ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1983, p. 379-384 Vol. 23, No.

3
0066-4804/83/030379-06$02.00/0
Copyright C 1983, American Society for Microbiology

In Vitro Antiplaque Activity of Octenidine Dihydrochloride

(WIN 41464-2) Against Preformed Plaques of Selected Oral
Plaque-Forming Microorganisms
ANDREW M. SLEE* AND JOHN R. O'CONNOR
Department of Microbiology, Sterling-Winthrop Research Institute, Rensselaer, New York 12144
Received 7 September 1982/Accepted 7 December 1982

The antibacterial activity of octenidine dihydrochloride (WIN 41464-2) against

intact preformed in vitro plaques of four indigenous oral plaque-forming microor-
ganisms, Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus,
and Actinomyces naeslundii, was studied. Both absolute (plaque bactericidal
index) and relative (chlorhexidine coefficient) indices of antiplaque efficacy were
established. Octenidine dihydrochloride compared favorably with chlorhexidine
digluconate with respect to overall antiplaque potency in this in vitro plaque

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bactericidal model. These data indicate that prudent selection of treatment
concentration and duration and frequency of exposure should provide an effective
means to aid in controlling dental caries and Actinomyces-associated disease in
vivo.
Dental plaque, the dense adhesive microbial be of greatest utility. In addition, dental plaque
mass that colonizes tooth surfaces, has been is representative of a slow-growing microbial
strongly implicated in both experimental animal system (27, 31). Therefore, for an agent to be
models and in humans as the key etiological efficacious, it must be able to permeate the
factor in dental caries (4, 7, 8, 13, 14, 21, 23) and plaque matrix and rapidly kill at least the patho-
various periodontal diseases (12, 18, 20, 25). genic members of the plaque microflora after
Notable among the various microorganisms as- only transient exposures.
sociated with dental plaque accumulations are In this context, the bispyridine octenidine
strains of Actinomyces viscosus, Actinomyces dihydrochloride (WIN 41464-2) has been shown
naeslundii (2, 9, 15, 16, 19, 24), Streptococcus to have utility in controlling bacterial plaque
mutans (10, 11, 26), and Streptococcus sanguis formation. Preliminary studies have demonstrat-
(3, 4). Infection of experimental animal models ed that octenidine can inhibit the colonization of
by representative strains of these microorga- a substrate such as ceramic hydroxylapatite
nisms has been shown to initiate the develop- (durapatite) by S. mutans OMZ-61 at a concen-
ment of enamel lesions and cause periodontal tration 10-fold less than that which promotes
tissue destruction, with the possible exception staining of such surfaces (J. O'Connor, D. A.
of S. sanguis, for which evidence of involve- Paris, and D. Bailey, J. Dent. Res. vol. 59,
ment in dental diseases is minimal (3, 4, 10). special issue A, abstract no. 743, 1980). In
Dental plaque removal from oral surfaces by addition, in vivo studies have demonstrated that
various mechanical means has been shown to octenidine at a concentration of 0.5% (wt/vol)
effect a short-term remission of the signs of inhibited the implantation and subsequent as-
gingivitis, periodontitis, and dental caries (12, cendency of S. mutans in the oral ecology and
25, 32). The effective control of dental plaque concomitant development of enamel lesions in a
and thereby of dental diseases of microbial etiol- rat caries model (R. Shern, E. Monell-Torrens,
ogy by such mechanical means is, however, W. Bowen, and A. Kingman, J. Dent. Res. vol.
dependent on patient compliance and appears to 59, special issue A, abstr. no. 186, 1980). Octeni-
be limited by failure of the patient to reach dine at a concentration of 1.0% (wt/vol) also
certain plaque-infected sites. Hence, there cur- appears to be able to inhibit the growth of
rently exists great therapeutic potential for the Actinomyces spp. in dental plaque in a primate
development of antiplaque chemotherapeutic model system (6).
agents to assist in the control of dental plaque- Our studies have extended earlier assess-
associated infections in humans (1, 22). Because ments of the in vitro antiplaque activity of
supragingival plaque adheres to tooth surfaces octenidine and have characterized the condi-
and is not bathed in either blood or interstitial tions required to kill preformed plaques of four
fluids, topical antiseptic agents would appear to indigenous dental plaque-forming microorga-
379
380 SLEE AND O'CONNOR ANTIMICROB. AGENTS CHEMOTHER.
nisms. An in vitro antiplaque model was em- this method were visually graded, and uniformly sized
ployed to determine the antiplaque activity of plaques of the target organisms were selected for
octenidine, the efficacy of which was compared assessment of agent efficacy (29).
with that of the extensively studied antiplaque Amessment of efficacy of octealdine. Efficacy was
assessed by using previously detailed techniques (29,
agent chlorhexidine. 30). Briefly, in vitro plaques were rinsed of growth
MATERIAULS AND METHODS medium by submersion in demineralized water for 2
min and then immersed in 10 ml of aqueous solutions
Antibacterial agents. Octenidine (WIN 41464-2; of the test or control agents for various durations and
N, N'-(1 ,1 0-decanediyldi-1 (4H)-pyridinyl-4-ylidene)- frequencies. After treatment, the in vitro plaques were
bis[l-octanaminel dihydrochloride), obtained from rinsed twice by immersion for 10 min in 15 ml of
Sterling-Winthrop Research Institute, Rensselaer, distilled water and then transferred to fresh broth
N.Y., and chlorhexidine digluconate, obtained from growth medium having the same composition as that in
Fermion, Finland, were used in these studies (Fig. 1). which the plaques had grown but which now contained
All agents were examined for purity before use and a pH indicator (bromocresol purple). Plaques were
were shown to contain no detectable impurities with judged to be killed if culture acid production and
bactericidal properties. turbidity increase ceased and if 24-h post-treatment
Microornsm. The microorganisms used in these plaque samples failed to grow when placed on appro-
studies were S. mutans NCTC 10449, S. sanguis priate agar media (29, 30).
ATCC 10558, A. viscosus M-1OOB, and A. naeslundii
631. These cultures were obtained from the collection
of the Oral Microbiology Division, Department of Oral RESULTS

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Diagnosis, School of Dental Medicine, University of MiI bacidal concentration. To deter-
Connecticut Health Center, Farmington, Conn., and mine the minimal concentration of octenidine
their identities were confirmed with standard micro- that maximally inhibited pre-
biological techniques before use. These bacteria form and chlorhexidine the four test microorganisms,
plaques both in vitro and in vivo and, with the possible formed plaques of
exception of S. sanguis, have been associated with the plaques were immersed in various concentra-
development of coronal caries or periodontal disease tions of these agents for 30 min at 37°C. Control
with attendant root surface caries or both (2, 3, 9, 16, plaques were similarly immersed in sterile de-
23, 24). All microorganisms were stored either lyophi- mineralized water. Both octenidine and chlor-
lized or frozen (-70°C). For use in experiments, hexidine were bactericidal as judged by the
working culture stocks were maintained by bimonthly criteria described in Materials and Methods (Ta-
passage in fluid thioglycolate medium containing 20%o ble 1). Octenidine at a concentration of 3.2 mM
(vol/vol) meat extract and excess CaCO3. plaques of a
In vitro plque formtion. In vitro plaques were effectively killed all preformed bacterial strain exam-
grown on ceramic hydroxylapatite slabs (1.6 by 1 cm; quadruplicate set for each
Sterling-Winthrop Research Institute) which were in- ined. At a concentration of 1.6 mM aqueous
dividually suspended on no. 26-gauge nichrome wire octenidine, acid production was perturbed; how-
by using a modification of methods previously detailed ever, upon extended incubation (36 h), the pH
(29, 30). Fluid thioglycolate cultures were used to fell to the same level as that of the control
inoculate a complex medium (17) supplemented with plaques. At octenidine concentrations of less
5% (wtlvol) sucrose and 0.005% (wt/vol) Na2CO3. The than 1.6 mM, there was little noticeable effect on
wire-attached ceramic hydroxylapatite slabs were acid production or plaque viability. Chlorhexi-
transferred daily to fresh medium for 3 days. All dine was bactericidal for plaques of S. sanguis,
cultures were incubated at 37°C under an anaerobic at 1.6 mM; how-
atmosphere (GasPak; BBL Microbiology Systems, A. viscosus, and A. naeslundii kill all plaques in
Cockeysville, Md.). The in vitro plaques formed by ever, 3.2 mM was required to

A
H H A
\N= =N-(CH)goONFN
CH3(CH2 \(CH2)7CH3
*2-HCI

NH NH NH B
/=\ H n H n H H I H IH \
CIN-C-CN-C-N-(CH2)6-N-C-N-C-N CI

*2 -
C6H120T
FIG. 1. Structural formulae of octenidine dihydrochloride (A) and chlorhexidine digluconate (B).
VOL. 23, 1983 OCTENIDINE, A NEW ANTIPLAQUE AGENT 381
TABLE 1. Minimal concentration of agents required to be batericidal to plaques of the test microorganismsa
Concn (MM)b bactericidal for:
S. mutans S. sanguis A. viscosus A. naeslundii
Octenidine 3.2 3.2 1.6 3.2
Chlorhexidine 3.2 1.6 1.6 1.6
a
Plaques were run in quadruplicate. Results represent the uniform behavior of the quadruplicate set.
b Concentrations tested were(
0.16, 0.8, 1.6, 3.2, 8.0, and 16.0 mM.

the quadruplicate set for S. mutans. These find- daily 1-min treatment, acid production by these
ings for chlorhexidine are similar to those previ- strains was greatly reduced in comparison with
ously reported by Tanzer et al. (29). the water-treated controls. S. mutans was effec-
Minimal exposure time. The minimal duration tively killed by both agents after four successive
of a single treatment which was bactericidal for once-daily, 2-min exposures, and acid produc-
these two agents was determined by immersion tion was also significantly reduced when the
of preformed plaques at the previously estab- treatment time was reduced to 1 min in a similar
lished minimal bactericidal concentrations (Ta- period. S. sanguis had to be treated with octeni-
ble 1) for periods of 1, 2, 5, 10, 15, 20, or 30 min. dine for 5 days at the 2-min exposure time to be

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These studies demonstrated that octenidine at a killed (Table 3); a once-daily, 1-min treatment
concentration of 3.2 mM was bactericidal for S. for 7 days failed to kill the plaques (data not
mutans after exposure times of 15, 20, or 30 min. shown).
The same organism had to be exposed to 3.2 mM To compare the therapeutic utility of octeni-
chlorhexidine for 30 min to achieve a similar dine with that of chlorhexidine, a plaque bacteri-
bactericidal effect. The other three plaque-form- cidal index (PBI) and a chlorhexidine coefficient
ing bacteria were susceptible to octenidine after (CC) were computed. The PBI is the integration
only a 10-min exposure, in contrast to chlorhexi- of the effects of concentration (millimolar), dura-
dine, which at a concentration of 3.2 mM re- tion (daily exposure time) and frequency of
quired an exposure time of at least 20 min to treatments (number of daily treatments neces-
achieve a bactericidal effect (Table 2). sary to kill plaques) required to achieve a plaque
Frequency of exposure. Assuming that a mouth bactericidal effect. The CC is computed by di-
rinse is the delivery method of choice, the time viding the PBI for chlorhexidine by that for
that such an agent could normally be expected to octenidine.
be retained orally would not likely exceed 2 min. These data indicate that, for the most potent
Studies were therefore conducted to evaluate cariogenic and periodontopathic components of
whether shorter but more frequent exposures in vivo plaque examined here (S. mutans and A.
could achieve the desired bactericidal effects. viscosus), the antiplaque activity of octenidine
Two types of experiments were performed to compares very favorably with that of bisbiguan-
assess this aspect of a potential treatment regi- ide chlorhexidine (Table 3). In addition, it is
men. In the first, preformed in vitro plaques of noted that higher concentrations or more fre-
the test microorganisms were treated for either 1 quent exposures to octenidine are required to
or 2 min once daily until plaque death occurred; kill the relatively innocuous plaque former S.
such a protocol was viewed as analogous to a sanguis, as compared with the conditions re-
single daily mouth rinse. In the second protocol, quired for chlorhexidine to kill this species.
in vitro plaques were treated for either 1 or 2 Table 4 shows the concentration, duration,
min, rinsed, incubated in growth medium for 6 h,
and then retreated. The procedure was repeated
daily until plaque death occurred, as judged by TABLE 2. Minimal exposure time required of agent
the criteria described above. This treatment at minimal bactericidal concentration to kill plaquesa
protocol was viewed as analogous to the use of a Minimum killing time (min) for
mouth rinse twice daily. agents at minimal bactericidal
Table 3 shows the concentration and frequen- Microorganism concn of 3.2 mM
cy of use required for each agent to be effica-
Octenidine Chlorhexidine
cious in a once-daily regimen. A. viscosus and
A. naeslundii plaques were killed after three S. mutans 15 30
successive once-daily, 2-min treatments with 3.2 S. sanguis 10 20
mM octenidine, but they were not killed after A. viscosus 10 20
five daily treatments if the exposure time was A. naeslundii 10 20
reduced to only 1 min (data not shown). It a
Plaques run in quadruplicate. Results represent
should be noted, however, that after the third the uniform behavior of the quadruplicate set.
382 SLEE AND O'CONNOR ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Comparison of the therapeutic utility of chlorhexidine with that of octenidine, one treatment
daily-
Chlorhexidine Octenidine
Minimal concn (mM) x Minimal concn (mM) x
Microorganism duration (min) x duration (min) x CC (chIorhexidine
frequency (no. of days) PBI frequency (no. of days) PBI PBI/octenidine PBI)
required for bactericidal required for bactericidal
effect effect
S. mutans 3.2 x 2 x 4 25.6 3.2 x 2 x 4 25.6 1.00
S. sanguis 3.2 x 2 x 3 19.2 3.2 x 2 x 5 32.0 0.60
A. viscosus 3.2 x 2 x 4 25.6 3.2 x 2 x 3 19.2 1.33
A. naeslundii 3.2 x 2 x 3 19.2 3.2 x 2 x 3 19.2 1.00
a Plaques run in quadruplicate. Results represent the uniform behavior of the quadruplicate set.

and frequency of treatment required to achieve a activity in vitro against preformed plaques of
bactericidal effect when preformed plaques were representative strains of four indigenous oral
treated twice a day. Octenidine was shown to plaque-forming bacteria. This agent was shown
compare favorably with chlorhexidine with re- to be efficacious and to compare favorably with

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spect to antiplaque efficacy for all test orga- chlorhexidine with respect to overall antiplaque
nisms, with the possible exception of S. sanguis. activity.
If used for 2 min twice a day, octenidine at a These studies were designed to evaluate the
concentration of 1.6 mM had a bactericidal conditions required for octenidine to kill pre-
effect for all plaque formers after four to five formed plaques in vitro and also to estimate the
twice-daily treatments, with the exception of S. conditions that are likely to be required for the
sanguis. If, however, the exposure time was agent to be effective in vivo (29, 30). The dense
reduced to only 1 min twice a day, a sufficient adhesive character of in vivo dentl plaque
number of organisms survived in the plaque to appears to constitute a diffusion barrier to many
grow and produce acid (Table 4). antiseptics (29, 30), and this probably accounts
for the inability of a nurflber of otherwise bacte-
DISCUSSION ricidal agents to be effective in controlling dental
A novel bispyridine, octenidine dihydrochlor- plaque development. Because freely suspended
ide (WIN 41464-2), was assessed for bactericidal cells of plaque-forming microorganisms are or-

TABLE 4. Comparison of the therapeutic utility of chlorhexidine with that of octenidine, two treatments
d"ya
Octenidine Chlorhexidine
Minimal concn (mM) x Minimal concn (mM) x
Microorganism durtion (min) x duration (min) x
frequency (no. of days) PBI frequency (no. of days) PBI
required for bactericidal required for bactericidal
effect effect
S. mutans 3.2 x (2 + 2) x 2 25.6 3.2 x (2 + 2) x 2 25.6
3.2 x (1 + 1) x 4 25.6 3.2 x (1 + 1) x 3 19.2
1.6 x (2 + 2) x 5 32.0
1.6 x (1 + 1)x >5 NCb
S. sanguis 3.2 x (2 + 2) x 3 38.4 3.2 x (2 + 2) x 2 25.6
3.2 x (1 + 1)x 5 32.0 3.2 x (1 + 1) x 3 19.2
1.6 x (2 + 2) x >5 NC
1.6x(1+1)x>5 NC
A. viscosus 3.2 x (2 + 2) x 2 25.6 3.2 x (2 + 2) x 3 38.4
3.2 x (1 + 1)x 3 19.2 3.2 x (1 + 1) x 4 25.6
1.6 x (2 + 2) x 4 25.6
1.6 x (1 + 1)x >5 NC
A. naeslundii 3.2 x (2 + 2) x 2 25.6 3.2 x (2 + 2) x 2 25.6
3.2x(1+1)x 3 19.2 3.2x(1+1)x3 19.2
1.6 x (2 + 2) x 4 25.6
1.6x(1+1)x>5 NC
a
Plaques were run in duplicate. Results represent the uniform behavior of the duplicate set.
b NC, Could not be computed.
VOL. 23, 1983 OCTENIDINE, A NEW ANTIPLAQUE AGENT 383

ders of magnitude more susceptible to agents and A. viscosus while allowing for the survival
than are intact plaques (22, 27, 28), efficacy was of the more innocuous S. sanguis. Thus, the
evaluated against intact bacterial plaques in this bispyridine octenidine appears to have utility in
study. Such a model is more representative of controlling dental plaque-related infections. In
the conditions and constraints an agent can be addition, selection of the correct dosage may
expected to encounter in vivo (22, 28, 30). allow manipulation of the plaque ecology to
Octenidine was found to possess bactericidal permit survival of the relatively nonpathogenic
activities similar to those of the well-character- organisms and thereby provide a mechanism for
ized bisbiguanide chlorhexidine. This may be sustained inhibition of recolonization and
readily seen by comparing the PBIs for the two growth of the more pathogenic members of the
agents against the various test microorganisms. dental plaque microflora. Furthermore, the
The PBI computation allows for a ready evalua- spectrum of antimicrobial activity displayed by
tion of the sum of treatment conditions (concen- this agent suggests that, at the indicated dose
tration, duration, and frequency) required to levels, organisms such as yeasts and molds
achieve a bactericidal effect on intact in vitro would not be given a selective advantage in the
plaques. The derivation and validity of this oral cavity, a problem of obvious concern for
index has been previously detailed (29). Briefly, agents used frequently. Clinical evaluations of
it should be noted that the index permits com- this agent for in vivo efficacy in controlling
parison of efficacy because dental plaque grows plaque-associated diseases in humans are cur-
slowly and, therefore, plaque as such represents rently under way.

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virtually a stable-sized mass of cells available for
killing. The effects of antiseptic treatment,
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