Note: Descriptions are shown in the official language in which they were submitted.
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USE OF GUAIFENESIN FOR INHIBITING MUCIN SECRETION
BACKGROUND OFTHE INVENTION
1. Field, of the tavenlion
"I'lte present invention relates to the use of a pharmaceutical compound for
the inhibitio x
_t'
of nnuetrs secretion in an individual. In particular, the present invention
relates to the us-'e of
gu.i enesin frrthe inhibition of mucus secretion.
2 Description of Related Art
Ã]uaifunesin, whose chemical name is 3-(2-methoxyypihernoxy)-1,?-fpropa
recliol, is an
expectorant. An expectorant is a drug that helps bring up mucus and other
material form th
lungs, bronchi, and trachea. Ouaifenesin is thought to act by thinning the
mucus, loosening
phlegm, and bronchial secretions, and also by lularieaitirrl; the irritated
respiratory tract. By
thinning the mucus, gua.r.fkncsirr reduces the viscosity of the rnucal
sccrctions, and as a result
increases the efficiency of the cough reflex and of ciliary action in removing
acciarnulat l
sccretions from trachea and rornchi. The effect felt by an individual is that
a nonproductive
cough becomes more productive and less frequent.
In the prior art there are disclosed methods of inhihiting rnaucirn. However,
these methods
are directed to tNw treatment of chronic conditions, such as ast:lizrrar, WO
2004/043392 discloses a
method of modulating nauc n synthesis and the thcrape?utic alrl licaticn of
c.otylpouncly in
controlling mucin over--production associated. with diseases such as chronic
obstructive
pul-r onary diseases (COPD), including chronic bronchitis, and,
intla.riinnatorr lung diseases,
asthrrrar, cystic fibrosis and acute or chronic respiratory infectious
diseases using compounds of a
defined fo nrrr.rla having at least two aromatic rings.
BR F SUMMARY OF THE INVENTION
The applicant has developed a rriethud of nhibitin4g the seoretion of 11-
rtrcus in am
individual which comprises administoril-ig an eftcot.ive armount of a
cornrpositicrrn which comprises
guaifenesin.
According to a first aspect of the present invention there is provided a
method of
inhibiting mucus secretion iii an individual which comprises administering an
efiictive amount
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of ; ;..ottipositlon which comprises The composition can contain fiorn
apprt)'\ irately (iO(3mg-1200m4g of guaifenesirl.
The guaifene in can be administ,-- -A in many suitable forms such as a tablet,
powder,
capsule, liquid or licluigel. The guaifenesiri can be adminisÃerrcd orally.
The rnucin can be produced in the ripper respiratory tract of an individual.
The composition can contain one or more additional active agents selected from
the
group i~ e:1udi? g, but not limited to. an autrtussive such as dextro?netho
han hvdrobmnndcõ t
decongestant such as phcnylephrine hydrochloride, pseudo epliedrine
hydrochloride or ephedrine,
an antihistamine such as chlorphen r-anun:. rrraleate, broraphertirainine
tnale.ate, plhenindarni e
tartrate, pyrilar nine nmaleate, doxylamine succinate, phenyltoloxarnine
citrate, diphenhydranaine
hydrochloride, promethazine, t nd cl i s ti,:t tumerate, flexofenadine or a
combination thereof:
The composition can Ii e. an immediate release portion and a sustained release
portions;
such that the inhibition of mucus secretion is therapeutically achieved for a
period of
approximately 12 hours.
The daily dose ofguaifenesin can be 2400mg.
According to a second aspect of the present invention there is provided a
method of
treating an individual h_a In a disease or condition characterized by
increased niucin secretion
with are effective amount of acotnposition which comprises guarfvnesin as
described in the first
aspect of the present invention.
The disease or condition characterized by increased mucin secretion infectious
can be
selected from inlarrrmatory conditions of the ~:ir avs.
BRIEF DESCRIPTION OF THE FIGURES
Example embodiments of the present invention will now be described in more
detail with
reference to the accompanying figures.
Fig. I illustrates the treatment protocol..
Fig. 2 is a graph showing the effect of gguaifertesinr on MUC5AC rnucin
secretion: 3(} ruin
Figs. 3a and 3b are graphs showing the effect of guaife resin on N4U 5AC r-
rucin
secretion: 6 hours
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.Figs. 4a and 4b are graphs showing the effiect of guaif nesin on MLICSAC
mucin
secretion. 24 hours
Figs. 5a and Sb arc graphs showing the e:{ t of guaifenesin on MUC5AC roue r
secretion: 48 hours
Fig, 6 is a graph showing the effect of gu ii- enesin on inw-c..: diary
clearance.
rigs. 7a and 7b are graphs showing metabolic activity.
Figs. 8a, 8b and 8c are graphs showing mucus rheolot y.
Figs. 9a and 9b are gr=aphs slaw ing the vector sum of lscosity and elasticity
against time
and dose,
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
MATERIALS AND METH.O.DS
Cens:
g
E pi: irway cu:lturcs, (normal human bronchial. epith=elial) cells roxvin on
Millipore
Transwells, I or 4.2 cnmr, surface area. The cells were purchased from MatTek,
and were cultured.
at air-liquid interface for two (mucus synthesis and secretion) or three (mut:
.: diary transport and
it ucus rheeolog ) weeks prior to use.
Guaifenesin (-.GE) Treatment:
For nrucocilrary clearance, a stock gu ir7cnt .sill solution of 2 rriginiL in
culture medium
was prepared in the morning of each e;\pcriment and lkgit cold until dilution
into aariried
rrrcd.iun7 to the target concentrations of 0.2, 2,220 or 200 .g1'ixmL.. The
medium in the basolateral
compartment of each culture was replaced with the (TG[ containing medium, and
the cultures
were returned totlre 37'C:, 51%) CO incubator.iir as the times indicated,
"l'hc experiments were
repeated three tires on independent, cultures.
The concentrations used in the in vitro experiments range from 0.2 l.r~g!'niL
to 20 nlg/mL-
and thus bracket the clinical doses used in hurrrans.
Measurement of Mucin Secretion:
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GUAII E:NE.STN solutions were prepared by dissolving in IBS (phosphate
buffered
saline) immediately be.bre treatment of the cells. V1UC5AC n-arucirls were
quantified by ELTSA
ttsin till antibody Lalauision, Fremont. CA) Confluent I ca l NHB yells grown
on ,in
air/liquid :interface were washed from the apical surface with 2Ã30 pL PBS and
incubated with
frdsh co fete rd)w,th naediu n added to the basal chamber. Cultures were
incubated 24 hours to
collect the apical fluid (pretreatment sample or PT) by adding 1Ã00 AL PBS to
the apical surface
of the cultures. PBS was added to dilute the highly viscous, Hain mucus layer
on the surface.
Because of the sindal I size of th . insert, it was not feasible to collect a
sufficient arrioutat of n-tuc is
for both pharmacology and Theology without the addition of PBS. \hea
collecting 100 Al., of the
1.0 diluted. nmiticus samples (PT), cultures were divided into three groups (6
hr= 24 hr and 48 lir=j, 16
inserts per grotip, and treated with varying concentrations ofguaifenesin (0,
0.21, 2, 20 AgrrriL)
for each ti rre group, 4 inserts per each dose. Thus' a total of 48 inserts
rver= : used .f r this study
(4 inset t~: dose x 4 doses!tirrae point x 3 time points), The apical fluid
was collected at 30
minutes li llowi.tig drug treatment ff=orri all the cultures to see whether
guaifenesin affects the
"secretion" of nTucins. 'I he. apical mucus sample was collected in two steps
first by adding 100
IA- PBS to the apical surface (I,r wash) and then by adding 100 1.tL PBS
containing 5 111M
d.ithiothreitol (DTT) i2.`1 1 v ar Ir;. Samples trorn each wash were assayed
for MUC5AC content
and the sum of the two valud (the I'a and 2nd wash) was expressed as the
"released MUCSA ::-'
of the culture. At the three different time points (Le, .. 24, and 48 hr),
cultures were washed to
collect the apical fluid as described above ("released naucin",) and lysed
using a lysis buffer (PBS,
pl-l 7.2, 1 mM Ttiton. X-100, 2 rail EDTA, 1 mM P.SN!ff and 5 mM ITT" IT)
("cellular mucin")..
The amount of'muc.in in each sample (either secreted, released or cell Mate)
was divided by the
amount of rauc _t7_ in the T'T sample collected fi=orn the same well, in order
to obtain a "secretory
ind ex" to compensate forte tar ations Sai11d3n the cultures. The treatment
protocol is depicted
2- i.n Fig. 1
Measurement of 1VTur ocili Clearance:
Cultures (4 .2 d titwere exposed to basolateral guaifenesin for I or 6 hr. 'f
he cultures
were r'errloved horn the incubator and placed on the stage of digital imaging
microscopy- systerta.
Video data were collected for 10 seconds using a. 25x objeectiv .. The rate of
movement of
endogenous cell, debris was aaalyred on the video images using a transparenÃ
template overlay
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on the video aar`~es and a stopwatch to measure.. at least 5 particles on each
culture, thr"a total of
between 30 and 45 measurements per condition.
Collection of It ucus:
Following the analysis of c. eau'ane , mucus was hat- :stt:d horn the apical
surtacc of the
Cultures, \ ithoUt dilution.
Viability:
The apical surfaces of the culture: were then washed with PBS and the
metabolic activity,
an indicator of viability.., was measured using the Water Soluble Tetrazolium
(WST) assay
(Boehringer).
Rheologic Meaasurenieu.ts.
The Theological properties of apical mucus; secretions (20 }r.L) were measured
using an
kRI000 controlled stress rheom tc_ (TA bnstrcnaents, Nev Castle, DE) using a.
parallel plate
geometry. The ds nanaic. linear viscoelastic behavior was cletermined horn the
strain response to
an oscillating stress and reported as a storage or elastic modulus (M. and
loss or viscous (C ' )
modulus, as a function of frequency to such that , sco ity, f - (" ta, l
heologk data can also
be preseritcd using vectorial notation as tangent ,,s hick is the ratio of
viscosity to elasticity and
* the vector sum of viscosity and elasticity d mechanical impedance j. When
stress in the linear
range is used to evaluate the materials, the material properties are
independent of stress.
In carder to Conduct a %-equenev seep from 0>1 to 1000 radr:5, we evaluated
Vis: taelasÃicrty usi n, a creep test at 0.5 Pa for 2. minutes. The strain -
response was fitted to a
discrete relaxation spectrum, tran.slbrmed to the retardation spectrum, and
then to the storage and
loss inoduli, as a function of #requency, using methods developed by the P1 We
evaluated the
linear- s isc.oelasticit at I and 100 ralrs and we used an oscillatory
s>.:~cep and. sÃe idy .}rata
floss experiments to evaluate the behavior M the nears-llinrear ranges. `l'be
oscillatory sweep data
15 xs ore analyzed by obsc:rx in= the stress where Ci' and G" crossed. This
point .incli'cates where the
material show n-aort: viscous behavior (Irreversible detorn:iat on and
.floras,) than recoil behavior.
All rhentogic measurements were made by technicians who were blinded to the
treatment
group origin.
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Statistics:
Fox !ti:r Ftl secretion, differences between control and gua1 G:i sm treatment
group' were
assessed by coin paring the means using Students t-i:est for unpt i; o d
samples and p<0.05 was
considered significant. X11 the clue in the figures rel r-c:scnt rt,eart SEM
of 4 cultures unless
1 otherwise stated. 4 p<il.O5, * lp <t1.C)1
For rucociliary clearance, differences between control and guaife aesin
treatment groups
were assessed by comparing the means rising AN OVA, with a Bonferroni post-
test to assess
differences from the controls tested at the same time after treatment. A p
value of <- O;()5 was
considered statistically significant.
For theology e peument5 data were analyzed using the Stat Ttew"'I 15 statistic
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
guaifenesi:tt.. Fisher's
protected least significant diffr,;rence test was done to determine
significance with multiple
comparisons, Data are presented as group rrrcans + I standard error unless
other,', is,-- it dicatcd,
1.5 By convention p < 0.05 is considered statistically significant.
RESULTS
In Fig, 2, Ep Airway cultures were treated with the indicated concentrations
of
t uaitenes n for 30 mitt. Secreted MUC5A.C was compa =ed witth the pr'e-tr t
atrmme r.t values.
wir:tc the 30 minute treatment period., there was no significant dif iir ence
(p <0.05)
between conÃ7 ol and guarfene:sin treatment groups.
In Fig. ;:a, the ' bite bw;cs sqyr<,sent the amount. of mucin associated vitl
the ccli,
whereas the black boxes represent the amount of trrucin used during the gu,cn
period of
tratrnent.. Therefore, the addition. of the white box and the black box
represents the total amount
of rrrucin produced during the given period. The total amounts of Vl_UC5AC
were compared for
statistical difi_creepces between control (no guaifen.csin) and gu:arfenesin
groups,
Treatment of NIil3E cells with guaifenesin for 6 hours did not affect the
amount- of
mucins released (Fig. 3h). However, the total amounts of nit cnt5 produced
during the t hour
treatment period were significantly (1)<0,01.) suppressed by the presence of
guaifc ncs _n. (both 2
~zg>'ml and 20 ~tg/tr l ).
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Twenty= four hour treatrlrertt with either 2 p~g/nrit_> or 20 g`/ML of
guaitenesin significantly
sut pr essir:d .u?ucin release (Fig,. 4b) as well as i -iucin production (F g
4a).
Treatine 2t with griai-fenesin (2 g, na.f_. and 20 ;!gr'inL) for 48 hours
significantly ('I:)<0,01)
')L 4,4:t ., ed the production of rnitcins (Fig.. Sa.). However, the arnotrlrt
ofniucirm released during
this period did not seem to be I .:rif_ cantfy affected:
Effect. of Gualfenesin un 1taicodiliaÃ=rr Clearance:
shm4n in Fig. 6, gua.dcnesirr: appeared. to increase the mobility of the
cellular- deb-j,-is
on the surfhc,e of cultures treated for 1 hr, but there was little evidence of
a dose-response and in
tarot, only the efkct of 2 ,u,g/ml ,vas statistically significant,
.ll.owetver, 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 Fig. 9
l piAir-\\v ay cultures were treated with the indicated concentrations of
guatfer.resin for 1. or
o firs;. Mucocili:ary clearane was assessed. by the rate of movement of
endogenous debris on the
surfaces. *,., indicates significantly di1 rent fi'ortr: the control cultures
at the saran: time, p
0.005.
V'iabi_lity
There was no adverse s ff'ect on the viability of the cells as indicated ley
the \'VST assay.
In t et, there appeared to he a trend to increased metabolic activity in the
cells treated with
gua'tf:_.',icnifr, however this did not reach statistical significance Data
from one of the three
replicate c-~laeiiari:rits is shown below.
As shown in Figs. 7a aid 7is.. EpiA_ir,~=ay cultures were treated with the
indicated
concentrations o ~ griaifenesin for I or 6 hr. Metabolic activity was assessed
using the WST
assay, separately added to the apical or basal surfaces ofthe cultures.
ÃheologY,
2.5 A total of 96 specie. mens from 5 sets, of experiments were analyzed. The
mucus fi om the
first tenor experiments was received at ambient tailiperature and analysis of
Theology Of these
sae ipl showed extreme hcterogerie 'ty and the iheologic sweep :urvc obtained.
were consistent
with degradation. The results shown in -figures 7 and 8 are thee' =ibre
derived from the 22
specimens received from batch. five. All specimens were non-Newtonian,
viscoelastic gels
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The results demon mist, a significant guaifenesia dose-dependent decrease in
vise l-V
elasticity, and complex nioduh.rs (G) of specimens at l hour (p 0,05) and
c.spcc:i-dlly at 6 hours.
l[ 0.01) when measured at f rad/s or roughly ciliary frequency but not sic,
ificantly at 100
rad/s corresponding to cough.
Mucus Rheology. Fig. 8a: G" viscosity, Fig. 8h: G' elasticity, Fig. Sc G* i
echan eal
impcdernce (sector sum of'viscosity and elasticity). Data shown are, the mean
and. standard error
oftlata from the 1 and 6 hr time points combined.
G*: vector sum of viscosity and elasticity, at I rad/s (Fig. 9a) and 100
rad/sec (Fig. 9b),
segregated by tiraac as well as dose.
In all three treatment time periods (6, 24 and 48 hours), guai.f rc in at both
2 FrgtrnL and
itg/niL suppressed the production cif inuz.ins by NI-HBE cells gi os-gin. in
to air/liquid interlace.
L kewi,e, treatment with both 2 fag/ni]L and 20 .ig/mL of guaitenesin for 24
hours showed a
Sifzcant (p<O.05) decrease in macro release.
To address the effects of guaife aesin on nruc ;..,limy clearance. m
a.ucociliar-y transport
15 rates wtrc navasured. The purpose:of'these experiments vas to investigate
potcaiaal alterations
in rnucoc diary clearance induced by exposure of differ ::mated prirnaiy human
tracheo-bronchial.
epitl-r. hal cells to Guaifc:ncsira. The original plan was to deposit
acerosoli :ed I Am diameter
tlt ai .. t.t?.Ã micL,ospher'es on the ,un'face of the cultures using a nebuli
er. Ho's cver, foir reasons
that ,ir.c nra~lc a although the inierospheres coruld be identified on the
cultures; there: was
?tl raacn mnent in oah a r<%ery fcw of the cultures, despite clear inovoincnt
of the endogenous cellular
debris. A switch to collecting video of the endogenous debris was nade. Z~I
\`:'cosity (loss modulus) is the loss of energy from a r-heolog.ic probe or
applied stress
and thus the resistance to fl_os . Elasticity (sÃor-a.ge modulus,) is the
recoil energy transmitted back-
to the probe. The cor:nplex modulus, G", is also kenos\n as the i.nechaui eal.
rnafl :.l t.r_ As the
,25 veetoral sum of the storage and loss naodrrli, (f measurejnent ind.ica yes
re i:stance to dcf a:rnratic n.
Viscoelasti its is a propren), of yarn-Nevdo i,an fluids (gels), Dynamic
viscoelrsticity measures
the strain response of mucus to an applied stress: Because mucus is subjected
to both loss stress
(cilh-to beat) and high stress (cough) comiuions we measure the strain
developed in response to
a dynamic stress.
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These results are consistent with. the secretions taken from the
differentiated cells being
rnzrrxetrs gels, Although degradation of specimens from. experiments 14
produced inconsistent
iv u ita sugggesting degradation (ra v results all available on request),
those from the tit al set of
cx;?c,riments were well preserved and the results were robust, The decrease in
complex modulus
l ;.:r: llcliri that of the ,viscosity= (loss modulus) ivo sld be consistent
with the ir1c.ro-aced ciliary
transport. The rhoologic characteristics of these specimens suggested a goblet
cell nrigm with
viscosity approximately equal to elasticity, rather than a submucosal gland
secretion where. Elie
elasticity is generally greater than viscosity. These results are consistent
with the reported
structure of the EpIA m ay culturc.s. It will be niurmatine to compare these
results with those
firom human tissue explants exposed to guaifenesi: i.
Guaifrn_esin suppressed nauein 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 mucociliar ,y
Iran, sport an d decreased r iscoclasticit-
y of the mucus..
Further modifications or improvements cane be made without departing firms the
scope
of the invention- herein described.
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