to facilitate an understanding of the principles and features of the various embodiments of the invention , various illustrative embodiments are explained below . although exemplary embodiments of the invention are explained in detail , it is to be understood that other embodiments are contemplated . accordingly , it is not intended that the invention is limited in its scope to the details of construction and arrangement of components set forth in the following description or examples . the invention is capable of other embodiments and of being practiced or carried out in various ways . also , in describing the exemplary embodiments , specific terminology will be resorted to for the sake of clarity . it must also be noted that , as used in the specification and the appended claims , the singular forms “ a ,” “ an ” and “ the ” include plural references unless the context clearly dictates otherwise . for example , reference to a component is intended also to include composition of a plurality of components . references to a composition containing “ a ” constituent is intended to include other constituents in addition to the one named . in other words , the terms “ a ,” “ an ,” and “ the ” do not denote a limitation of quantity , but rather denote the presence of “ at least one ” of the referenced item . also , in describing the exemplary embodiments , terminology will be resorted to for the sake of clarity . it is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose . ranges may be expressed herein as from “ about ” or “ approximately ” or “ substantially ” one particular value and / or to “ about ” or “ approximately ” or “ substantially ” another particular value . when such a range is expressed , other exemplary embodiments include from the one particular value and / or to the other particular value . further , the term “ about ” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art , which will depend in part on how the value is measured or determined , i . e ., the limitations of the measurement system . for example , “ about ” can mean within an acceptable standard deviation , per the practice in the art . alternatively , “ about ” can mean a range of up to ± 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 % of a given value . alternatively , particularly with respect to biological systems or processes , the term can mean within an order of magnitude , preferably within 2 - fold , of a value . where particular values are described in the application and claims , unless otherwise stated , the term “ about ” is implicit and in this context means within an acceptable error range for the particular value . by “ comprising ” or “ containing ” or “ including ” is meant that at least the named compound , element , particle , or method step is present in the composition or article or method , but does not exclude the presence of other compounds , materials , particles , method steps , even if the other such compounds , material , particles , method steps have the same function as what is named . throughout this description , various components may be identified having specific values or parameters , however , these items are provided as exemplary embodiments . indeed , the exemplary embodiments do not limit the various aspects and concepts of the present invention as many comparable parameters , sizes , ranges , and / or values may be implemented . the terms “ first ,” “ second ,” and the like , “ primary ,” “ secondary ,” and the like , do not denote any order , quantity , or importance , but rather are used to distinguish one element from another . it is noted that terms like “ specifically ,” “ preferably ,” “ typically ,” “ generally ,” and “ often ” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical , essential , or even important to the structure or function of the claimed invention . rather , these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention . it is also noted that terms like “ substantially ” and “ about ” are utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison , value , measurement , or other representation . the dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited . instead , unless otherwise specified , each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value . for example , a dimension disclosed as “ 50 mm ” is intended to mean “ about 50 mm .” it is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified . similarly , it is also to be understood that the mention of one or more components in a composition does not preclude the presence of additional components than those expressly identified . the materials described hereinafter as making up the various elements of the present invention are intended to be illustrative and not restrictive . many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention . such other materials not described herein can include , but are not limited to , materials that are developed after the time of the development of the invention , for example . any dimensions listed in the various drawings are for illustrative purposes only and are not intended to be limiting . other dimensions and proportions are contemplated and intended to be included within the scope of the invention . epiairway cultures ( normal human bronchial epithelial ) cells grown on millipore transwells , 1 or 4 . 2 cm 2 surface area . the cells were purchased from mattek , and were cultured at air - liquid interface for two ( mucus synthesis and secretion ) or three ( mucociliary transport and mucus rheology ) weeks prior to use . for mucociliary clearance , a stock guaifenesin solution of 2 mg / ml in culture medium was prepared in the morning of each experiment and kept cold until dilution into warmed medium to the target concentrations of 0 . 2 , 2 , 20 or 200 μg / ml . the medium in the basolateral compartment of each culture was replaced with the gge - containing medium , and the cultures were returned to the 37 ° c ., 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 μg / ml to 20 mg / ml and thus bracket the clinical doses used in humans . guaifenesin 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 , calif .). confluent 1 cm 2 nhbe cells grown on an air / liquid interface were washed from the apical surface with 200 μl pbs and incubated with 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 μl 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 pharmacology and rheology without the addition of pbs . after collecting 100 μl of the 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 μg / ml ) for each time group , 4 inserts per each dose . thus , a total of 48 inserts were used for this study ( 4 inserts / dose × 4 doses / time point × 3 time points ). the apical fluid was collected at 30 minutes following drug treatment from all the cultures to see whether guaifenesin affects the “ secretion ” of mucins . the apical mucus sample was collected in two steps — first by adding 100 μl pbs to the apical surface ( 1 st wash ) and then by adding 100 μl pbs containing 5 mm dithiothreitol ( dtt ) ( 2 nd wash ). samples from each wash were assayed for muc5ac content and the sum of the two values ( the 1 st and 2 nd wash ) was expressed as the “ released muc5ac ” of the culture . at the three different time points ( i . e ., 6 , 24 , and 48 hr ), cultures were washed to collect the apical fluid as described above (“ released mucin ”) and lysed using a lysis buffer ( pbs , ph 7 . 2 , 1 mm triton x - 100 , 2 mm edta , 1 mm psmf and 5 mm dtt ) (“ cellular mucin ”). the amount of mucin in each sample ( either secreted , released or cell 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 in fig1 . cultures ( 4 . 2 cm 2 ) were exposed to basolateral guaifenesin for 1 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 25 × objective . the rate of movement of endogenous cell debris was analyzed on the video images using a transparent template overlay on the video images and a stopwatch to measure at least 5 particles on each culture , for a total of between 30 and 45 measurements per condition . following the analysis of clearance , mucus was harvested from the apical surface of the cultures , without dilution . 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 ). the rheological properties of apical mucus secretions ( 20 μl ) were measured using an ar1000 controlled stress rheometer ( ta instruments , new castle , del .) using a parallel plate geometry . the dynamic linear viscoelastic behavior was determined from the strain response to an oscillating stress and reported as a storage or elastic modulus ( g ′), and loss or viscous ( g ″) modulus , as a function of frequency ω such that viscosity , η ′= g ″/ ω . rheologic data can also be presented using vectorial notation as tangent δ which is the ratio of viscosity to elasticity and g *, the vector sum of viscosity and elasticity ( mechanical impedance ). when stress in the linear range is used to evaluate the materials , the material properties are independent of stress . in order to conduct a frequency sweep from 0 . 1 to 1000 rad / s , viscoelasticity was evaluated using a creep test at 0 . 5 pa for 2 minutes . the strain response was fitted to a discrete relaxation spectrum , transformed to the retardation spectrum , and then to the storage and loss moduli , as a function of frequency , using methods developed by the pi . the linear viscoelasticity was evaluated at 1 and 100 rad / s and an oscillatory stress sweep and steady shear flow experiments were used to evaluate the behavior in the non - linear ranges . the oscillatory sweep data were analyzed by observing the stress where g ′ and g ″ crossed . this point indicates where the material shows more viscous behavior ( irreversible deformation and flow ) than recoil behavior . all rheologic measurements were made by technicians who were blinded to the treatment group origin . for mucin secretion , differences between control and guaifenesin treatment groups were assessed by comparing the means using student &# 39 ; s t - test for unpaired samples and p & lt ; 0 . 05 was considered significant . all the values in the figures represent means ± sem of 4 cultures unless otherwise stated . * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 for 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 & lt ; 0 . 05 was considered statistically significant . for rheology experiments , data were analyzed using the statview ™ 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 &# 39 ; s protected least significant difference test was done to determine significance with multiple comparisons . data are presented as group means ± 1 standard error unless otherwise indicated . by convention p & lt ; 0 . 05 is considered statistically significant . in fig2 , epiairway cultures were treated with the indicated concentrations of guaifenesin for 30 min . secreted muc 5 ac was compared with the pre - treatment values . during the 30 minute treatment period , there was no significant difference ( p & lt ; 0 . 05 ) between control and guaifenesin treatment groups . in fig3 a , 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 mucin produced during the given period . the total amounts of muc5ac were compared for statistical differences between control ( no guaifenesin ) and guaifenesin groups . treatment of nhbe cells with guaifenesin for 6 hours did not affect the amounts of mucins released ( fig3 b ). however , the total amounts of mucins produced during the 6 hour treatment period were significantly ( p & lt ; 0 . 01 ) suppressed by the presence of guaifenesin ( both 2 μg / ml and 20 μg / ml ). twenty - four hour treatment with either 2 μg / ml or 20 μg / ml of guaifenesin significantly suppressed mucin release ( fig4 b ) as well as mucin production ( fig4 a ). treatment with guaifenesin ( 2 μg / ml and 20 μg / ml ) for 48 hours significantly ( p & lt ; 0 . 01 ) suppressed the production of mucins ( fig5 a ). however , the amount of mucin released during this period did not seem to be significantly affected . as shown in fig6 , 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 effect of 2 μg / ml was statistically significant . however , at the 6 hr time point , 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 fig6 . 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 & lt ; 0 . 005 . there was no adverse effect on the viability of the cells as indicated by the wst assay . in fact , there appeared to be a trend to increased metabolic activity in the cells treated with guaifenesin , however this did not reach statistical significance . data from one of the three replicate experiments is shown below . as shown in fig7 a and 7 b , epiairway cultures were treated with the indicated concentrations of guaifenesin for 1 or 6 hr . metabolic activity was assessed using the wst assay , separately added to the apical or basal surfaces of the cultures . a total of 96 specimens from 5 sets of experiments were analyzed . the mucus from the first four experiments was received at ambient temperature and analysis of rheology of these samples showed extreme heterogeneity and the rheologic sweep curves obtained were consistent with degradation . the results shown in fig7 and 8 are therefore derived from the 22 specimens received from batch five . all specimens were non - newtonian , viscoelastic gels . the results demonstrate a significant guaifenesin dose - dependent decrease in viscosity , elasticity , and complex modulus ( g *) of specimens at 1 hour ( p & lt ; 0 . 05 ) and especially at 6 hours ( p & lt ; 0 . 01 ) when measured at 1 rad / s or roughly ciliary frequency but not significantly at 100 rad / s corresponding to cough . mucus rheology . fig8 a : g ″ viscosity , fig8 b : g ′ elasticity , fig8 c g * mechanical impedence ( vector sum of viscosity and elasticity ). data shown are the mean and standard error of data from the 1 and 6 hr time points combined . g *: vector sum of viscosity and elasticity , at 1 rad / s ( fig9 a ) and 100 rad / sec ( fig9 b ), segregated by time as well as dose . in all three treatment time periods ( 6 , 24 and 48 hours ), guaifenesin at both 2 μg / ml and 20 μg / ml suppressed the production of mucins by nhbe cells grown on an air / liquid interface . likewise , treatment with both 2 μg / ml and 20 μg / ml of guaifenesin for 24 hours showed a significant ( p & lt ; 0 . 05 ) decrease in mucin release . to address the effects of guaifenesin on mucociliary clearance , mucociliary transport rates were measured . the purpose of these experiments was to investigate potential alterations in mucociliary clearance induced by exposure of differentiated primary human tracheo - bronchial epithelial cells to guaifenesin . the original plan was to deposit aerosolized 1μm diameter fluorescent 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 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 mechanical impedance . as the vectoral sum of the storage and loss moduli , g * measurement indicates resistance to deformation . viscoelasticity is a property of non - newtonian fluids ( gels ). dynamic viscoelasticity measures the strain response of mucus to an applied stress . because mucus is subjected to both low stress ( ciliary beat ) and high stress ( cough ) conditions , the strain developed in response to a dynamic stress was measured . 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 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 . 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 . while several possible embodiments are disclosed above , embodiments of the present invention are not so limited . these exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the invention , but instead were chosen and described in order to explain the principles of the present invention so that others skilled in the art may practice the invention . indeed , various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description . such modifications are intended to fall within the scope of the appended claims . further , the terminology employed herein is used for the purpose of describing exemplary embodiments only and the terminology is not intended to be limiting since the scope of the various embodiments of the present invention will be limited only by the appended claims and equivalents thereof . the scope of the invention is therefore indicated by the following claims , rather than the foregoing description and above - discussed embodiments , and all changes that come within the meaning and range of equivalents thereof are intended to be embraced therein . disclosed are methods and compositions that can be used for , can be used in conjunction with , can be used in preparation for , or are products of the disclosed methods and compositions . these and other materials are disclosed herein , and it is understood that combinations , subsets , interactions , groups , etc . of these methods and compositions are disclosed . that is , while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed , each is specifically contemplated and described herein . for example , if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed , each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary . likewise , any subset or combination of these is also specifically contemplated and disclosed . all patents , applications , publications , test methods , literature , and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification .
a method of inhibiting mucus secretion in an individual that includes administering an effective amount of a composition which comprises guaifenesin .