Molecular basis for mucolytic therapydownloads.hindawi.com/journals/crj/1995/578696.pdfMolecular basis for mucolytic therapy BONNIE DASGUPTA MSc, MALCOLM KING PhD FCCP Pulmonary Research

REVIEW

Molecular basis for mucolytic therapy

BONNIE DASGUPTA MSc, MALCOLM KING PhD FCCP

Pulmonary Research Group, University of Alberta, Edmonton, Alberta

B DASGUPTA, M KING. Molecular basis for mucolytic therapy. Can Respir J 1995;2(4):223-230.

Ai rway m ucus is a complex. viscoelastic ge l that has a three-d imens ional structure . It is composed of water, mucous glycoprote ins, low molecular weight ions , proteins and lipids . The three-dimens ional structure of the mucous gel depends on a number of fonns of bonding, such as ionic bonds and d isu lph ide bridges. Airway obstruction in cys tic fib rosis (CF) lung di sease is accompan ied by the accumu lation of th ick and viscous sec retions result ing from chron ic infect ion and inflammation, promoting recurrent exacerbations. The normal, free-flow ing airway mucus becomes thick and purulent in pat ients suffering from CF lung disease . Therefore, curre nt approaches to the treatment of CF incl ude strategics for changi ng the physical properties of pulmonary secretions wi th the goa l of improving airway c learance. Some of the same strategies may be applicab le in the larger group of patients with chronic obstructive airway diseases, includi ng bronchiectasis and chronic obstructive pulmonary di sease . T hi s paper rev iews various approaches to m ucolysis based on the mo lecular natu re of cross link ing and bonding in rnuc in gel s. A brie f review of the structure and biochemistry of airway m uc us is fo llowed by a discuss ion of the various physical and b iochemical approaches to muco lys is. Seven representative mucotropic modalities are presented: N-acetylcysteine: urea: hy pertonic sal ine: recom binant hu man DNase; gelsolin: oscillation: and surfactants. Each of these rnucotropic modalities acts on a different component within the mucous ge l. Finally, the poss ibil ities of rnucolytic synergism among these various agents are cons ide red.

Key Words: Acetylcys1ci11e, Airway nbsrruction. Cystic fihrosi.,. Gelso/i11. Mucolysis. Recomhina111 /11111wn DNase . Urrn

Bases moleculaires en faveur d'une therapie mucolytique

RESUME : Le mucus bronchiquL· est un gel visco-clastiquc complcxc possedant une slructur(' tridimensionncllc. II sc compose d 'eau, de glycoproteines muqucuses, d ' ions de faiblc poids molecu laire, de proteincs ct de lipides. La structure tridimcnsionnd le du ge l muqucux repose sur plusieurs fom1cs de liaisons telles que !es liaisons ioniqucs ct !es pants <.fou lfures. L'obstruction bronchique dans le cas de la fib rose kystique du pancrtas (FKP) s'accompagne d'une accumulation de sec retions epaisses et visqueuses resultant de I 'infection chronique ct de I 'inflammation ct qui fav orise des exacerbations rccurrentcs. Le mucus bronch iquc, qui normalement circule libremcnt. dcvicnt alors cpais ct purulent chez les patients souffrant de FKP. Done. lcs approchcs courantes cnvcrs le tra itcrnent de la FKP comprenncnt des strategics pour changer les proprietes physiques des secretions pulmonai res dnns le but d'ameliorer la clairance bronchique. Ce1taines :,tratcgics peuvent ctn: applicables au groupc de patients plus large souffrant de maladies obstructives chroniqucs des voies acriennes, notamment de bronchectasies et de maladies pulmona ires obstructives chrnniqucs. Cct article examine Jes dil'fcrentes approches envcrs la mucolyse basees sur la nawre molcculai re de la reticulation et des liaisons des gels des mucines. Une revue breve de la structu re ct dl:' la biochimie du mucus hronchiquc est suivic d' une discuss ion sur !es diffcrcntcs approchcs biochimiqucs ct physiques envcrs la mucolyse. Sept modali tes mucotropcs representatives sont prcscntces : la N-acetyl-cysteine, l'urcc. la solution saline hypcrtoniquc, la Dnase humainc recombinante. le gc lsolin, l 'osci llation ct lcs surfactants. Chacune de ces modalites mucotropes agissent sur un composant different du gel muqueux . Enfin , les possib il ites d 'une synergic mucolytiquc cntrc ccs diffcrcnts agents sont envisagccs.

Correspo11dc11cc ,111d rcpriws: Dr Malcolm King. Pulmonary Rescal'<'h Cro1111. 173 f lcrirage Medical Research Ce111re. U11il'asit_1· of Alberw. £dmo111011, A/11erta T6C 2S2 . Teleplume ./03-492-6703. Fw: 403 -492 -4878. e-mail [email protected]

Can Respir J Vol 2 No 4 Winter 1995 223

DASGUPTA AND KING

A IRWAY MUCUS IS THE FIRST LI NE OF PROTECrION IN THE

lungs. It traps inhaled dust particles and bacteria and

removes them from the airway by c iliary clearance or by coughing. Airway mucus is a complex viscoelastic gel, composed mainly of water (lJO to 95%) and mucins (or mucous

glycoprolL'ins) ( I to 2% ). Under normal conditions, less than

I% of airwa y rnucus i.\ composed of proteins, lipids and low molec ular WL' ight ions. In patients suffering from cystic fi brosis (CF). 3 to 4'k of airway mucus is composed of the latter (I).

The viscoelastic properties of airway mucus can be attributed predominantl y to its muc·in content (2). Muc ins arL'

g lycoproteins composed of a polype ptide core, rich in serine. threonine and proline, with numero us oligosaccharide s ide ch,tins connected to the core thro ugh 0-glycos idic li nkages

via N-acdylgalactosamine (3). Mucins are polydi perse, with a large si ze (3 to 7 MDa) and an unusual molecular structure that distingu ishes them l"rom other components of mucus , as well as from mos t other glycoproteins. Ai rway

mucins are synthesized and secreted by a nu mber of spec ial

ized ce lls (ie, mucous and serous cells of the submucosal glands. and goblet and C lara cells of the surface epi thel ium).

SOL AND GEL PHASES OF MUCUS The airways of the respiratory tract arc li ned with a double

layer o f lluicl, a superficial gel layer and an underlyi ng sol layer. The ge l layer is produced from the mucous glands and

the goblet ce lls in the a irways. The notion of the mucous bilaye r (gel and sol phases)

makes it s imple to understand muL"us transport , especially the velocity of transport that is re lated to the frequency of beat and leng th of cilia. In 1961. Hilding (4) demonstrated till'

optimal rate llf nrncociliary c ll'arance in animals to be ahllut 15 mm/min. It was explained that thi s vdocity is clue to the gel layer. which interacts with the ciliary tips du ring the

effective phase of the beat and perhaps during the peak velocity or that phase. During the recovery phase. the cilia interact only w ith the sol layL'f. and each cilium is; sl ightly

ahead in thl' phase of the cycle l"rom the one immediately bchiml it. In o ther words, Hild ing demonstrated the reverse direction taken by the waves of the ,rcove ry stroke in the gel

phase . However. Sande rson and S le igh (5) suggested that tht

sol laye r shows little net movement.

CROSSLINKS IN THE MUCOUS GEL LAYER The hi gh ly viscoelastic airway muc us secretions exhibit

characteri stics of a ge l. A ge l is desc ribed as a solution in which the macrnmokculcs arc all li n1'ed together hy at least one crosslink per chain to produce ve ry large macroscopic

aggregates (6). T he mo lec ul ar nature of cross linking of airway mucus depends on a number of different types o f bonds. T herefore. to reduce the viscosity and elasticity of the puru

lent muc us. the crosslinking and bonding within thl' mucous gel must be broken.

A normal muL·ous gel is composed of numerous bllmls

(Figurl' I). incl uding covalent bonds (such as disulphide bonds). hydrogL'n bonds (which hind hydroxyl groups), ionic

224

Figure I) Diaw am illustrating the 1,v11cs of bonding i11 a 11111r ·o11s gel ( each subject tu n111colysis ). a/Ill site ,faction o( l'<ll'ious 11111co/ytic tffatmcnts. I . Col'l.1le11t howls (f)ri111arily diwl11hidc honds. hoth intra- t1!1(/ inter1110 /cc11/t1r) : 2. lonit hond.,· (11111cin 11wcro111,,/cc11/cs ll'ith hot!t positi1 ·e and negatil'e fixed clwrgcs ca11ah!e of interacting): 3. Hydrogen honds (c/11(' to t!t c ol(~ o.,ucclwride sidcc!tuins) : .J. l'l/11 der Waals ' jr1rces (with fJOSsih/e i1111)(>r/a11cc of i111cnligi1a tion hetwec11 uligosacclwride 1110/crnles): 5 . /11tcn11i11gling (phl'.lica / c11ta11glcme11ts): 6. Extraccllulur DNA ullll F-at tin (parallel 11etworkfomwtio11 in infectio11). OTT Dith it>1hrcito/; NAC N-acc1y/cystci11e; rhDNasc Rcco111hina11 t h11111un DNasc

bonds (which bind su lphated sugar units to amino groups, inter a li a) , van dcr Waa ls ' forces (weak, attracti ve forces that contribute to inte rdigita tion between neighbouring mole

cules) and physica l en tang lements (mcchanil'al entrapment between po lypeptide chains) (7). In short, all of these bonds

con tribute to c rosslinkages within the mucous gel and a ll are subject to m ucolysis. In norma l, noninfected mucus. the

majority of the bonding is due to muci ns (mucous g lycoprotcins). Addit ional facto rs rela ted to crossl inking within the mucous gel mod ify the phys ical properties of the ge l: hi gh wate r content lowers vi scosi ty (8) wh ile the add ition of DNA

(9 ) o r se rum a lbumin ( I 0) raises it. In mucus producL·d hy chronic irritation , such as smoking or sulphur dioxide exposure . the mucin macromolec ules within thL~ mucous ge l de

velop an excess of fixed negative charges , as indicated by the histochemical shift to acidic rnucin ( 11 ). This excess o f

negative charges develops a net repubion, making the mu cous gel highly acidic .

In chronic a irway infect ion . such as in CF airways infected by Psc11dornonas species, in addition to the mucin macromolecules , the mucous ge l contains DNA and F-actin

(both of which arc rel eased from dying cells and bacteria) ,

and bacterial alg inate . T hese affix additional cross linkages to

Can Respir J Vol 2 No 4 Winter 1995


TABLE 1 Approaches to mucolysis

Sources of bonding Disulphide Hydrogen Suface Physical

Mucotropic agents DNA F-actin bonds Ionic bonds bonds tension entanglement rhDNase X ? X Gelsolin ? X X

N-acetylcysteine X X

Hypertonic saline X X

Urea X X X X

Exosurf/bLES X ?

Oscillation ? ? ? X

X Expected interaction: ? Possible interaction: bLES Bovine surfactant; rhDNase Recombinant human DNase. Exosurt (Burroughs Wei/come)

the gel network. The DNA from the host leukocytes has a very high molecular weight and is re latively in11exiblc , thcrehy forming a rig id network. In CF, DN A concentrat ions may CVL'n be sufficient to make the parallel DNA network stronger than the mucin matrix.

MUCUS RHEOLOGY Mucus can he considered as a viscoelastic 11uid, since it

exhibits both liquid-like (viscous) and solid-like (elastic) properties_ Viscosity is the resistance to flow and represents the capacity of a material to absorb energy as ii moves. Elasticity is the capacity of a material to store energy used to move or deform it. The relative proportions o f e lasticity and viscosity are as important in describing how a material such as mucus behaves when it is subjected to ex ternal forces as are the absolute values of either parameter ( 12).

The effects of different approaches to mucolysis can be tested by measuring the d ifferent phys ica l prope11ies of airway mucus. \iiscusity and elasticity, the basic properties describing the liquid-like and solid-like properties of the mucous ge l, can be measured by means of a magnetic micrnrheomcter. Spinnahility, the capacity of mucus to fonn threads under distraction, corre lates with mucus viscoelasticity and clearability. S pinnability is measured by a filanccmctcr. Swfacc /J/'O/ll'rfics arc important in determining how mucus adheres to epithelial lining and how it inleracts wilh airflow. Mucociliary and cough cfeamhilitv, the effect on clearance of airway mucus by the two major relevant mechanisms. are measured to determine ciliary action and airflow.

GENERAL APPROACHES TO MUCOL YSIS AND MUCOKINESIS

To change the physical properties of the viscous and rigid mucous gel, the direct strategy is mucolysis, but two indirect strateg ics may also be involved, namely secretory inhibition and mucokinesi s. Mucolysis refers to the disruption or the mucous gel, generally by altering the degree of crosslinking or the interactions between the macromolecules that form the gel. Normally it is desirable to reduce the crosslinking and viscoelasticity in the mucous gel to improve clearance. Occasionally, the mucus is too thin for effective transport ( I 3, 14 ): hence, increasing the crosslinking. of the mucus by a muco-


spissic agent could he apprnprialc. Mucolylics and mucospissics are known collect ively as mucotropic agents.

The second strategy is mucokincsis, where 1hc ohjcctivc is to move 1he mucus physically from smaller to larger airways, from where it can be eliminated hy cough clearance. Mucok inesis does not necessarily imply mucolysis, especially if the mucokincsis occurs primarily through stimulation of the cilia, or through airflow or gravily mechanisms (the lwo primary mechanisms used to move mucus). However, agents that alter ciliary beating often also alter mucus rheology. For instance, physiotherapy methods, such as high frequency chest compression (HFCC), involve airflow regimens that have the potential to al ter mucus rheology and to stimulate clearance ( 15 ).

MUCOTROPIC AGENTS The aforementioned are general approaches 10 mucolysis.

To attain the optimal e ffects of mucolysis, it is essential 10 look. al the complex mucous gel. Breakage or reduction of the honds within the mucous gel can he achieved through disrup-1ion of the ge l network, the process k.nown as mucolysis. Mucoly.,is can be. achieved either through physical inlcrvcntion, such as high-frequency oscillation ( 16), or by biochemical or pharmacological agents (mucotropic agents), such as recombinant human DNase (rhDNasc) or N-acctylcysteinc (NAC). Thus, by breaking the bonds, rnucoly.,is reduces the vi.-coclasticity of the mucous gel. If this process is carried out to the proper extent, it will enable the patient lo improve muc us clearance (12).

The array of mucotropic agents described below arc primary agents that ei ther directly affect the production of mucus or change the characteristics of the mucus. There are many different lypes of primary mucotropic agents. Seven representative examples are examined here: NAC, urt>a, hypcrtonic saline, rhDNase, gelsolin, mechanical oscillalion and surfactants. Each of these mucotropic modalities arc meant lo disrupt or mod ify a separate bond within the mucous gel (Tahlc l ).

N-acetylcysteine NAC is a derivative of cysteine and is a thiol reducing

agent. NAC has been widely used as a mucolytic agent in many countries. although not in Canada or the United States.

225

DASGUPTA AND K ING

It has been reported to reduce the vi scosity of purulent sputum in both CF and chronic bronchit is patients ( 17). thus enhancing the removal of pulmonary secretions by ciliary action or cough.

The chemical structure of disu lphide-containing proteins and peptides connected by disulphide linkages is altered by thiol compounds. Phys ical changes in the bronchia l glycoprote in are the result of thiol reduction; they are associated with reduction in molecular size, sedimentation coefficient and viscosity ( 18). NAC reduces the disu lphide bond (S-S) to a sul fh ydryl bond (-SH), which no longer participates in cross li nki ng, thus reduci ng the elasticity and viscosity of mucus ( 19). Previous studies have shown a correlation between the reduction in mucous vi scosity and the reduct ion of disulphide bonds (20). These mucolytic properties of NAC were tested and confi m1ed (by using a magnetic microrheometer) in in vitro experiments (21 ), where treatment with NAC resulted a reduction in the e lastic shear modulus of canine tracheal mucus .

In vi tro studies of the effect of NAC on the viscosity of sputum report a dose-re lated activity: the greater the concentrat ion of NAC administered the greater the decrease in vi scosity (20.22). In terms of purulent and nonpurulent sputum, NAC demonstrated similar effects on both types of sputum. Other in vitro studies reported inc reasing mucolytic activity of NAC in solutions over the pH range of 5.5 to 8.0 (23).

Nevertheless, NAC has di sadvantages, particu larly in topica l or aerosol administration. It is difficult for the drug to reach more peripheral ai rways or poorly ventilated areas in therapeutic concentrations; there is also the risk of bronchospasm in hyperreacti ve patients. On the other hand , NAC can be too potent in its ac tion - it can overliquify the mucus in central ai rways yet underliqu ify the mucus in the periphery, both instances leading to suboptimal clearance ( 12) .

Urea Urea has long been recognized as a dissociat ing solvent

that can break ionic and hydrogen bonds (24). In mucin ge ls, urea disrupts the hydrogen bonds that provide li nks between the oligosaccharide side chains of the neighbouring mucus !llolecules. The disru ption of these bonds consequently reduces the phys ical entanglements between macromolecules, thereby reducing the viscosity of the mucus. Urea may also reduce the interaction between DNA molecules, thus fu rther decreasing the degree of physical entanglement.

In 1966, Waldron-Edward and Skoryna (25 ) carried out an in vit ro experiment using urea as a mucotropic agent. They reported that urea was effec tive only at 3 M concentration. Concentra tions less than 3 M had no significant mucolytic effect, while concentrations higher than 3 M overliqu ified purulent sputum (26,27). For effective mucolysis, 0.2 g of urea/mL of sputum was used, although th is is an immense amount of urea for nebulization. In 1974, Marriott and Richards (28) confim1ed Wald ron-Edward and Skoryna's find ings, explain ing that when administered in vitro, the mucolytic action of urea increases as its concentration in-

226

creases from 5 to 8 mol/L. Because signifi cant effects result only at such high concentrations, urea would not be considered appropriate for human use. However, since toxic effects are generally concentration-dependent. urea toxicity might be minim ized by combining it with another mucotropic agent.

Hypertonic saline As suggested by Figure I , hypertonic saline breaks up the

ionic bonds wi thi n the mucin gel, thereby reducing the effective degree of cross linking and lowering the viscos ity and elasticity (29). Hypertonic sali ne is also believed to promote mucus clearance by extracting flu id from the respiratory tract

pi theli um, thereby increasing the volume and water content of mucus (30). Upon administration of hypcrton ic sal ine by means of a nebulizer, water from the tissues is shi fted across the epithelia l membrane to dilute the saline. The diluted fluid is then eliminated from the respi ratory tree by the mucocil iary esca lator or by cough ing. Administering excess ive quantities of hyperton ic saline to the patient must be avoided, because it could result in hypematremia. particularly in patients with renal insufficiency or impaired sa lt secretion (29).

In infected mucus, hypertonic sa li ne separates the DNA molecules from the mucoprote in, making the mucoprotein suscept ible to proteolytic enzyme digestion (31 ). Liebennan (32) reported that DNA cleavage by hypertonic saline increased as the ion ic strength of the solution inc reased beyond 0. 15 M. In 1978, Pavia et al (33) investigated the effec t of hypertonic sa line on chronic bronchitis pat ients. They found that. compared with normal saline, hypertonic saline ( 1.2 1 M) doubled the rate of mucociliary clearance with an increase in the weight of sputum expectorated. Wanner (34.35) noted that th is stimulation of mucociliary transport by saline solutions occurs in both normal individuals and chron ic bronchitis patients. Recent ly, hypcrtonic sal ine has been reported to enhance the removal of lung secretions in patients with CF (36,37).

Recombinant human DNase I High concentrat ions of DNA (up to 15 mg/mL) have been

shown to be the leading cause of the tenacious and vi scous nature of the sputum (9,38,39). By cleav ing the DNA molecules, rhDNase reduces sputum viscosity (38) transfom1ing the tenacious sputum from a nonflowing gel to a fl owing liquid within minutes. This change in the physical property of the sputum (non flowing gel to a flowi ng liquid) he lps patients to remove sputum from their lungs, thereby improving lung function. This reduction in viscosity of sputum is associated with a decrease in the size of the DNA molecules with in the sputum (38). In theory, rhDNase reduces only the excess viscoelastici ty. leaving the glycoprotein gel intact (quasi normal). Hence, it is believed not to overliquify mucus, although this could happen in some c ircumstances (40) .

Recent mult icentre stud ies of exacerbations in CF patient s reported that short and long term administration of rhDNase correlated with an improvement in pulmona ry function (41.42). The use of rhDNase (Pulmozyme, Genentech) for


the treatment of C F patients has been approved by Health and Welfare Canada and by the Food and Drug Administration in the United States. The application of rhDNase in non-CF patients is under investigation (43,44).

Gelsolin F-actin is released from dis integrating inflammatory cells.

It constitutes 10% of the total leukocyte protein (45) and devl.'lops long, protease-resistant, eminently viscoelastic filaments (46). Plasma gclsolin, a natural component of extracellular fluid s, reduces the length of F-actin filaments (47). However, with the overwhelming degree of infection in CF airway disease. there may he insufficient levels of natural gelsolin in the inflamed airways of the CF patient, analogous to the small amounts of DNase also present in their a irways (47i. Thus, this provides the rationale for administering exogenous gelsoliu or DNase in this disease.

Accumulation of the long F-actin filaments can cause mucus to he quite purulent and viscous, similar to the excess concentration of DN A networks. Vasconcellos et al (47) reported a 58% decrease in the elastic shear modulus (the rate of refom1ation after distortion of the mucous gel) of CF sputum after treatment with gelsolin. Another study reported a signi ficant decrease in the viscoelastici ty index (G*, the complex shear modulus) and cough clearnbility index after treatment with gelsolin (48). Recently , in an in vitro study, we demonstrated that combined treatment with rh DNase and ge lsolin was more effective in reducing the viscoelasticity of CF sputum than individual treatments with either agent, indicating mucolytic synergism between the two (49). A like ly cxplan;,ition for this action of gelsolin is that after disassocia tion , gebolin obstructs the monomers from adding back onto filaments. These results substantiate reports of mucolytic action of gelsolin and confi rm its efficacy in reducing the length of F-actin filaments. This shortening of F-actin filaments not only reduces the viscoelasticity and increases the cough clearability of mucus, but also reduces the rate of formation of the distorted mucous gel. Further work is needed in this area because clinical studies with gelsnlin have not yet been carried out.

Oscillation As illustrated in Table I, high frequency oscillations break

up the physical entanglements by reducing the crosslinkages within the mucous gel. This disruption and d isentanglement of tht' mucou., macromolecu les reduces the cross linkages that hind the mucous glycoprotei ns, and thereby loosens the mucus to facilitate rnucociliary c learance. However, oscillations may also break up the DNA molecules because these molecules have an inflexible helical structure, which make them more vulnerable to be broken hy high frequency oscillation. Oscillation is most probably unable to break hydrogen and ionic bonds because both types of bonds are reve rsible and, even if broken, they will reform.

Earlier studies reported that the rate of mucoeiliary clear::mce depends on the degree of cross linking ( 12); however. a large reduction in crosslinkages makes the muc us too fluid.



thereby obstruct ing mucociliary clearance. High frequency oscil lation transmits airllows through the lung ti ssue, vibrating the chest wall, and physically d isrupting and dise ntangling the mucous macromolecules. High frequency oscilla tions could also enhance ciliary beating ( 15).

Our own studies indicate that oscillating CF sput um (16)

results in a decrease in spinnability. This decrea e in spinnability may be explained by the reduction of polymer size, induced through lysis of macromolecular hackbonL' linkages (Figure I). The dec rease in spinnability of CF sputum correlated in pre viuus studies (50) with increased transportability of mucus hy ciliary and/or cough mec hanisms . These changes in the physical properties of the sputum may therefore be considered potentially beneficial in the treatment of CF patients .

T he clinical value of HFCC is due to the high frequency airflow it generates through the lungs. This airflow not only reduces the viscosi ty of sputum but it also provides ·ignificant oscillatory airflow at the mouth through mucus-airflow interaction, consequently provoking the patient to cough a11d expectorate a s ignificant amount of sputum . Hence, the high frequency airllows are partially analogous to cough in provoking the movement of mucus mouth ward, which then leads to e xpectoration of sputum. King ct al ( 15) and Warwick and Hansen (51) propose that HFCC is an excdknt altL·rnatiVl' to standard chest physiotherapy.

Surfactants The principal biological function uf pulmonary surfact-·

ants is to keep the alveoli open during breathing. In the airways, surfac tants can provide lubrication for cough clearance. They also improve interaction with cilia, thereby facilitat ing ciliary beating. Hence. surl'actants have the potential to improve both mucocili ary and cough clearance, and to reduce surface tension and phys ical entanglements within the muco us gel (52).

Various studies have shown that the viscoelastic properties of airway muc us are altered by airway I ipids (53-55 ). Sputum from CF pat ients demonstrates a corrl'lation between changes in rheological properties and changes in phospholipid content (53). De Sanctis e t a l (52) re ported no change i11 th1: vi scoelasticity of dog tracheal mucus after administration of porci ne su rfactant (Curosurf, Chies i, Pamu, Italy), bur a four- to sixfold increase was observed in mucus clearance, partially Jue to stimulation of ciliary beat frequency, and poss ibly due to changes in the surface properties of secretions and their interaction with the cilia. However, Festa e t al (55) showed that direct application of ho vine surfactant (bLES ) tn CF sputum reduced mucus rigidity and improwd mucus clearability on the isolated frog palate.

Modification of the mucous bilayer (gel and sol) hy exogenously applied surfactants could improve rnucocili ary clearance by enhancing the sliding of the gel phase over the sol phase (56). lt has been suggested that surl'actants facilitate the formation of interfacial bilayers between the mucus and the periciliary fluid (57). Enhanced bilayer formation between the two airway fluid phases would serve to augment the transfer of kinetic energy between the cilia and the mu-

227

DASGUPTA AND KING

16

14

12

10

8

6

4

2

O min 15 min

Control

Osc.

DNase

DNase + Osc.

30 min

Figure 2) Combined c/fcc/s of oscilfa1ion (Osc) and rccom/Ji110111 lu1111a11 DNase on spilllwhi/i1y (mean± SD) of cys1icji/Jrosi.1 s1111111111.

COIII/Jllf'Ccl ll'i{/i cjfec/s of i11dil'id11al {f'C<l{f//Cl/{S c111d Ci///{/'()/

cous layer hy improving the no-slip condition necessary for effic ient momentum transfer between the layers (58).

COMBINED TREATMENTS WITH MUCOTROPIC AGENTS

As shown above. treatments with a variety of individual

mucotropic agents have successfully improved the properties

of mucus. leading to increased mucous clearance. However, a combination of mucotropic approaches may produce even greater e ffects upon mucolysis, as illustrated by the poss ibi li ties in Tab le I. For example, both oscillation and the enzyme

rhDNase can break apart the fragil e DNA molec ules found in CF sputum. Although the independent results of studies of high frequency oscillation (51) and rh DNase (42) are very

encouraging, these two the rapies have not been researched to detennine the ir additive effect on mucus clearance in CF patients. Results from in vitro studies of the ind iv idual and

combined effects of oscillation, saline and rhDNase on the spinnability of CF sputum (16,59) (Figure 2) indicated that additive treatment with rhDNase and osc illation decreases

spinnabili ty of CF sputum significantly more than with either trea tment alone (eg, saline alone or oscillation alone) (P<0.01 ).

As another example. hoth rhDNase and gelsolin are in

volved in breaking up the excess crosslinks resulting from

infection byprod ucts released into the mucus. Gelsolin. which binds to monomeric ac tin, is a protein capable of shortening the length of actin filamen ts (45), analogous to the action by rhDNase on DNA. In an in vitro study, Vasconce llos et al (47) comhincd these two mucotropic agents and

reported that DNase may also reduce the viscos ity of CF sputum through its ac tion as an actin-binding prote in (45 ). Actin a lso inhibits the DNA-hydrolyzing activity of rh DNase

228

(60); therefore. by binding actin. the action ofrhDNase in CF

sputum may be enhanced. By reducing the length of actin

filaments, ge lsolin has been shown 10 reduce vi scoe lasticity of CF sputum in vi tro (47). Additionally, a recent in vitro study (49) showed that combined treatment with gelsolin and

rh DNase was more effective in reducing the viscoelasticity of CF sputum than individual treatments with e ither agent al one, indicating synergy between gelsolin and rhDNase.

As shown above, these in vitro results (16,47,49) demon

st rate an incremental or optimal effect from the additive effects of m ucotropic agents . This may suggest potential superiority of combinations o f chemical (eg, rh DNase) and

physical (eg, oscillation) trea tments on C F patients. Further studies arc requi red to confi rm these findings, to asse s mucus dearability directly and to extend the observations, not

only to a larger number of CF patients, but also to patients with other ty pes of pu lmonary d iseases.

SUMMARY Airway mucus has a number of functions within the hu

man body. In proper balance. airway mucus is generally beneficial in its effects to humans. The advantage of airway mucus is that it serves as a protective layer, trapping bacteria.

viruses and foreign bod ies in the conducting airways of the respiratory tract. T he primary drawback is that the accumulation of ai rway mucus blocks the small airways, causing airway obstruc tion. People suffe ring from diseases charac

terized by mucus hypersecretion (eg, chronic bronchitis , CF) have e xcess mucus obstructing the ir ai rways and will benefit from measu res designed to enhance its rate of elimination.

A variety of mucotropic modalit ies arc involved in reducing the viscoelasticity and improving the clcarability of the m ucous gel. Previous in vitro and in vivo studies, using

indi vidua l mucotropic agents (42,51 ), have been successful. and a few studies , inc luding ours ( 16,49), indicate that combined mucolytic treatments have greater potential. Neverthe

less, addit ional studies are required to confirm these findings and to define further the additive effects of mucolysi s with

combined treatments of physical and biochemical muco Lropic agents.

ACKNOWLEDGEMENTS: This paper was adapted in pa1t from the MSc thesis of B Dasgupta. The authors thank Drs A Dean Befus. Marek Duszyk, Ne il E Brown and Bruce K Rubin for their critical review of the original manuscript. B Dasgupta was supported by studentship awards from the Alberta Lung Association and the Canadian Cystic Fibrosis Foundation.

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Molecular basis for mucolytic therapydownloads.hindawi.com/journals/crj/1995/578696.pdfMolecular basis for mucolytic therapy BONNIE DASGUPTA MSc, MALCOLM KING PhD FCCP Pulmonary Research