, although the microspheres could be identified on the cultures, there was 20 movement in only a very few of the cultures, despite clear movement of the endogenous cellular debris. A switch to collecting video of the endogenous debris was made Viscosity (loss modulus) is the loss of energy from a rheologic probe or applied stress and thus the resistance to flow. Elasticity (storage modulus) is the recoil energy transmitted back to the probe. The complex modulus, G*, is also known as the mechanica] impednce As the 25 pectoral sum of the storage and loss moduli, G* measurement indicates resistance to deformation. Viscoehsticity is a property of non-Ncwtoni an fluids (gels). Dynamie viscoelasticitv measures tUe strain response of mucus to an applied stress, Because mucus is subjected to both low stress (cilary beat) and high stress (cough) conditions we measure the strain developed in response to a dynamic stress. 8 These results are consistent with the secretions taken from the differentiated cells being mucus gels. Although degradation of specimens from experiments 1-4 produced inconsistent results suggesting degradation (raw results all available on request), those from the final set of experiments were well preserved and the results were robust. The decrease in complex modulus paralleling that of 5 the viscosity (loss modulus) would be consistent with the increased ciliary transport. The rheologic characteristics of these specimens suggested a goblet cell origin with viscosity approximately equal to elasticity, rather than a submucosal gland secretion where the elasticity is generally greater than viscosity. These results are consistent with the reported structure of the EpiAirway cultures. It will be informative to compare these results with those from human tissue explants exposed to guaifenesin. 0 Guaifenesin suppressed mucin production from confluent human bronchial epithelial cells grown on an air-liquid interface in a dose-dependent manner in vitro at concentrations that are clinically relevant. The reduction in mucus production correlated with increased mucociliary transport and decreased viscoelasticity of the mucus. Further modifications or improvements cane be made without departing from the scope of the 5 invention herein described. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. !O All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or 25 elsewhere before the priority date of each claim of this application. 9

Claims (21)

1. A method of inhibiting Inucus secretion of bronchi and trachea in an individual comprising administering an effective amount of a composition comprising gualfenesin wherein the composition coinprises an iminiediate release portion and a sustained release portion, such that the inhibition of mucus secretion is therapeutically achieved for a period of approximately 12 hours and furiher wherein the compositon comprises from 600mg-1200mg of guaifenesin and further wherein the composition inhibits mucus secretion of bronchi and trachea.

2. A method as claimed in Claim 1, wherein the composition comprises approximately 600mg of guaifenesin,

3. A method as claimed in Claim 1, wherein the composition comprises approximately 1200m of uaifenesin.

4. A method as claimed in any one of the preceding Claims, wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a tablet.

5. A method as claimed in any one of Claims I to 3, wherein administerinag an effective amount or a composition comprising gluaifenesin comprises administering the composition as a powder.

6. A method as claimed in any one of Claims I to 3, wherein administering an effective amount of a composition comprising guaifenesirn comprises administering the composition as a capsule, A method as claimed in any one of Claims 1 to 3 wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a liquid 8, A method as claimed in any one of Claims 1 to 3, wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a liqu~igel.

9. A method as claimed in any one of the preceding Claims, wherein the composition further comprises one or more active agents,

10. A method as claimed in Claim 9, wherein the one or more active agents are selected from the group consisting of an antitussive, a decongestant, and an antimistamine.

11. A method as claimed in Claim 10, wherein the antitussive comprises dextromethorphan hydrobronuide.

12. A method as claimed in Claim 10, wherein the decongestant is selected from the group consisting of phenylephrine hydrochloride, pseudoephedrine hydrochloride and ephedrine. 1(l 13 A method as claimed in Claim 10, wherein the antihistamine is selected from the group consisting of chlorpheniramine naleate. brompheniramine maleate, phenindamine tartrate, pyrilanine maleate. doxylamine succinate, phenyltoloxamine citrate, diphenhydramine hydrochloride, prometlazine clemastine fumerate, and fexofenadine,

14. A method of treating an individual having a disease or condition characterized by increased mucin secretion of bronchi and trachea with an effective amount of a composition comprising guaifenesin wherein the composition comprises an immediate release portion and a sustained release portion, such that the inhibition of mucus secretion is therapeutically achieved for a period of approximately 12 hours and further wherein the composition comprises from approximately 600mg-1200mg of guaifenesin and further wherein the composition inhibits mucus secretion of bronchi and trachea. 15, A method as claimed in Claim 14, wherein the composition coinprises approximately 600mg of guaifenesi.

16. A method as claimed in Claim 14, wherein the composition comprises approximately 1200mg of guaifenesin. 17,.A method as claimed in any one of Clairns 14 to 16, wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a tablet. 18 A method as calmed in any one of Claims 14 to 16, wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a powder.

19. A method as claimed in any one of Claims 14 to 16, wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a capsule. 20, A method as claimed in any one of Claims 14 to 16, wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a hiuid, 21, A method as claimed in any one of Clairns 14 to 16, wherein administering an effective amount of a composition comprising guaifenesin comprises administering the composition as a liquigel.

22. A method as claimed in any one of Claims 14 to 21, wherein the composition further comprises one or more active agents,

23. A method as claimed in Claim 22, wherein the one or more active agents are selected from the group consisting of an antitussive, a decongestant, and an antihistamine. I i

24. A method as claimed in. Claim 23, wherein the antitussive comprises dextromethorphan hydrobronide.

25. A method as claimed in Claim 23, wherein the decongestant is selected from the group consisting of phenylephrine hydrochloride, pseudoephedrine hydrochloride and ephedrine.

26. A method as claimed in Claim 23, wherein the antihistamine is selected from the group consisting of chlorpheniramine maleate, brompheniramine ntaleate, phenindamine tartrate, pyrilamine nialeate, doxylamine succinate, phenyltoloxamine citrate, diphenhydramine hydrochloride, promethazine, clemastine fimerate., and fexofenadine.

27. A method as claimed in any one of Claims 14 to 26, wherein the daily dosage of guaifenesin is 2400mg.

28. A method as claimed in any one of Claims 14 to 27., wherein the condition a disease or condition characterized by increased mucin secretion is selected from infectious and inflammatory conditions of the airways.

29. A method as claimed in any one of Claims I to 13 wherein the daily dosage of guaifenesin is 2400mg, 30, A method as claimed in either Claim I or Claim 1 4 and substantially as herein described. 12