On the Mechanism of the Plaque Inhibition by Chlorhexidine G. ROLLA and B. MELSEN Royal Dental College, 8000 Aarhus C, Denmark
The marked plaque-inhibiting activity shown by chlorhexidine1 is a unique property demonstrated only by the bis-biguanides.2,3 Initial killing of the bacteria by the chlorhexidine mouthrinse seems not to be the decisive factor in the plaque inhibition, as antibacterial agents with as high or higher activity against salivary bacteria did not show corresponding clinical effects.2 Considerable evidence has been accumulated indicating that a retention of bisbiguanides in the mouth is the basis for the plaque inhibition.4-9 About 30% of the chlorhexidine introduced into the mouth during a mouth rinse is retained.9,10 The teeth are not a major retention site, at least not quantitatively, but plaque-covered teeth have been shown to bind limited quantities of the drug."1 Results of studies that used mouthrinses at different pH levels strongly indicated that acidic groups, presumably on the macromolecules of the mucous secretions covering the oral surfaces, are the receptors responsible for the major share of the retained chlorhexidine. Electrostatic binding between the acidic protein groups and the basic bis-biguanide molecules seems to be the mechanism involved (Fig 1) .7,8,10 In the current study, the interaction of chlorhexidine with carboxyl, sulfate, and phosphate groups was studied in vitro, including factors that might interfere with these interactions. The properties of cetylpyridine in the same systems also were investigated for comparison, as this is a basic antibacterial agent with virtually no plaqueinhibiting effect.2 It was believed that such data could supply information that might shed light on the mechanism of adsorption and release of chlorhexidine in the mouth and possibly indicate the properties essential for a substance to be retained onto and released from the oral surfaces. Such substances may be of interest not only in dental
research, but could possibly also serve as carriers for other drugs when a gradual, slow oral release is needed. The chlorhexidine-binding properties of protein extracts from the major salivary glands at different pH levels were also investigated.
Materials and Methods Sephadex SP (C25), Sephadex CM (C25), Sephadex DEAE (A50), and Sepharose 6 B (inert control) a and Whatman P11 Cellulose phosphateb were used in the experiments. The chlorhexidine in 2 ml of a 2.2 Ml aqueous solution was completely adsorbed to 50 mg of Sephadex Sp and Whatman P1, whereas 100 mg of Sephadex CM was needed to obtain this effect. Two milliliters of chlorhexidinec or cetylpyridined ionic exchange material as described, and different concentrations of cations (acetate salts) or other potential inhibitors were incubated for one hour at room temperature and centrifuged for ten minutes at 3,000 x g and then the supernatant was tested for loss of chlorhexidine or cetylpyridine. Chlorhexidine was quantified by adding a l-ml sample to 6.5 ml of Dimilune scintillation liquid (Packard) and counted on an I.D.L. (Liquid Measuring Head 2022) scintillation counter. Cetylpyridine chloride was measured by the optical density at 260 nm. Human salivary glands were obtained from fresh autopsy material. The glands were homogenized and extracted as de-
scribed previously.12 The drug-binding capacity of the different gland extracts was evaluated as described Pharmacia Fine Chemicals, Uppsala, Swed. Whatman Biochemicals Ltd., Maidstone Kent, Eng. e ICI, Macclesfield, Eng. d Norsk Medisinal-depot, Oslo, Norway.
a
b
BS7 Downloaded from jdr.sagepub.com at UNIVERSITE DE MONTREAL on July 9, 2015 For personal use only. No other uses without permission.
B58
ROLLA AND MELSEN
J Dent Res Special Issue B
elsewhere.13 The pH was adjusted by adding acetic or hydrochloric acid to the extracts. Hydrochloric acid in the amounts used did not precipitate chlorhexidine.
Results ADSORPTION
OF CHLORHEXIDINE TO ANIONIC
AND CATIONIC IONIC-EXCHANGE MATERIAL.The results are shown in the table. As expected, chlorhexidine was strongly bound to the anionic materials, whereas no binding
could be observed to the cationic DEAESephadex or to the inert material (agarose). Extensive washing with distilled water or weak buffers did not release any of the bound drug. INFLUENCE OF PH AND UREA ON THE BIND-
ING.-The bound chlorhexidine was immediately released at a pH of 1. The presence of up to 30% of urea did not interfere with the binding of chlorhexidine to carboxyl, sulfate, or phosphate groups in our systems. The influence of urea was tested because previous experiments had indicated that this substance interfered with the pro-
tein binding of chlorhexidine.13 BINDING OF CHLORHEXIDINE TO EXTRACTS FROM THE MAJOR SALIVARY GLANDS.-The extracts bound about the same amount of drug at pH's of 6.4, 4.5, and 3.0. Only the sublingual gland extract retained chlorhexidine below a pH of 3.0. The bound drug could be recovered at a pH of 1.0. EFFECT OF DIFFERENT CATIONS ON THE BINDING.-The results are shown in Figure 2. It can be seen that Ba++, Ca++, and Cd++ all interfered with the chlorhexidine binding of the acidic groups. The effect .was most noticeable on the phosphate binding and only moderate on the sulfate. Cadmium did not interfere with the binding to sulfate in the concentrations tested. Zn++ and Mg++ were also tested and had an effect comparable with Ca++, whereas Hg++ was more like cadmium. Sodium had only a slight effect. NH4+ had an effect comparable to that of sodium. Column chromatography with CM and SP Sephadex saturated with chlorhexidine and a gradient of calcium acetate gave displacement of chlorhexidine adsorbed to carboxyl groups (Fig 3) whereas chlorhexidine adsorbed to sulfate was not displaced. Similar experiments in which calcium was adsorbed to the- ionic exchangers and chlorhexidine was applied as a displacement agent showed that chlorhexidine could displace calcium from sulfate but not from carboxyl. PROPERTIES OF CETYLPYRIDINE.-Cetylpyridine chloride is a quaternary ammonium salt with well-established antibacterial activity against salivary bacteria but with no plaque-inhibiting effect.2 The drug was adsorbed to the cation-exchangers in a manner similar to chlorhexidine. Calcium could displace cetylpyridine from carboxyl groups ,
Electrostatic bonds between
negatively charged
and
macromolecules
-
chlorhexidine molecules
SO3-
+
_1
- coo-
TABLE INTERACTION OF CHLORHEXIDINE WITH AcIDIC AND BASIC IONIC EXCHANGE MATERIAL (2 ml of 2.2 M Chlorhexidine in Distilled Water) Material -
P03
...... fl
FIG 1.-Postulated interaction between chlor.hexidine and oral macromolecules.4.5'78'l4l
CP-ll Cellulose (phosphate) CM-Sephadex (carboxyl) SP-Sephadex (sulfate) DEAE-Sephadex (basic) 6 B-Sephadex (inert)
Amount Binding of Needed Chlorhexi(mg) dine (%)
50 100 50 100 100
Downloaded from jdr.sagepub.com at UNIVERSITE DE MONTREAL on July 9, 2015 For personal use only. No other uses without permission.
100 100 100 0 0
PLAQUE INHIBITION BY CHLORHEXIDINE
Vol 54 1975
at significantly lower concentrations than those needed for displacement of chlorlhexidine (Fig 4). The binding of this quaternary ammonium base to carboxyl groups thus seems to be weaker than that of the bis-biguanide.
Discussion The results in the table show that chlorhexidine was firmly bound to acidic ionic exchangers, wlhereas basic or inert material exhibited no such binding. The bound drug was displaced by hydrogen ions as observed in corresponding clinical experiments,7,8 and the acidic ionic-exchange materials thus seem to be an interesting model system where some aspects of the interaction between chlorliexidine and oral retention sites can possibly be studied. The effect of external factors such as ionic strength or competing ions on the binding of chlorhexidine by acidic groups could be conveniently evaluated in these systems. Our experiments showed that up to 30% of urea did not interfere with the chlorhexidine binding to carboxyl, phosphate, or
sulfate. It has been shown in our laboratory (unpublished results) that 30% urea added to chlorhexidine mouthwashes reduced the amount of drug initially bound, without interfering with the clinical effect. It was also shown in vitro13 that urea redissolved albumin precipitated out of serum by chlorhexidine. These results, combined with our present data, thus indicate that part of the drug retained in the mouth is bound by mechanisms other than electrostatic interaction, presumably hydrophobic or hydrogen bonding.14,15 These do not, however, seem to be clinically important receptor sites. Free carboxyl groups are present in the sialic acid which is known to be abundant in salivary glycoproteins. Carboxyl groups in acidic amino acids are presumably involved in intramolecular interactions to a higher degree, but aspartic acid and glutamic acid are found in high amounts in some salivary glycoproteins.16 Sulfate is present in sulfated glycoproteins, a group of substances present in the mucous salivary secretions.17-19 These substances have recently been ascribed important roles
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The marked plaque-inhibiting activity shown by chlorhexidine1 is a unique property demonstrated only by the bis-biguanides.2,3 Initial killing of the bacteria by the chlorhexidine mouthrinse seems not to be the decisive factor in the plaque inhibition, as antibacterial agents with as high or higher activity against salivary bacteria did not show corresponding clinical effects.2 Considerable evidence has been accumulated indicating that a retention of bisbiguanides in the mouth is the basis for the plaque inhibition.4-9 About 30% of the chlorhexidine introduced into the mouth during a mouth rinse is retained.9,10 The teeth are not a major retention site, at least not quantitatively, but plaque-covered teeth have been shown to bind limited quantities of the drug."1 Results of studies that used mouthrinses at different pH levels strongly indicated that acidic groups, presumably on the macromolecules of the mucous secretions covering the oral surfaces, are the receptors responsible for the major share of the retained chlorhexidine. Electrostatic binding between the acidic protein groups and the basic bis-biguanide molecules seems to be the mechanism involved (Fig 1) .7,8,10 In the current study, the interaction of chlorhexidine with carboxyl, sulfate, and phosphate groups was studied in vitro, including factors that might interfere with these interactions. The properties of cetylpyridine in the same systems also were investigated for comparison, as this is a basic antibacterial agent with virtually no plaqueinhibiting effect.2 It was believed that such data could supply information that might shed light on the mechanism of adsorption and release of chlorhexidine in the mouth and possibly indicate the properties essential for a substance to be retained onto and released from the oral surfaces. Such substances may be of interest not only in dental
research, but could possibly also serve as carriers for other drugs when a gradual, slow oral release is needed. The chlorhexidine-binding properties of protein extracts from the major salivary glands at different pH levels were also investigated.
Materials and Methods Sephadex SP (C25), Sephadex CM (C25), Sephadex DEAE (A50), and Sepharose 6 B (inert control) a and Whatman P11 Cellulose phosphateb were used in the experiments. The chlorhexidine in 2 ml of a 2.2 Ml aqueous solution was completely adsorbed to 50 mg of Sephadex Sp and Whatman P1, whereas 100 mg of Sephadex CM was needed to obtain this effect. Two milliliters of chlorhexidinec or cetylpyridined ionic exchange material as described, and different concentrations of cations (acetate salts) or other potential inhibitors were incubated for one hour at room temperature and centrifuged for ten minutes at 3,000 x g and then the supernatant was tested for loss of chlorhexidine or cetylpyridine. Chlorhexidine was quantified by adding a l-ml sample to 6.5 ml of Dimilune scintillation liquid (Packard) and counted on an I.D.L. (Liquid Measuring Head 2022) scintillation counter. Cetylpyridine chloride was measured by the optical density at 260 nm. Human salivary glands were obtained from fresh autopsy material. The glands were homogenized and extracted as de-
scribed previously.12 The drug-binding capacity of the different gland extracts was evaluated as described Pharmacia Fine Chemicals, Uppsala, Swed. Whatman Biochemicals Ltd., Maidstone Kent, Eng. e ICI, Macclesfield, Eng. d Norsk Medisinal-depot, Oslo, Norway.
a
b
BS7 Downloaded from jdr.sagepub.com at UNIVERSITE DE MONTREAL on July 9, 2015 For personal use only. No other uses without permission.
B58
ROLLA AND MELSEN
J Dent Res Special Issue B
elsewhere.13 The pH was adjusted by adding acetic or hydrochloric acid to the extracts. Hydrochloric acid in the amounts used did not precipitate chlorhexidine.
Results ADSORPTION
OF CHLORHEXIDINE TO ANIONIC
AND CATIONIC IONIC-EXCHANGE MATERIAL.The results are shown in the table. As expected, chlorhexidine was strongly bound to the anionic materials, whereas no binding
could be observed to the cationic DEAESephadex or to the inert material (agarose). Extensive washing with distilled water or weak buffers did not release any of the bound drug. INFLUENCE OF PH AND UREA ON THE BIND-
ING.-The bound chlorhexidine was immediately released at a pH of 1. The presence of up to 30% of urea did not interfere with the binding of chlorhexidine to carboxyl, sulfate, or phosphate groups in our systems. The influence of urea was tested because previous experiments had indicated that this substance interfered with the pro-
tein binding of chlorhexidine.13 BINDING OF CHLORHEXIDINE TO EXTRACTS FROM THE MAJOR SALIVARY GLANDS.-The extracts bound about the same amount of drug at pH's of 6.4, 4.5, and 3.0. Only the sublingual gland extract retained chlorhexidine below a pH of 3.0. The bound drug could be recovered at a pH of 1.0. EFFECT OF DIFFERENT CATIONS ON THE BINDING.-The results are shown in Figure 2. It can be seen that Ba++, Ca++, and Cd++ all interfered with the chlorhexidine binding of the acidic groups. The effect .was most noticeable on the phosphate binding and only moderate on the sulfate. Cadmium did not interfere with the binding to sulfate in the concentrations tested. Zn++ and Mg++ were also tested and had an effect comparable with Ca++, whereas Hg++ was more like cadmium. Sodium had only a slight effect. NH4+ had an effect comparable to that of sodium. Column chromatography with CM and SP Sephadex saturated with chlorhexidine and a gradient of calcium acetate gave displacement of chlorhexidine adsorbed to carboxyl groups (Fig 3) whereas chlorhexidine adsorbed to sulfate was not displaced. Similar experiments in which calcium was adsorbed to the- ionic exchangers and chlorhexidine was applied as a displacement agent showed that chlorhexidine could displace calcium from sulfate but not from carboxyl. PROPERTIES OF CETYLPYRIDINE.-Cetylpyridine chloride is a quaternary ammonium salt with well-established antibacterial activity against salivary bacteria but with no plaque-inhibiting effect.2 The drug was adsorbed to the cation-exchangers in a manner similar to chlorhexidine. Calcium could displace cetylpyridine from carboxyl groups ,
Electrostatic bonds between
negatively charged
and
macromolecules
-
chlorhexidine molecules
SO3-
+
_1
- coo-
TABLE INTERACTION OF CHLORHEXIDINE WITH AcIDIC AND BASIC IONIC EXCHANGE MATERIAL (2 ml of 2.2 M Chlorhexidine in Distilled Water) Material -
P03
...... fl
FIG 1.-Postulated interaction between chlor.hexidine and oral macromolecules.4.5'78'l4l
CP-ll Cellulose (phosphate) CM-Sephadex (carboxyl) SP-Sephadex (sulfate) DEAE-Sephadex (basic) 6 B-Sephadex (inert)
Amount Binding of Needed Chlorhexi(mg) dine (%)
50 100 50 100 100
Downloaded from jdr.sagepub.com at UNIVERSITE DE MONTREAL on July 9, 2015 For personal use only. No other uses without permission.
100 100 100 0 0
PLAQUE INHIBITION BY CHLORHEXIDINE
Vol 54 1975
at significantly lower concentrations than those needed for displacement of chlorlhexidine (Fig 4). The binding of this quaternary ammonium base to carboxyl groups thus seems to be weaker than that of the bis-biguanide.
Discussion The results in the table show that chlorhexidine was firmly bound to acidic ionic exchangers, wlhereas basic or inert material exhibited no such binding. The bound drug was displaced by hydrogen ions as observed in corresponding clinical experiments,7,8 and the acidic ionic-exchange materials thus seem to be an interesting model system where some aspects of the interaction between chlorliexidine and oral retention sites can possibly be studied. The effect of external factors such as ionic strength or competing ions on the binding of chlorhexidine by acidic groups could be conveniently evaluated in these systems. Our experiments showed that up to 30% of urea did not interfere with the chlorhexidine binding to carboxyl, phosphate, or
sulfate. It has been shown in our laboratory (unpublished results) that 30% urea added to chlorhexidine mouthwashes reduced the amount of drug initially bound, without interfering with the clinical effect. It was also shown in vitro13 that urea redissolved albumin precipitated out of serum by chlorhexidine. These results, combined with our present data, thus indicate that part of the drug retained in the mouth is bound by mechanisms other than electrostatic interaction, presumably hydrophobic or hydrogen bonding.14,15 These do not, however, seem to be clinically important receptor sites. Free carboxyl groups are present in the sialic acid which is known to be abundant in salivary glycoproteins. Carboxyl groups in acidic amino acids are presumably involved in intramolecular interactions to a higher degree, but aspartic acid and glutamic acid are found in high amounts in some salivary glycoproteins.16 Sulfate is present in sulfated glycoproteins, a group of substances present in the mucous salivary secretions.17-19 These substances have recently been ascribed important roles
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