CA2125576A1 - Oral compositions for inhibiting plaque and calculus formation - Google Patents

Abstract

Oral compositions and methods effective in promoting oral hygiene containing in an effective amount at least one cyclic alkylamine or cyclic amine as an antiplaque and/or antigingivitis agent.

Description

~,WO 93/11741 PCI/US9~/10897 21~576 ~`

ORAL COMPOSITIONS FOR INHlBlTlN~:i PLAQUE
AND CALCULUS FORMATION

S BACKGROUND OF TtlE INVENTION

Thepresentinventionrelatestooralcompositionscontaininganarltiplaque agent or an anticalculus agent.

"~ral composition" means a composition for topical appiiCatiQns to ~he oral 10 cavity to clean and care for the teeth as well as th~ oral cavity surfaces. Representati~es of such compositions are oral hygiene products and dentifrices such as mouthwashes or rinses, toothpaste, dental gels, tooth powder, chewing gum, lozenges and similar produ~s. In addition to cleaning teeth to remove dental plaque, the function of oral hygiene prep~rations is tQ stop the formation of dental calculus, to prevent d~ntal di~orders such as caries, ~ ;
15 periodontosis and gingivitis, and also to eliminate halitosis.

Dental calcuius, or tartar as it is sometimes called, is a hard mineralized material which forms on teeth that consists of inorganic and organic components The inorganic portion is largely calcium and orthophosphate arranged in a crystal lattice called 2G hydroxyapatite (HAP~. The organic por~ion is derived mainly from microorganisms (i.e., bacteria, yeast, etc.) as well as epithelial cel Is, white blood cells and food debris.

Fvrmation of dental calculus OCCU7S in two steps. In the first step"~laque is deposited on the teeth. "PlaqueU is a ~eposit which forms on teeth and consists of inorganic 25 and organic components derived from saliva, food and bacteria which are present in ~he oral cavity. Most of the plaque consists of dead and living bacteria surrounded by a gel-like rnatrix derived from the bacteria and saliva. In the second phase, plaque undergoes calcification to form dental calculus. tnitially, amorphous deposi~s of calcium phosphate begins to appear on and within the matrix of the dental plaque As the aggregates of calcium phosphate become :

212557~
WO 93/11741 P~/US92/10 sufficiently closely packed together, they crystallize to ~orm HAP. The amorphouslcalcium phosphate, although related to hydroxyapatite, differs from ;t in crystal structure, particle morphologyand stoichiometry The presence of bo~h the bacteria and the plaque deposits is detrimental to the health of the teeth and gums. If the bacteria and the plaque formation are not checked, they may result in infected gingivai tissue, the formation of den~al caries and periodontal disease A wide variety of chemical and biological agents have been suggested in the art to re-tard calculus formation or to remove calculus after it is formed. Mechanical removal of 10 this material periodically by the dentist is, of course, routine dental office procedure. ;;

In addition to being an integral step forthe formation of calculus, consequencesof the presence of plaque include gin~ivitis, periodontitis, tsoth decay (dental caries) and denture associated problems. Inhibition of oral bacteria involved in the formation of plaque r5 by antibiotics or antiseptic agents is one meansto retard the formation of plaque, thus aiding in preventing or controlling the formation of dental calculus and other plaque related diseases.; see, for example, P.S. Hull, J. ain~ Periodontology, 7, 431-~2 ~1980). Examples of ~ntiseptic agents include amidines, such as chlorh0xidine and alexidi ne, and numerous antibacterialty active guaternary ammonium compounds, such as cetylpyridinium chloride or 20 the quaternary ammonium ~ompounds described in IJ.S. Patent 3,369,046; U.S. Patent 4,820,507; and quaternary ammonium organosiloxane compounds described in U.5.
Patent4,161,518.

Although the quaternary ammonium compounds are rapidly adsorbed onto the tooth surface, they exhibit only a moderate degree of efficacy as antiplaque agents as they are rapidly r~leased from the tooth surface and thus retained in the oral cavity for only a short period of time. Chlorhexidine has been the most successful antiplaque agent and is retained in the oral cavity by binding to anionic groups ~mainly on the oral mucosa. The use of chlorhexidine in oral preparations however, suff~rsfrom the following disadvantages: ~1) a prolonged bitter aftertaste iasting up to several hours; (2) a~ter prolonged use they produce stains on the teeth, tongue, gums, oral mucosa and dental restorations; and (3) local irritation of the oral mucosa and tongue. ~ ~

Another means to prevent plaque and calculus formation is to coat the teeth witha material to prevent the release of previously applied therapeutic agents or to coat the teeth 35 with a material contai ni ng an anti microbial agent. U.S. Pa~ents 4,243,658; 4,428,930;
4,470,964 and 4,485,û90 disclose a dentifrice composition containing a water-dispersible, membrane-forming material which, when~applied to~tooth surfaces forms a hydrophobic :
' WO 93/1 17ql PCir/USg2/1~8~7 2125S71~
barrier thereon which substantially reduces elution of a previously applied therapeutlc agent.
A varnish containing an antimicrobial agent, which provides the sustained release of the antimicrobial agent over a period of at least four days was disclosed in U.S patent 4,496,322 The use of ~he therapeutic agent or varnish coating are not entirely satisfactory as thel r 5 application requires a qualified professional, making their use as part of toutine oral hygiene maintenance impractical.

As the formation of calculus requires the crystallization of HAP, agents which effectively interfere with crystal grovvth, including HAP, can be effectiv~ as antiplaque/anticalculus agents. Inhibition of crystalline HAP formation can therefore be achieved by compounds which chelate calcium ions, which prevents the calculus from forming and/or breaks down mature c~lculus by removing calcium. It is known in the prior art that water soluble hexametaphosphates, tripolyphosphates and pyrophosphates and the like, are effectivecalcium and m~gnesium ion threshold agents, sequestrants ~nd/or chelatir1g agents ;
A threshold agent has the ability to prevent the precipitation of certain scale forming salts (e.g., calcium carbonate) at concentrations that are much lower than the amount needed for .
sequestration. 5ee, for example, U.S. P3tent 3,488,419 which discloses oral compositions containing polyphosphonate and U.S. Patent 4,215,105 which discloses oral compositions containing phosphonoacetic acid. The effectiveness of polyphosphonates as antiplaque agents has been limited as they are significantly hydroly~ed by salivary enzymes ;
(phosphatases) to orthophosphates which are ineffediye as inhibitors of HAP formation. The amount of enzymatic hydrolysis of the polyphosphate has been reduced by the use of a linear molecularly dehydrated polyphosphate salt ~ombined with fluoride as described in U.S.
Patent 4,808,410. ~ ;

~5 It would therefore be desirable to have an oral composition containing an effective antiplaque agent to aid iri the prevention of caries and gingivits whicll does not stain the teeth and is not subject to inactivation by enzymatic hydrolysis It would also be desirable to provide an improved means whereby antimicrobial compound can be re~ained vvithin the oral cavity for a longer period of time.
Itwould also be advantageousto have improved anticalculus oral compositions which inhibit the transformation of amorph~ous calci4m phosphate to HAP crystal structure normaliy associated with calculus.
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SUMMARY OF THE INVENTION
~ . ~
The present invention relates to an oral composition containing an antiplaque agent In particular, the present i nvention relates to oral com position comprising an orally acceptable vehicle containing therein an effective amollnt as an antiplaque sr anticalculus . ~ .
agent a cyclic alkylamine or a cyclic amine selected from one or more of the compounds represented by the Formulae I to IV:

N--CH~CH~--N~

1 !!i ' N--CH2 ~ CH2--N~ (II) ~

~, , :~
~ `, (III) .
N (CH2)~ ~ m : :, , ~ :
:, .
`. . . `:
~ .
.: `

. ~ , .
;;:

4~

: ~ .

;WO ~3ti1741 PCI/US92/11)8~7 2125576 :~
. " .
~'' N ~ ~ .

R--N N--P~ (IV) ;~

( N ) ,...
R

wherein each R is independently selected from hydrogen, and C1~Cg hydrocarbon radical, C2~C6 hydroxyalkyl :

~ X ~ ' ~
C~ Z ~,, wherein Z is independently -PO3H2, -COOH, -H, C1-C18 alkyl or a physi~logically accept2ble salt of the acid radicals; and X a,ld Y are independently -H, Cl-C~ hydrocarbon radicals, with ~he proviso that each cyclic alkylamin~ orcyclic amine contain at le~s~two Z groupswhich are -PO3H2 or a physiologically acceptable salt thereof;
each n is independently 2 or 3;
~ m is 3 to 6, inclusiYe; and . ~
n and m being selected such that the tota~ number of carbon and nitrogen atoms in the cyclic alrjne ring does not exceed i 8. .

As used herein, the term "al kyl " means a I in~ar or branched alkyl; and thus, secondary and tertiary amines are included. The aikyl terms up to C20 include, for example, t-butyl, sec-bwtyl, isobutyl, and in like m-nner all such branched or straight chain alkyls. ;

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WO 93/11741 2 1 2 5 5 7 6 PCI'/llS92/lOf As used herein the term "hydrocarbon" includesthe fc~llowing organic radicals:
alkanes, alkenes, alkynes, cycloalkanes, cyclozlkenes, and benzene raclicals.

In another embodiment of the present invention there is provided an oral composition containing a cyclic amine ancl an antimicrobial quaternary ammonium compound.
The combination provides that the antimicrobial quaternary ammonium compound is retained within the oral cavity, especially on the tooth surfac~, for a longer period of time. The enhanced retention of the antimicrobial quaternary ammonium compound within the oral cavity increases the ti me period during which the compound will be effective as an an ti plaque agent and/or antigingivitis agent.

A further embodiment of the present invention takes the forrn of an oral composition containing a cyclic amine, antimicrob;al quaternary ammonium compound and a metal ion for enhanced retention of the quaternary ammonium compound within the oral cavity.

In yet another embodiment of the present invention, there is provided an improved method of inhibiting the formation of d~ntal plaque and gingivitis ;.

The present invention also provides an oral cornposition which inhibits the transformation of amorphous calcium ~hosphate to the hydroxyapatite crystal structure normally associated wîth dental calculus.

The present invention further provides an improved method of inhibiting the ; :.
formation of dental calculus and an improved method forthe reduction of undesirable dental ~ :
plaque andlor gingivitis.
-BRIEF DESCRIPTION OF THE DRAWINGS
.
Figure 1 shows the inhibition of hydroxyapatite formation as observed by a : .:
decrease in the amount of titrant consumed in the presence of various concentrations of ethylenediaminetetramethylene-phosphonic acid (EDTMP).

Figure 2 shuws the i nhibition of hydroxyapatite formation as observed by a decrease in the amount of titrant consumed in the presence of various concentrations c~f 1,4,7,1 ~tétraa~acyclododecane-1,4,7, 1 O-tetramethylenephosphonic acid (DOTMP).
Figure 3 shows the i nhibition of hydroxyapatite formation as observed by a ;:
decrease in the amc~untof titrant consumed in the presence of 3.3 x 10 5 M DOTMP, hydroxyethyiidenediphosphonic acid (HEDP), EDTMP, ~W(~93/117~1 212~S76 PCr/lJS92/10897 norbornanediaminetetramethylenephosphonic acid (NDATMP), or ~.
dicyclopentadienediaminetetramethylenephosphonic acid ~DCDATMP).
Fi gure 4 shows the inhi bition of hydroxyapatite formati on as observed by a decrease in the amount of titrant consumed in the presence of 3.3 x 10 5 M DOTMP over a 4 hour time period.

Figure 5 shows the inhibition of hydroxyapatite formation as observed ~y a decrease in the amount of titrant consumed in the presence of 2.71 x 10 5 M EDTMP, 3,6,9,15-tetraazabicyclol9 3.1]tetradeca-1-(15~
0 11,13-triene-3,6,~-trimethylenephosphonicacid (PCTMP)or ~ ;;
1,4,7-triazacyclononanetrimethylenephosphonic àcid (NOTMP) Figure 6 shows the inhibition of hydroxyapatite formation as observed by a decrease in the amount of titrant consumed in the presence of 2.73 x 10-~5 M DOTMP and .,!'.
1~ DOTMP pl us cetylpyridinium chloride (CPC) both presen~ at a 2.73 x 10-5 M concentration.

This invention relates to an oral composition containing in an orally acceptablevehicle an effective amount of of a cyclic alkylamine or cyclic amine as an anticalculus agent or as an antiplaque andlor antigingivitis. l~n "orally acc~ptable vehicle" means a medium in which an anticalculus or an antiplaque agent may be administered to the oral cavitywithout substantial harmful effects to the oral cavity surfaces. An 'effective amount" is that which will inhibit or reduce the formation of dental calculus, plaque and/or gingivitis in the oral cavity.

As bacterial plaque is a main etiological factor in gingivitis, periodontitis, tooth decay (dental caries~ and other dental a#ociated problems, the ability to control dental plaque 25 aids in preventing and/or controllinç~ gingivitis, periodontitis and dental caries. Thus, as used herein, "antiplaque~ means antiplaque andloranti9ingivi~isand/or antiperiodontitisand/or anticaries. In addition, as the volatile sulfur compounds associated with oral malodor are related to the gingival heaith, as well as being pr~duced by the putrefactive activity of microorganisms, as used herein, an antiplaque ag~entwill also aid in the control of oral ; ;;
30 malodor. ~

Cyclic alkylamines of the present invention are derivatives of bicycloheptacliene as ~;
given in Formula (I) ;' ' .
~ ~ ~

1'' ': ' .

WO 93/11741 2 1 2 5 5 7 6 P(~/IJS92/10~

N--CH2~CH2--N~ (I) or derivatives of dicyclopentadiene as given i n Formula (li) N--CH2~3CH2--N~

wherein each R is indr~pendently-H, Cl-Cc hydrocarbon radical, C2-Cc hydroxyalkyl or / X ~ ~ .
~ l ~ Z , :, Y

wherein Z is independently PO3H2, -COOH, -H, Cl-C18 alkyl or a physiologically acceptable salt of the acid radicals; and XandYareindependently-H,C~ hydrocarbon;

with the proviso that each cyclic alkylamine or cyclic amine contain at least two Z
groups which are -PO3~2, or a physiologically acceptable salt thereof A physiologically acceptable salt refers to the acid addition sal~s, or rnixtures thereof, of those bases which will 30 form a salt which does not callse a significant adverse physiological effqc~ when used in an orai product consistent with~good pharmac4togical practice Examples of suitable salts include those of the a3kali metal, alkaiine-earth metal and mixtures thereof.

Methods for preparing compounds of Formulae l-lll are krlown in the art and referencemadetheretoforthepurposeofthisinvention. Forexample,thecyclicalkylamines ~r of Formulae l and ll can be prepared from commerciaily available dicyclopentadiene (DCPD) and bicychheptadiene (BCHD). Electrophilic addition reactions are known to form the nitrile or dinitrile by reacting the double bonds of DCPD or BCHD with hydrogencyanide ~HCN). The ::
', ,.

WC~ 93/l 1741 21 2 5 5 7 6 PCl /US92/108~7 nitrile can then be hydrolyzed to form the carboxylic acid derivative. The DCPD or BCHD can also be reacted with HCN followed by reduction to obtain the bis methylamine derivative; this product in turn can be reacted with glycolonitrile in the presence of caustic to give the sodium salt of tetraacetic acid ofthe bisamine.

Cyclic amines of the present invention are macrocyclic amines of Formula (lil):

:

( R t) (III) ~N (c~12)~
":.., or Form~a (lV):
, R- N N R (IV) N _) I `
R

.
wherein each n is independently 2 or 3 and m is 3 to 6, inclusive, n and m being selected such that the total number ~f carbon and nitro~en atoms in the cyclic amine ring does not exceed 30 18; and each R is as defined her~inbefore. ;

Polyazamacrocycles useful as starting materials which can be derivatized to formthe cyclic aminophosphonates of Formula lll of the present invention include ~:
1,4,7-triazacy~:lononane, 1,4,7,10-tetraazacyclododecane, 1,4,8,11-tetraaza-cyclotetradecane, 1,4,7,1û,13-pentaazacyclopenta~ecane, 1,4,7,10,13,16-hexaa~acyclooctadecane, and the like 35 These polyazamacrocycles may be prepared by docurnented methods such as T.J. Atkins et al., J. Amer. Chem. Soc., 96, 22~8-70 (1974) and T.J. Richman et al., O~g. Synthesis, 58, 8~91S (1978) The preferred starting material, 1,4,7,10-tetraazacyclododecane, for pol~Jazamacrocycles c,f 9. .

: : .
'' ~

WO93/11741 ~1 2 5 5 76 PCI/US92/10~ .~

Fom1ula lll is commercially available. Derivatization of the polyazamacrocycles means the reaction of the amine with the appropriate reactive compound to give an R group as defined hereinbefore.

Polyazamacrocycles useful as starting materials for the cyclic aminophosphonatesof Formula IV of the present invention are prepared by the procedures set forth in U.S. Patent Application 07/805,551 filed December 10, 1991, filed on even date herewith and entitled ~Bicyclopolyazamacrocy~lophosphonic Acids, and Complexes and Conjugates Thereof for Use as Contrast Agents", by Garry Kiefer, Jamie Simon and Joseph R Garlich (Attorney Docket No. C-38,662), the disclosure of which is hereby incorporated by reference. In general, compounds of f ormula IV are synthesized by the following reaction scheme.

~, 2~
,,,;~, ~ ,;
'~

; ~:

~ ~

!
~ ,'.
~""', ~ ~

WO 93/11741 ` ' P~/US92/1~8g7 212~576 `

æ~
o < z z~ r ~

P <~Z ~ ~ i"' ~rz-~t~ zL~ ~
=~ ~ O
~ ~ P~
--æ--E~ ~E: , ~ :

U N
~ ~o,l ~.

U , ¢ ''~ ~ Z-~ , ~

~ U ~--Z ~ ~

J ~
~_ ~ X~
<~Z :~ ~ ~ ,:

~' ,''~

-11- ', WO93/11741 212S5 7 6 PCI/US92t10~

Aminophosphonic acids can be prepared by a number of known synthetic techniques. Of particular importance is the reaction of a compound containing at least one reactive amine hydrogen with a carbonyl compound (aldehyde or ketone) and phosphorous acid or derivative thereof as described in U.S. Patent 3,288,846, the disclosure of which is 5 hereby incorporated by reference, and clescribed by Moedritzer and Irani, J.Org. Chem., 31, 1603 (1966). For e%ample, ~nitrobenzyl ethylenediamine reacted with formaldehyde and phosphorous acid can be converted to the p-nitrobenzyl ethylenediaminetetramethylenephosphonic acid. Reduction of the nitro group would yield p-aminobenzyl ethylenediaminetetrarnethylenephosphonic acid. The preparation of cyclic 10 aminophosphonates of Formula lll of the present invention can also be found in U.S. Patent Application Serial No. 071284,876, the disclosure of which is hereby incorporated by reference.
In a preferred embodiment of the present invention, all the i~ substituents of Formulae l-IV are methylene phosphonic acid.

Carboxymethylation of the amines of the present invention may be performed by the method of Desreux using bromoacetic acid derivativesand a suitable i~ase iJ.F. Desreux, Inorg. Chem., 19,.1319-24 ~1980)1.

Methods for carboxyalkylating to give amine derivatives containing a carboxyalkyl group are well known, see, for example, U.S. Patent 3,726,912, the disclosure of 20 which is hereby incorporated by reference, as are the methods which give alkyl phosphonic and hydroxyalkyl substituents on the amine nitrogens, see, for example U.S. Patent 3,398,198, the disclosure of which is hereby incorporated by reference.
. . .
While not v~/ishing to be bound by theory, it is believed that the advantageous 25 results as in inhibiting calculusformation are obtained because the phosphomethylene amines t~
act as antinucleating agents inhibiting the formation of crystalline hydroxyapatite from amorphous hydroxyapatite.

Thecycdicalkylaminesorcyclkaminesofthepresentinventionaretypically presem in an oral composition in a concentration of from about O.S millimolar (mM) to about 20 mM, preferably in the range of about 1 rnM to about 10 mM, and more preferably about -.;;
t mM to about 2 mM.

In certain highly preferred forms ~theinvention, the oral compositions are substantially liquid in character, such as a mouthwash or rinse. In such a preparation the 35 vehicle can be water or is a water-alsohol mixture,~ a!though alcohol is not required. When alcohol is present, the weight ratio of water to alcohol is in the range of from about 1:1 to about 20: 1, preferabiy about 3:1 to 10:1 and~ more preferably about 4:1 to about 6:1. The '. '' WO 93/l 1741 2 i 2 5 ~i 7 ~ PCI /US92/10897 ~ .

total amount w~ter or water-alcohol mixture in this type of preparation istypically in the range of frorn about 70 percent to about 99.92 percent by weight of the preparation.

The pH of such liquid and other preparations of the invention is generally in the range of from 4.5 to 9 and typically from about 5.5 to about 8. The pH is preferably in the range of from about 6 to about 8.

A variety of other ingredients may be added to the dentifrices of ~he present invention. Thus, for example, prophyla~ic agents, polishing agents, soaps or detergents, ;
flavoring and sweetening agents, thickening agents and humectan~s may be included using 10 techniques which are known to the art.

In certain other desirable forms of this invention, the oral composition may be ;
substantial Iy solid or semisolid in charactçr, such as toothpowd~r, a dental ~ablet, a toothpaste, gel or dental cream. The vehicle of such solid or semisolid oral preparations generally contains added polishing material more fully described hereinafter. ~ -Representative prophylactic agents include supplemental caries-preventing materials such as sodium fluoride, stannous fluoride, potassi um fluoride, hexyl~mine hydrofluoride, myristylamine hydrofluoride, betaine fluoride, glycine pOta#ium fluoride, etc. ~;
A particularly preferred fluoride is sodium fluoride. Typically these prophylactic ag~nts ar~
present in sufficient concentrations so as to provide an available fiuoride ion concentration of up to about 2 percent by weight, and preferably in the range of about 0.~2 percent by weight, o~th~ dentifrice composition.
: ' Sui~able polishing agents indude, for example, abrasive materials such as 25 insoluble condensed phosphates such as calcium pyrophosphate, insoluble caleiurn polyphosphate (also known as calcium polymetaphosphate) and highly polymerized soclium ~ -polyphosphate: and water impervious cross-linked thermosetting resins. Other suit3ble -polishing agents will be obvious to those skilled in the art.

The polishing materlal is generally~present in the solid or pasty compositions in !weight concentrations of about 10 percentto about 99 percent. Preferably, it is present in amountsrangingfromabout20percenttoabout75percentintoothpaste,andfromabout 70 percent to about 99 percent i n tooth powder.
:
Soaps, detergents or surfa~an~s may also ~e employed in the present inventic~n to lowerthe surface tension to achieve increased prophylactic a :tion, assist in achieving . ; I
thorough and complete dispersion of the antiplaque agent and render the instant c ompositions more cosmetically acceptable. Suitable soaps include, for example, the soaps of ~;

WO 93/11741 - , PCI~/US92/10 highmolecularweightfattyacidssuchassodiumandpotassiumsoapsofmyristic,stearicor palmitic acids and fatty acids mixtures of palm oil and coconut oil. Typical synthetic detergents include alkyl sulfates and sul fonates having alkyl groups of from about 8 to about 18 carbon atoms, such as sodium lauryl sulfate, the sulfated fatty alcohols derived from coconut oil and 5 palm oil, etc. The soaps typically comprise up to about 5 percent by weight of the dentifrice composition An example of a particùlarly suitable nonionic surfactant is poly(oxyethylene), .
poly(oxpropylene) block polymers known as poloxamers and available, for example, under the trademark ~PLURONICS" (BASF Wyandotte Co, Parsippany, NJ) Another example of a particularly suitable nonionic surfactant is polyethylene oxide sorbitan esters, available for 10 example, under the trademark "lWEENS" (ICI American Inc., Wilmington, De.). Suitable aninoic surfactants include, for example, anionic surfactants produced from fatty acids and the amino acid sarcosine, such as N-lauroyl sarcosine, available for example, under the trademark ~ HAMPOSYL~ by W. R. Grace and Co. ~CT). Surfactants ~ypically comprise from about 0 05 ts about 5 percent by weight of the dentifrice composition.

Any suitable flavoring or sweetening material may also be employed Examples - $
of suitable flavoring constituents are flavoring oils, e.g., oil of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon and orange and methyl salicylate. Suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, APM (aspartylphenylalanine, methyl ester), saccharine and the like. Suitably, flavor and sweet~ning agents may together comprise from about 0.1 percent to 5 percent of the preparat~on :`

Toothpastes, creams and gels typical!y contain a natural or synth0tic thickener or gelli ng agent in proportions of about 0 1 percent to about 10 percent, preferably about 0.5 to 25 about S percent, by weight. Suitable gelling or thickening agents include for example, water--soluble salts of cellulose etherssuch 2s sodium carboxymethyl cellulose and sodium carboxymethyl hydroxy ethyl cellulose; natural gums such as gum karaya, gum arabic, and ~um tragacanth; and colloidal magnesium aluminurn silkate or finely divided silica.
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Suitable humectants which may be employed in compositions of the invention include glycerine, propylene glycol, sorbitol, polypropylene glycol and/or polyethylene glycol and other polyhydric alcohols. The humectants may comprise 10 to 90 percent by weight of the dentifrice composition. --It has also beén surprisingly found that in the presence of a cyclic alkylamine or cyclic amine of the present invention, the inclusion of a cationic antimicrobial compound in the dentifrice results in the antimicrobial compound being retained in the oral cavity for a longer period of time than in the absence of a cyclic alkylamine or cyclic amine. While not wishing to `:
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W{) 93~11741 ~ s PCr/US92/10897 212~76 -;;
be bound by theory, it is bel ieved that the cycl ic alkylamines and cyclic amines of the present , invention have an affinity for the tooth surface creating an anionic surface on the teeth to which cationic antimicrobial compoundsare beneficially attracted. The antimicrobial is ~,, `
therefore retai ned within the oral cavity for a longer period of time due to the electrostatic , 5 forces between the anionic tooth surface and the cationic antimicrobial, The abiiity of an antimicrobial compound to be retai ned and remai n active withi n the oral cavity to exer~ an ~;
antiplaque effort for a longer period of time is referred to as ~he "subs~antivity" of the compound, The terms "antimicrobial ~ and "antibacterial " as used herein refer to the abi lity of a cationic compound to inhibit the growth, reproduction or metabolism of microorganisms, The enhanced substantivity of the antimicrobial compound aids in the prevention of dental calculus by inhibiting microorganisms responsible for the initi31 formation of dental plaque. The inhibition of microorganisms will also reduce the amount o~ volatile sulfur '~
compounds produced by the putrefactive activity of the microorganisms, thus helping to control mouth malodor. It has also been surprisingly fc und that inclusion o~ an antimicrobial ";
does not affect the calculus inhi biting ability of the cyclic alkylamin~s or cyclic ami nes used in the dentifrices of the present invention. ~ r;~, Antimicrobial compoundswhich are particularly useful in dentifrice compositions of the present invention are compounds which contain an organic amine where the nitrosen is 20 capable of being positively charged in an aqueous environrnent,~referably organic amines which are capable of being protorlated in an aqueousenvir~nmentand quaternary ammoniumcompounds. ~, ,;, ;l Among the most common antibacterial quaternary ammvnium compounds used 25 in oral compositions are cetylpyridinium chloride, benzethonium chloride, also known as ,, Hyamine 1622 or di-isobutyl(phenoxy-ethoxyethyl~ dimethylbenzyl ammonium chloride) and sanguinarine. Antibacterial quaternary ammonium compounds useful in the present ~,, , invention include those represented bythe formula : ; ~ R3 ~ R~ X~

~-:
~ ` ~

WC~ 93/1 17~1 2 I 2 5 5 1 6 PCr/~JS9~/lOB `
. -~ , . , and formula _ _ , ~ ~ Rs ~ X (~

;
10 whereln R1 is a C8-C20 alkyl;
RZ is benzyl or C1-C12 alkyl;
;--R3 and R4 are independently a C1~C7 alkyl or -(CH2-CHOH-CH2-O),lH wherei n n is 1 to 6;
F~s is -H, a C1-C7 alkyl or -(CH2-CI lOH-CH2-O)nH wherein n is an integer,from 1 to 6;
and . . .
X- is chloride (Cl ), brornide ~Br ), iodide (I ~ or fluoride (f~ ion.
. .
The quaternary ammonium eompounds useful in the present invention are ;:
commercially available or may be obtained by ~h~se ~F ordinary s~ill in the art without undue ~;
experimentation. For example, they may ~e produced by reacting alkyl halides with ammonia . ::
or primary amines, or by reacting a tertiary amine, pyridine or pyridine derivative with an alkyl ^:
halide. See, for example, Zo!tewicz and Deady, Adv. Heter~cy~l. Chem" 22, 71-1~1 (1978); U.S.
Patents 2,446,792; 2,295,504 and 4,g94, 1 9g, the tea~hings of which are hereby incorporated by : ~:
reference.
:
", Other quatern~ry ammonium compounds which can be employed in dentifrices of the present invention includethe followlng~
Pyridinium chlorides contai ning alkylthiomethyl or alkoxymethy hydrophobic :
groups as disclosed byWeglowski et al., J. Phar. Sci., ~0, 91-85 (1991~, the disclosure of which is .
hereby incorporated by reference, the quaternary ammonium compounds having the formula . ~ -WO 93/11741 PCI/US~2/10897 212~57 6 .

,",.;, ~CH20CH2CH

N~ :.
CH2X 1 R6 X~

`' 'i;

wherein X is as clefined herein before and X' i5 oxygen or sulfur; and ~;
R6 is a C4-Cl6 alkyl or benzyl.

Quatemary ammonium compoundsthat are esters of betaine and fatty alcohols, asdisclosed by Linstedtetal.,AntimicrobialAgentsandChemother~py,39, 1949-1954(1990), 2~ the disclosure of which is hereby incorporated by reference, the quaternary ammonium ~;
compounds having the formula ~ ~ -:, .
(CH3)~N~-CH2C~O)ORt;

whereinR7isaCI~Clgallcyl; andphysiologicallyacceptablesaltsthereof. i~

Sanguinarine and sanguinaria, sanguinaria being an extract from the bloodroot plant Sanguina~ia candensis, the extract containing benzophenanthridine alk21Oids such as sanguinarine, chelerythrine, protopine, homochelidoni ne and physiologically acceptable salts thereof asdisclosed in U.S. Patents4,145,412and4,406,8~1,thedisclosuresof whichare `~ ~;
herebyincarporat~dbyr~ference. Sanguinariaisavailableindentifricesunderthetrademark Viadent~ brand sanguinaria;~ the major active ingredient sanguiharine chloride salt having the fbrmula~
- ,, :, j. .
`

. ~;
1 7~

,.,, :.

WO 93/1 1741 2 1 2 ~ ~ i 6 PCr/U~92/lQ;: ` :

o > '~

N ~

O o CH3 Cle \/ , ;: , ~" ,.~ .

Dodecyltrirnethylammonium bromide, benzyl dimethylstearylammonium ;~
chloride,cetylpyridiniurn chloride, N-tetradecyl~4-ethylpyridirlium chloride, sanguinaria and 5-amino-1,3,bis(2-ethyl-hexyl)~5-methylhexahydropyrimidine are pref~rred quaternary ;5 ammonium antibaçterial agents used in dentifrices of the present invention. .
.,i.., ;, Examples of antibacterial organic amines which can be protonated in aqueous . ; .
environments and are use~ul in dentifrices of the present invention indude the following: ,. . ' ., .',, ~.. .:
Morpholine compounds as disc!osed in U.S. Patent 4,894,221, the disslosure of ~ :
which is hereby incorporate by referen e, the morpholine compounds having the fcrmula t~H2 -~C~2 : ., / ~ :
- o ~N-R9 , . . :. , CH2~--CH2 : ,, ,'', : ~8 ;~;

wherein : :
, R8:is a Cg^C16 alkyl atthe 2~or:3:position of the morpholino riny;
. : : : ,:: ' .' ','.:
R9isaC2-C1Oalkylsubstitutedwith:ahydroxygroupatotherthanthe alpha^position;
. ...

^ 1 8- ~

: . ;~; .
. .,~, . .
.. . ~.

" ,.
.

~WO 93/11741 2 1 2 5 5 7 6 PCr/~JS92/10897 the surn of R8 and R9 being greater than or equal to 10 and preferably! 10-20; and physiologically acceptable salts thereof. ~:
Antibacterial secondary amines and amides as disclosed in J Antibacteri~l and Antifungal Agents, 17, 371 (1989), the disclosure of which i5 hereby incorporated by reference, wherein the antibacterial compounds have the following formula ,/, .
: .:

R 1 0-NH/\~ H ~ NH2 ;~
~.
'; "' wherein ~l is a C,0-Cl8 alkyl;

R 1 1 -NH /\~ H ~ N--R 1 1 ~ , ". . :, wherein each R1' is independently C8Hl7 or C,oH2l;
~,, .:
O s~

R13Ib-NH/\~NH~NCR13 ~
:

wherein Rl3 is a Cg-Cl7 alkyl;
3~ or : ,~
.:

,; ,.
û
1 1 .; ~, - : ` : 01 ~ NH~NCR13 -~ R 1 3G-NH : ~ ~

: ~ .
1 9 ~ , "~:, :~ .
:

WO g3/11741 PCl/US92/10~

wherein each R13 is independently C7H1s or CgH19; and physiologically acceptable salts ~;
thereof.
Diaikyl amines and N,N'-dialkylpolymethylene-diamines as disclosed in J.
~ntibacterialandAntifungalAg~nts, 17, 579 (1989), the disclosure of which is hereby incorporated by reference, having the formula Rl4-NH-Rl4 wherein each R14 j5 independently 4Ht7 or Cl2H25; or formula R1s-NH(cH2)nNH~ s .
whereln each R15 is independently a C7-C10 alkyl;
n is an integer from 2-5; and pharmaceutically acceptable s21ts thereof.

N'-Alkyl~ (2-aminoethyl)piperidine compounds as tlisclosed by Murata et al., J.
Pharm. Sci., 80, 2~28 (1991), the disclosure of which is hereby incorporated by reference, the compounds having the formula ;

R16-NH(CH2)2 wherein R16 isa Clo-c18 alkyl; and pharmac0utically aFceptable saltsthereof.
The ammonium compound 4-(2-propylenepentyl)-1-piperidinoethanol having the structure '' ' ~.' ~ , . ,~.
CH3CH~H2 \ /--\ ~ OH
CH-CH2 ~N ~ ~ ;
CH3CH2cH2 _ X- .
_ ,.,.
"

..:
-20~

'''~.;

iWO 93/117~1 212 5 5 7 6 PCT/US92/10897 wherein X is as defined hereinbefore. This antimicrobial is described in J. Periodontal Research, 18, 429-437 ~1983), as the Octapinal Tn brand 4-(2-propylenepentyl)- --1-piperidino~thanol (Ferrosan AB, Sweden). . ~ `
, ., .~
Alkyl-N-betaine in combination with an alkyl-N,N-dimethylamine oxide; the alkyl-N-betaine having the structure fH3 R17 N~ /\C00-l:H3 wherein R17 is a C10-cl8 alkyl;
15' the alkyl-N,N-dimethylamine having the structure fH3 ~ R

CH3 . :
:, wherein R18 is a C10-C18 alkyl;
-:
as disclosed in U.S. Patent 4,839,158, the disclosure of which is hereby incorporated by reference.

Other antimicrobial agentswhich can be employed in the dentifrices of the ` :`
30 presentinventioninciudebi~uanidessuchas`chlorhexidine(1,6-bis,~N5-(~chlorophenyl~
-N'biguanidolhexane; N'-(4-chlorobenzyl~N5-(2,4-~ichlorobenzyl)biguanide; ,' ~chlorophenyl biguanide; 4 chlorobenzylhydrylbiguanide;
.
N-3-lauroxypropyi-Ns-p-chlorobenzylbiguanide; 5,6-dichloro-~-guanidino ..
benzimidazole;N'-~chlorophenyl-Ns-laurylbig~3anide;

2~ .:

~ :, WO 93/11741 PCI/US92/10~ ~

and their non-toxic acid addition salts. Chlorhexidine being the preferred biguanide ; 1:
antimicrobial agent used in the dentifrices of the present invention.

The antibacterial agents are typically employed in amounts such that the oral product contains between about 0.001 percent and l 5 percent by weight of the agent.
Preferably, for desired levels of antiplaque effect, the finished oral product contains about `;
0.01 pèrcent to about 5 percent and most preferably about 0.025 percent to 1.0 percent by weight of the agent. `
:
The molar ratio of the qùaternary ammonium compound i n the oral composi tion "
1~ tothecyclicaminesofthepresentinventionispreferablyfrornabout5:1 toabout l:5, more ~:
preferably from about 3:1 to about 1:3, and rnost preferably about about 1:1.

The cyclic amines of Formula lll are the preferred arnines of th~ present invention to be used in oral compositions in csnjunction with c~tionic antimicrobial cc~mpounds. A
1~ preferred cyclic amine of Formula 111 is when n = 2, m = 4 and each P~ group i5 methylene phosphonic acid. The preferred cationic antimicrobial compound to be used in conjunction with the cyclic amine is cetylpyridinium chloride. .

The dentifrices of the present invention may also be in a kit form, the kit ;~
comprising in a first compartment an orally acceptable vehicle containing one or more cyclic alkylamines or cyclic amines and in a second cornpartment an orally acceptable vehicle containing one or more cationic antimicrobial compounds. When the dentifrice is in a kit form, the compounds in the separate compartments rnay be applied to th~ oral cavity sequentially or mixed prior to application. When ~pplied sequentially, it is preferred that the cyclicalkylamineorcyclicaminebeapplied~otheoral cavitypriortothecationicantimicrobial -compounds.
~'~
When mixing the cyclic alkylamine or cyclic amine with the cationic antimicrobial , .
compound prior to application to the oral cavi ty, it may be nec~ssary to i ncrease their concentration to accouni ~or diiution effects which carl occur upon mixing. Whether applying 30 the cyclic alkylamine or cyclic amine and cationic antimicrobial compound sequentially or mixir!g prior to juse, the eoncentration of the cyc!ic alkylamine, cydic amine and ~ationic antimicrobial compound to which the ora! cavity is exposed should be in the range given hereinbefore fortheir concentration in the final dentifrice product. for ease of use, it is desirable for the dentifrice to contain both the antirnicrobial and cyclic alkylamine or cyclic :;
35 amine in one composition.
. .
~', -~2- :
~ ,.

iwo93/117~ 2~57~ PCl/US~2/10897 A Yariety of other ingredients may be added to the separate compartment of a dentifrice kit, such as, p~lishing agents, soaps or detergents, flavoring and sweetening agents, :;
and the like as described hereinbefore.

Surprisingly, it has also been found that inclusion of a polyvalent metal ion such as,forexample,calcium(Ca2+),magnesium(Mg2~)or,mixturesthereof,intheoral composition with a cyclic amine and cationic antimicrobial compound does not interfere with ;~
the cationic antimicrob;al compound being retained within the orai cavity for a longer period of time than when using an oral composition containing only a cationic antimicrobial compound The inclusion o,f a rnetal ion:also does not interfere with the ability of the cyclic amine to inhibit calculus formation. The molar ratio of the metal ion to cyclic amine is preferablyfromabout5:1 to 1:5,morepreferablyfromabout3:1 to 1:3,andmostpreferably . ~
about 1:1. .
i ....
In a preferred embodiment of the present inventi'on, the cyclic amine is a cyclic 1~ amine of Formula lll wher~ n = 2, m = 4 and each R group is m~thylene phosphonic acid, the cationic antimicrobial compound is cetylpyridinium chloride and the metal ion is strontium (Sr2 + ), magnesium tMg2 + ), tin (Sn2 + ), zinc (Zn2 + ), calcium (Ca2 + ) or mixtures thereof Preferably, the metal ion is Sn ~ or CaZ +, more preferably the metal ion is Ca2 + .

In th~ practice of this invention, an oral composition accordi ng to this i nvention, ~;
such as a mouthwash containlng the cyclic amin~ or cyclic alkylamine and/or a cationic antimicrobial compound and/or a metal ion, may be prepared by unifying the components in conventional mannerand applied tothe te~th and gingiva. .;
~ .
The invention wlll~ be ~further clarified by a consideration of the following exampiesj which are intended to be purely exemplary of the present invention , : ~ ~ 30 ., .

~ 35 ~
....

, -~. .
: : ~ .. ,!, WO 93/11741 2 1~ a ~ ~ ~i PCr/US92/10~ ;

Abbreviations CPC = cetyipyridiumc~loride DOTMP = 1,4,7,1 0-tetraazacyclododecane- 1,4,7,1 0-tetra-methylenephosphonic acid PCTMP = 3,6,9,15-tetraazabicyclol9.3.1.]pentadeca- ~
-1(15),11,13-triene-3,6,9-trimethylenephosphonicacid ;~;

HAP = hydroxyapatite ~;

DCDATMP = dicyclopentadienediaminetetramethyiene-phosphonicacid `:

DOTMP - 1,4,7,10-tetraazacyclododecane-1,4,7,10- te~ramethylenephosphonic acid `~ :

DTPMP = diethylenetriaminepentamethylenephosphonic acid 15' ~ ;
EDTMP = ethylenediaminetetramethylenephosphonicacid HAP = hydroxyapatite -.
, HEDP = hydroxyethylidenediphosphonicacid `

NDATMP = norbornanediaminetetramethylenephosphonicacid PCIIVlP = 3,6,9,15-tetraazabicyclol9.3.11pentadeca--1(15),11,13-triene-3,6,~trimethylenephosphonicacid ~ ;~

NOTMP = 1,4,7-triazanonanetrimethylenephononicacid Preparation of Stock Solutions i :
The water used in preparing the stock solutions and in the follawing exampies was distilled, deionized water.
::
0.0045 M DOTMP. Astock solution of 0.0045 M 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra- ~ ;
methylenephosphonic acid (DOTMP) was prepared by adding 0.6168 ~ .0001 g of solid i",, ~

, ' ,, DOTMP to a 60 mL beaker and bringing into solution with the addition of 20 mL of water by the dropwise addition of 1.0 N sodiun7 solution which raised the pH o~ the solution to 7.4 This solution was'transferred quantitatively to a 250 mL volumetric flask and diluted to the mark with water, 0,0015 M DOTMP. Twenty mL of a 0,0045 M DOTMP stock sotution was loaded into a four oùnce jar and then diluted with 40 mL of water to produce a 0.0015 M DOTMP solution, 0.0045 M CPC, A 0,0045 M sol ution of cetylpyridi nium chloride (CPC) was prepared by addi ng , ~ ~ .
40mLcfwatertoa60mLbeakercontaining0,4026 ~ 0.0001 gCPC(Ald-ichChemical ~;
10 Company, Inc.). This solution was transferred quantitatively to a 250 mL volumetric flask and diluted to mark with water. The final pH of this solution was 7.

~5~. A four ounce jar was loaded with 20 mL of 0.0045 M CPC stock solution and . .
diluted with 40 mL of water to give a O.0015 M CK solution.

,9~, T~,venty rnL of a 0.0045 M DOTMP sol ution and 20 mL of a 0.0045 M CPC ~ ', '; ;, solution were lo,aded into a four ounce jar and then diluted with 20 mL of water to produc~ a , 0.0015 M solution. ' ,;, : :~
0.0015 M Chlorhexidi ne. A 100 mL beaker was tared and loaded with 0.0455 ~ .0001 9 of chlorhexidine (CHj (Aldrich; FW 505.46) and 60 mL water added to give a 0.0015 M ,, chlorhexidine solution at pH = 7.4.

0.0045 M PCTMP. A O.0045 M solution of 3,6,9,15-tetraazabicyclo-[9,3,1]-tetradeca--1(15),11,13-triene-N,N',N"-trimethylenephosphonic acid (PCTMP) was prepared by adding 25 0.0224 ~ 0.0001gofPCTMPtoa10mLvolumetr;cflaskanddilutingtomarkwithwater. The ; ~ pH of this solution was adjusted to 7.0 with~addition of a few drsps of 1.0 N NaOH.

0.0015 M PCTMPICPC. A 2.0 mL a!iquot of a O.0045~M stock solution of PCTMP was added to a four dram vial. To this vial was then added 2.~ mL of water and a 2.0 mL aliquot of 0.0045 M
3 ~ ~CPCstocksolution. Thefinal~concentrationof~thisfotmulationwasO.0015MPCTMPand "'~
0.0015 M CPC.

O.û015 M HEDP A 0.0045 M solution ~ HEDP was prepared by adding 40 m L of water to a 60 mL beaker containing 0.0618~ ~ ~ Q.Oû1 g of 60 percent active HEDP ~MAYO Chemical Co" FW
; 2~6). ~his solution was adj usted to pH 7.6 using few' drops~* 1.0 N sodium hydroxide. A 20 mL
35 ~ aliquot of the solution was removed and~placed in~a four oun~e jar where it was dil uted with 40 mL of water to produce a 0.0015 M HEDP~solution. The procedure for making solutions of , -... ......

WO 93/117~1 2 1 2 5 5 7 ~ PCl`JUS92/10~ ;

EDTMP, NDATMP, DCDATMP, DTPMP and NOTMP were done in a similar manner as fo~
DOTMP, PCTMP and HRDP. ~ ;~

Crvstal Growth Inhibition Determination ~ ;

The efficacy of the compositions of this invention in calculus prophylaxis was demonstrated by crystal growth inhibition tests. Inhibition of HAP formation was evaluated by a pH stat method, the procedure being as follows: ~ ;

To 50 mL of water in a 100 mL disposable beaker was added 2.0 mL of 0.1 M
10 phosphate solution (KH2PO4, adjus~ed to pH 7.4 with NaOH). An inhibitor, if desired, was added, typically t û to 30 microliters (~L) of a O.Og M solu tion The beaker was secured to an automatic titrator and stirring begun. A constant temperature water bath was raised around the beaker to keep the temperature of the reaction constant at 25C. After stirring lFor several minutestoallowfortemperatureequilibration,therunwasstar~edbytheadditionof2.0mL
of a 0.175 M calcium solution (CaCI2.2H2O, pH 7.4). A pH stat titration was immediately begun, keeping the pH at about 7.4 by the addition of 0.01 M NaOH when needed. The volume of :
titrantconsumedversustimeduringtherunwasrecorded. Runsweretypicallystoppedafter one hour.

An initial rise in titrant consumption was observed due to the formation of amorphous calcium phosphate. A later rise in titrant consumption (approximately 22 minutes in the control) was due to the forma~ion of hydroxyapati~e. The presence of inhibitors delays or prevents this second rise in titrant consumption.
'.' ~.,~
Preparation of Metal Chloride Solutions Several 0.004275 molar metal chloride solutions were prepared by adding the listed number of grams of the metal chlo rides given in Table I to 40 mL ~f water. ~

' ~' ' , . ..

W O 93~11741. s PCT/US92/108g7 212~76 ~;
TABLE I
PREPARATION OF METAL CHLORIDE SOLUTIONS
_ _ ~t. ~g) Solution Solthtiopc Metal Formula added to Fwa __ _ ___ _ __ 1 2 Calciwm CaC12 H2 0.0252 147.02 ~:
_ _ ., 3 4 Zinc ZnCl2 0.0234 136.28 __ ~ ~ _ 6 Iron* FeCl3 0.0278 162.22 7 8 Strontium SrC12:6H20 0.0456 266.62 ~_ .,, 9. 10 Tin Sn(II)Cl2:2H 0.0386 225.63 __ _ 11 12 Magnesium MgCl2:6H20 O.0348 203.31 , 13 14 Copper* CuCl2 0.0230 134.45 , : ~
aFW = ~ormula weight *Thes~ metals produced a cloudy solution when CPC
was added.
To prepa~e each solution listed in Table I (numbers 1-14), 40 mL of a 0.00a,275 M
metal chloride solution prepared as described above, was added to 40 mL of a 0.0045 M
DOTMP stock solution. The pH o~ this solution was adjujted to 7.4 with a few drvps of 1.0 N
sodium hydroxide solution and the solution then divided into two 4û mL aliquots. To one solution,20mLofwaterwasaddedtoproduceaO.0015molarconcentrationof l)C)TMP/.0014 25 molar concentration of metal. To the other 40 mL aliquo~, ~0 rnL of CPC stock solution (0.0045M)wasaddedtoproduc~a 1:0.95:1 ratioo~DOTMP:metal:CPCinsQlutionwitha 0.0û15 molar concentration of DOTMP and CPC. Thus, solutions 1, 3, 5, 7, 9, 1 1 and 13 contained metallDC)TMP in solution and solutions 2, ~, 6, 8, 1 O, 1~ and 14 contained metal/DOTMP/CPC in soluei;on.: ~The 0.004275 molar concentration of metal insured that a five 30 percent excess of DOTMP ligand was present when these solutiGns were mixed.

GlvcolYsis DH Test Asucrose solution was prepared by loading l.O g cf sucrose ~Imperial Pure Cane Sugar) into a 60 mL beaker~and then adding 20 mL of water. To this solution was added 8 0 ml ~;
of pooled~whole human saliva. The saliva was collected From donors who had been permi~ted to eat or drink anything prior to collection period, but had ~oregone any aral hygiene on the ~ ~ .
day of ~ollection. Prior ta the collection, each donor rinsed thelr mouth for thirty seconds with .......
; -27- - :
: ~
: : : : .

WO 93/11741 ~ 12 5: 5 7 6 PCl /US92/lOh approximately 30 mL water, and afterwaiting 5 minutes, began collecting saliva for 30-40 minutes, keeping the collected saliva on ice.

To the saliva/sucrose solution was added 1.0 mL of brain/heart infusion broth containingStreptococcusmutants(AmericanTypeCultureCollectionsNo.25175,ATCC)and 1.0mLof brain/heartinfusionbrothcontaining Streptococcussanguis (ATCC#10556). These ~;
cultures had been inoculated into 40 mL of broth and grown at 37C for sixteen hours prior to adding to the salivalsucrose solution. (Each broth contained approximately 60 million colony forming units atthetim~ of addition.) Aliquots of 0.75 mL of the above saliva/sucrose/bacterial solution was added to ~;
thetesttubes containing variousvvashed H~P suspension. Thesetesttubeswere capped and attached to a tube r~tator and placed in a 37C incub~tor for sixteen hours. Following this incubation period, the rotator was removed from the oven and allowed to cool to ambient temperature. The pH of the solutionswere checked with a pH meter using a pH electrode 5 calibrated with pH 4, 7 and 10 buffers.
ExamDle 1: Preventic n of bacterial adherence to HAP surfaces Hydroxyapatite disks, (7.5 x 2.7 mm), o~tained frorn Calcitek, Inc. were prepared ` ~;
by tying a short length of chromel wire around then and placing the other end of the wire into ~ :
20 a rubber stopper to allowthe disk to be suspended within a test tube. Separate disks wh~re then treated by placing separate disks into 2 mL of the foll~wing test solutions for two minutes with occasional mixing: water (control); 0.0015 Nl chlorhexidine; 0.0015 M cetylpyridinium chloride; 0.00l M DOTMP; DOTMP/CPC0.0015 M each; and 0.0015 M Dow Corning DC-5700, a 42 percent by weight suspension 3-(trimethoxysilylpropyldimethyloc~adecylammonium 25 chloride in methanol. The disks were than each washed by placing them into 2 mL of water, and the washing repeated a second time. ~ ~

Each of the six test samples were than placed into a test tube containing 8 mL of a 'f'~""
growth medium of brain-heart infusion broth with S p~rcent sucrose, the growth medium having been inoculated with 100 ~L of Streptococcusmutans, ATCC #25t25 (approximately ;;
5.5 x 107 colony forming units). The tubes~were then incu6ated at 37C. Each day for a week, :
t he sample disks were transferred to a new test tube ontai ning fresh medium and;S. Mutans.
After one week, the samples were taken out, rinsed by dipping into water, and s~ai ned with Butler Red Coat, a dental plaque clisclosing agent.
.
~5 :~ ~ ~
"
;, ,:, -2~-:'`' WO 93/11741 2 I 2 5 5 7 6 PCl /US92/11)897 ~he amount of plaque on each disk was determined visually and rated'according to the ~ollowin~ scale: ~
O = nopJaque ~:
1 = slightplaque 2 - moderatecoverase 3 = completecoverage,lightplaque 4 = complete coverage, heavy plaque.

The pla~ue rating For each of the test samples was as follows: ;

~ water(control) = 4;
chlorhexidine = 3;
CPC_ 2;
- DOTMP = 1;
DOTMP/CPC = 2.
The heaviest plaque growth was found on the control disk Formulations containing an antimicrobial agent (chlorhexidine, CP~, and DOTMP/CPC all showed a reduction i n plaque. The sample with DOTMP alone shows that the HAP surface has been modified so as to inhibit bacterial adherenc~ without the use of an antimicrobiai agent.

Example 2: Absorbence of DOTMP onto a HAP Surface A 0.3 mM solution of DOTMP at pH 7 was prepared and injected on an I IPLC (high performance liquid chromatography) system. The HPLC system was a Hamilton PR X10~ anion exchangecolumn(4.1 mmxSOmm)withO.016Msulfuricacidat1 mUminaseluent,Dionex 25 gradient pump, Dionex variable wavelength detector (set on 210 nm~ and a V :i PDP- 1 1 data collection system. The DOTMP at this concentration exhibited a peak with a 6.25 minute retention time and an area of 9.16.
. .;.. ~
A 13.45 g portion of a hydroxyapatite suspension (24.5 percent by weight 3~ suspension of hydroxyapatite ;in phosphate buffer from Sigma Chemical Co.l was washed with 20 m L of water and then mix~d with 20 mL of 0.3 mM DOTMP. After a contact time of about one minute, the hydroxyapatite was removed by fi!tràtion and the filtrate analyzed by HPLC
for DOTMP. The analysis showed that less than 0.7 area units could be attributed to DOTMP, indicating that 93 percent o~ the DOTMP lef~ the~solution to adhere tothe hydroxyapatite 35 surface , . .
.....
., ' ,.

,:

WO 93/11741 212 5 5 7 6 PCI/US92/108~ ~

Example 3: Absorbence of CaDOTMP onto a HAP surface .:- .
. . .
The calcium salt of DOTMP (1 mM Ca-DOTMP) was prepared by mixing DOTMP
andcalc;umchlorideinwa~erandadjustingthepHupto7,4with0.1NNaOH. At.1184gram portion of hydroxyapatite suspension as descri bed in Example 2 was washed with 12 m L of ~ ;
vvater and then resuspended in 10 mL of 1 mM Ca-DOTMP After stirring overnight (about ;
16 hours~ the suspension was filtered and the filtrate analyzed by HPLC dS described in Example 2. This analysis showed that 98.5 percent of the ~OTMP had come out of solution, :
presumable ontothe hydroxyapatite surface. ~ :

1~ The filtered hydroxyapatite with DOTMP attached to the surface was resuspended i n 10 mL of water with sti rri ng for 2 hours at room temperature. The suspension ~ t vvas then filtered and the filtrate analyzed by HPLC. This analysis showed ns evidence for any DOTMP in solution indicating the DOTMP was not coming off of the hydroxyapatite surface ~;
with a large volume of ~,vater wash. The results also show that the addition of calcium ions ;
15' does not interfere with the ability of DOTMP to at~ach to hydroxyapatite surfaces.
Example4: Reduction in adherence of salivary proteinsto hydroxyapatite in the presence of DOTMP : .
.,, -.:
Hydroxyapatite disks (7.5 x 2.7 mm, Calciteck, Inc.) were separately placed in glass vialsto which wasthen added either 2 mL of water (control) or ~ mL of 0.001 M DOTMP. The 20 vials were capped and placed on an end-over end rotator For 30 minutes. The supernatant was removedandthediskswashedthreetimeswith 1 mLwaterusedasawasheachtime. Into each vial was then added 2 mL of salivary supernatant and th~ vials capped and placed on an end-o~er-end rotator for 19 hours. The s~livary supernatant was obtained by centrifuging saliva, which had been collected from human volunteerswho had refrained from oral hygiene 25 after eatin~, at full speed on a IEC-HN-SII centrifuge (about 4,00û rpm). After the 19 hour period, the supernatant from each vial was gentiy removed and each disk washed gently for a brief time with 2 mL of water. The disks were then separately place in vials containing 1 mL of .-0.1 N NaOH and sonicated for 2 minutes A 200 11L aliquot of the supernatant from each vial was then mixed with 3.0 mL of water in a quartz cuvette and the absorbence measured at 30 600 nm ~water reference) for each sample.
, ~ I ; j~ i The control showed an average absorbence reading ~7 readings) of 0.0167 ~
absorbence units and the DOTMP exposed disk (~6 readings) 0.00588 absorbence units. Thus, ~i ;
the DOTMP treated disks showed a 2.84 fo!d reduction in the amount of sal ivary material 35 accumulated on the DOTMP treated disk versus a water treated disk.

-30- .

~, WO 93/11741 212 5 5 71~ PCI/US92/10897 Example 5: Substantivity of DOTMP/CPC versus CPC using hydroxyapatite powders.
As the surface portion of a tooth is composed of about 97 percent inorganic substances, about 1 percent organic substances and about 2 percent water, the inorganic substances being mainly hydroxyapatite, hydroxyapatite powder was used as a model for tooth enamel.

A 24.5 9 portion of a hydroxyapatite suspension (purchased as a 24.5 percent by weight suspension of HAP in phosphate buffer from the Sigma Chemical Co., St. Louis, MO) wasweighed out and washed with 3-3û mL portions of water to remove the phosphate buffer.
The solid was resuspended i n 60 mL of water to give an HP~P suspension in water of about 100 rng HAP/mL of water. In each of two test tubes was placed 100 11L of this suspension which :; .
contained 10 mg of solid HAP. Into tube 1 was placed 4.5 mL or 0.0015 M cetyl pyridinium chloride (CPC). Into tube 2 was placed 4.5 mL of a solution containing CPC at û.O015 M and . .~., DOTMP at 0.0015 M, pH 7.75. Both tubes wer~ capped and mixed by end-over~end rotation for ~ O minutes. After this 10 minute period a SOO ~L portion of each suspension was removed and filteredthroughaO.22micronsyringefilterintoacleanpolystyrenetesttube. A20011Laliquot of the filtrate was mixed with 3.0 mL of water in a quartz cuvette and the ultraviolét (UV) absorbence at 260 nanometerswas measured. The tubes containing the suspension were then put back on the rotator for an additional 20 minutes at which ti me they were sampled as .
before. The tubes were then put back on the rotator for an addltional 35 minutes and sampled ;
again. The UV absorbence of the filtered solutions at these times points as well at time zero, which isthe absorbence of the 0.0015 M solutions before exposure to the hydroxyapatite surface, are shown in Table ll.
TABLE I I
ADSORPTION OF CPC TO~ HYDROXYAPATITE OVER TIME -~
_ ~ . ~ , ~
U~ Absorbance ~ . ~
Compound ~ : ~ ~` Decrease ~ . :
O m~n.:~ 10 min. 30 min. 50 min. : ;1 - ~~ : . : .. , ~ ,~
1 cPc o . 3892 o . 3159: o . 3 108 o . 30B2 20, 8 2 DOTMP J~PC o . 3810 a . 2~10 O . 2718~ O . 2562 32 . 8 ~
' I i- ' ' ~ - -- ~ ~ -- - --, .........

These results show that the equimolar~addition of DOTMP to CPC gives a .
58 percene increase in the amount of CPCthat is absorbed onto the hydr~xyapatite.

To determine the retènti~on of CPC~on the HAP from repeated washing, the two treated HAP samples from above, which~ now contai n CPC loaded in~differing amounts, were isolated by centrifuging and discarding the supern-tan~ To each of the solids was added WO 93/11741 ~ 1 2 ~ 5 7 6 PCI /US92/10~

3.1 mLofwaterwhichrepresentsthefinalvolumebeforecentrifuging. Thetubeswere ~, agitated by end-over-end rotation for 10 mi nutes after which SOO }lL was removed and filtered ~ `
through a 0.22 micron syringe filter. A 200 ~lL portion of this filtrate was added to 3.0 mL of water in quartz cuvette and the solutions absorbence at 260 nanometers was measured . The 5 suspensions were then mixed end-over-end and samples taken as described above after an additional 20 and 45 minutes of mixing. The UV absorbence readings for this equilibrating wash of the two suspensions as a function of wash time is given in Table lll. Also shown in Table lll is the percentage of CPC calculated to be present on the hydroxyapatite that was found during these exposure times.
TABLE I I I ~ , LOSS OF CPC FROM HYl~ROXYAPATITE INTO A WATER WASH
Max. UV 10 Min. X of 30 Min. ~ of 75 Min. X of - Compound Absorb*Con~act Max. Contact ~ax. Contact Max. ::;
_ . _ ~, 1g CPC ~ . 13100 . 0415 32 (~ . ()435 33 . 0400 31 ~;
_ DOTMP/CPC 0.,17000.0146 9 0.0183 11 0.0175 10 ~ . _ , ~ .:, *This i~ the maximun UV ab~orbanoe ~xpected if 100% o~ -:
the CPC that waQ found attached to the hydroxyapatite :;:
came of ~ and went :into ~oluti on .
. . ~, 20 ~
From this washing experiment, it is clear that the CPC is washed off of the hydroxyapatite by water to a much smaller degree (68 percent reduction) in the case of the DOTMP/CPC treated hydroxyapatite versus CPC treat:ed alone. Letting the wash equilibrate for 30 and 75 minutes longer did not change these results. ~:
25. ExamPle 6: Substantivity of DOTMP/CPC verws CPC using hydroxyapatite spheroids (SHAP) Hydroxyapatite spheroids (SHAPj were purchased from BDH Chemicals Ltd, Poole, England. These were specially developed hydro? yapatite~in the form of mechanically stable, porous spheroidai particles~of unknown surface area. -Into each of two test tubes was~ placed~200 mg of SHAP. To tube 1 was added 2.0mLofO.ûO15MCPCandto~be2wasadded~2.0mLof3.0015MCPCwhichalsocontained 0.0015M DOTI~IP. Thetwotubesweremixedbyanend-over-end motionfor 10minutesand ~then centrifuged briefly to settle the~fine particles which arose after agitation. A 200 IlL
aliquot frorn each tube was thèn removed and;added to 3.0~mL of v~ater in a quartz cuvette.
35 The UV absorbence at 260 nanometers was then measured. ~ The tubes were then piaced back on the rotator and aliquo:ts removed~for measurin~ the UV absorben~e after 4 and 24 hours of ~; -32~
.

WO 93/1 1741 2 1 ~ 5 5 7 6 PCT/US92/10~97 mixing. The initial W absorbence of the 0.0015 M solutions and the solutions a~er exposure to SHAP as a function of time is shown in Table IV
TABLE IV
ADSORPTION OF CPC ONTO SPHEROIDAL HYDROXYAPATITE
_ __ I
UV Absorbence % of Compound r-~~~~~ I - CPC uMoles*
O min. 10 min. 4 hr~. 24 hrs. Bound _ _ _ ~ ~ , .
1 CPC Oc3747 0.3384 0.3316 0.3343 10.8 0.324 ...
_ . _ ~ . .
DOTMP/CPC 0.3832 0.2258 0.2377 0.2243 41.5 1.245 _ ~ _ ".
*Mioromoles of CPC ad~orbed onto 200 mg of Spheroidal `-Hydroxyapatite , ,, These results indicate that the use of DC)TMP at equimolar ratios to CPC causes a 1~ 3.84 fold increase in the amount o~ CPC that is adsorbed from solution by spheroidal hydroxyapatite as well as increased its affinity toward the hydroxyapatite surface relative to water washes.

To determine the retention of CPC on SHAP from repeated washing, each o~ the 20 two hydroxyapatite solids from above were transferred to a disposable polypropylene column equippedwithafr;t(Bio-RadLaboratories). Thesolutionwasallow~dtogravi~yfilterthrough the fritwhich holds back the solid spheroids. The lasttracesof solutionwere removed by blowing 10 mL of air through the tube which exp~ls most of the solution. The remainin3 hydroxyapatite was then exposed to 2.0 mL of water for 10 minutes by an end-over-end 2~ rotation. After 10 minutes, a 200 ~L aliquot of the supernatant was taken and added to 3.0 mL
of water in a quar~z cuvette. Th~ UV absorption was then determined for these two solutions whichcorrespondstothefirstwash,lOminutesexposuretime.Thespheroidcontaining ~
solutions were then mixed for an additional 4 hours, at which time another 200 IlL aliquot was ~ ~;
taken for an LIV measurernent. A third 20~ ~uL aliquot wastaken for UV measurement a1~er 30 24 hours of mixing. The spheroidal hydroxyapatite was then separated from the supernatant using the disposable colurnn method described above and placed in 2.0 ml of waterforthe sec~nd wash. The second wash was sampled as previously descri bed after rnixing for 10minutesandaftermixingfor4hours. Athirdwashwasalsoperformed,againusing~.OmL ~`-of water and a 10 minute exposure time The UV absorbence data frs~m these samples is shown 35 in Tabl~ y . . .
' ~., ,' ,. ...
'~'`"'';

WO 93/11741 212 5 !~ 7 6 pcr/uss2/1o: , TABLE V -:
DESORPTION OF CPC FROM SPHEROIDAL HAP AS A FUNCTION
OF TIME AND DEGREE OF WATER WASHING ~;
. _ _ _ ;~' 2nd ~a~h, M~ l~t ~ta~h, ë~po3ure Tia~e 3rd ABS if E!~cposure Time (ABS) ~ABS~ ~h, 10 Compouuld all CPC _ min~.

Soilzuebdil min./% 4 hrs /Z hrs./Z 10 40fhrMBa/Z ~(2~(sm)elr~ :
of Ma~. of Ma~.. of Ma~. of Ma~.
_ _ _ . , CPC O . 0404O . 0372/O . 0384/O . 03gO/ O, 0065/ O . oo36/ o/o 9~ . 1 95 . O 96 . 5 1 6 . 0 8 . 9 _ _ _ _~ __ DOTMP/CPC O . 1589O . 0222/O . 0274/O . 0367/ O . 0249/ O . 0341/ O . 0256/
_~ 14.0 17.2 23.1 15.7 21.5 15.5 *Thi~ is the maximun UV absorban¢e if all the CPC on the 1~ hydroxyapati'le wa~ de~orbed.

Theseresultsindicate~hattheuseof DOTMPatequimolarratiostoCPCcaused over a 10 fold ;ncrease in the amount of CPC retained on the spheroidal hydroxyapatite.

20 Example 7: Improved antimicrobial substantivity An 8 g sample of a HAP suspension (as described in Example 1) was weighed out ~:
and washed with 3-20 mL portions of water using a sintered ~lass filter funnel. The washed-solid HAP was then resuspended in ~0 mL of water to give a milky white suspension containing about 1 û0 mg solid HAP per rnL of suspension.
SamPle A :
One mL of this HAP suspension was placed in each of 2 test tubes. Two mL o~ a 0.0015 M CPC solution wasadded to tube 1 and 2.0 rnL of a solution composed of CPC
(0.0015 M~ and DOTMP (0.0015 M) at pH 7.44 was added to tube 2. The resulting suspensions 30 were mixed thoroughly for 10 minutes on an~end-over-end ro~ator, the tubes were then fentrifuged for 3 minutes and the supernatant removed. The solid HAP in each tubewas then resuspended in 3 mL of water by Yigorous agitation with a plastic pipette, the suspensions centrifuged and the supernatants discarded. The solid HAP in each tube was resuspended in 2.0 mL of water and a 50Q }IL aliquot from each suspension was removed and placed into ~ :.
35 separate tubes labeled 1A and 2A. The remaining 1.5 mL of each suspension was recentrifuged and the supernatant discarded.

34.

Iwo 93/11741 212 5 5 7 6 PCII/US92/10897 ~;

Sa m pl e B
The solid H~P remai ning in each tube after removing Sample A, was resuspended in 3.0 mL of water, centrifuged, and the supernatant discarded. The HAP solid was resuspend~d in 3.0 mL of water a second time, centrifuged, and the supernatant discarded 5 The HAP from each tube was then resuspended in 1.5 mL of water and a 0.5 mL aliquot removed and labeled as 1 B and 2B. The remaining 1.0 mL suspensions are centrifuged down and the supernatant discarded.

Sample C ;
The HAP remaining in each tube after the removal of Sample B was resuspended;~
in 3.0 mL of water, centrifuged, and the supernatant discarded. The HAP solid was resuspended in 3.0 mL Qf water a second time, centrifuged, and the supernatant discarded.
The resulting HAP in ~ach tube wasthen resuspended in 1 0 mLof water and a 0 5 rnL aliquot removed and labeled 1 C and 2C. The remaining O.S mL of suspension was diluted with 3.0 mL
of water, centrifuged, and the supernatant discarded.

samDle D i;
The HAP solid in each tube after the removal of Sample C was resuspended in .; -3.0 mL of water, centrifuged, and the supernatant discarcled. The HAP was ~hen resuspended 2~ in 0.5 mL of water and labeled as 1 D and 2D. ~ i The HAP sarnples labeled as ~A" received 3 mL of water wash, those labeled as ~B" rec~ived 11 mLofwaterwash,thoselabeled~as~C" received 18.5mLofwaterwash,and those labeled as ~ Dn received 25.5 mL of water wash. ' ;
Into each of the 8 tubes, each containing about 0.5 mL of HAP ~2S mg) suspensionwas added 0.5 mL of a 5 percent sucrose solution and 0.5 mL of saliva. The saliva was pooled 2 hours after breakfast from fouf human volunteers which had refrained from oral hygiene .. ..
aftereating. 1hetubeswereshakentomixthecontentsandthepHofeachsolutionmeasured using a pH meter. The tubes are then apped and placed in an end-over-end rotator at about 30 50C overnight. The next day (t _ 13 hours) the tubes were altowed to cool and the pH of each suspension was measured. The resl~lts are shown in Tab!e Vl. ~;

:
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:.................................................................................................. .. , '~

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;,, :.

W 0 93/11741 212 S 5 7 6 PCT/US92/10' ~ ;

TABLE VI
pH DROP AS A FUNCTION OF HAP WASHINGS :~.
Samp~e A Ssmple B Sample C Sample D
(3 mL wash) (ll mL wash) (~8.5 mL wash) (25.5 mL wash) . /, Compound _ _ _ _ _ . .
O 13 * O 13 * O 13 * O 13 JA :
hrs. hrs. hrs. hrs. hr~. hrs. hrs. hrs.
_ __ _ _ __ ____ _ ,:.' 1 CPC 7.62 7.63 ~.Ol 7~60 6~31 -1.29 7.69 6~0 -1.49 7.75 6.41 -1.34 :~
_ _ _ _ .. _ _ __ _ 2 DOTMP/CPC 7.52 7.49 0.03 7.58 7.64 0.06 7.55 7.70 0.15 7.58 7.62 0.04 _ _ _ _ _ _ __ *pH chang~ ;~

A drop in pH meansthatthere is littl~ or no inhibition of bacteria suchthatthe bacteria are able to n~etabolize the sucrose into organic acids, such as lactic acid, which then lowers the pH. The l~ck of a pH drop indicates that the bacteria are either dead or unable to 1 S metabolize sucrose which is a measure of how much CPC has been carried through the water washing on the hydroxyapatie solid.
, These results shc,w that the addition of DOTMP to a CPC solution causes more re~ention of CPC to hydroxyapatite which can then exert antimicrobial acti~Jity after longer 20 amounts of washing. This test also shows enhanced substantivity of antimicrobial activity when using DOTMP together with CPC. s Exam~leB ~ ~
:
To further determine the substantivity of various combinatiorls of cyclic 25 amines/CPClmetal ions, the following washing procedure of the hydro~yapatite was done prior to performing a glycolysis pH test:
~ .
TwentyfivemLofwaterwereaddedtoa60mLbeakercontaining 12.0gof ~;~
hydroxyapatite (HAP) in a buffer suspension (25~ percent by weight solits from Sigma Chernical Co.). The HAp suspension was~filtered through~a meclium;glass fitted filter to obtain a HAP ~;
filter cake. The H~P filter cake was washed a second time with an additional 25 mL of water. . ~ .
, ...
The whi~e soliq filter cake containinQ 3.0 9 of HAP without the b~ffer was resuspended with ~ -30.0 mL of water to produce a 3.0 9/30.0 mL or 100 mglmL suspension.
: . :
Two mL o~ the HAP suspension was trans~erred to each of several sterile-35 disposable polystyrene 5 mL test tubes labeled DI-~Dn (where n = number of test solutions).
Two mL of a test solution were then added to each test;tube. :

-36-~

.....
;~

WO 93/11741 2 1 2 5 5 7 6 PCl/US92/l~g7 .;
.. , . ~, ~.
The tubes con~ining the HAP and test solution were cappecl and attached to a Tube Rotator and rotated end-over-end to allow the test solutions to contact the ~IAP for a total of ten minutes.
:
A~ter mixing, the test ~ubes were placed in an Industrial Equipment Company ~IEC) model K centrifuge and spun at setting 25 (rnid-range) forten minutes. The tubes were removed and the liquid layer decanted. A macropipettor wasthen used to add 3.0 mL of water to each test tube containing the centrifuged hydroxyapati~e. The HAP solids were resuspended by vigorous i~and-out flowing action through the pipette. The tubes were again centrifuged at setting 25 forten minutes and the liquid lay~r decanted. Following the three milliliter wash step, the HAP solids were resuspencied in 2.0 mL of water to produce the original 1 ûO mg/mL suspension concentration. A 0.5 mL sample (containing 50 mg HAP) of this HAP suspension was removeri and placed in each of several S mL polystyrene test tube labeled A~--An. This sample A contains one fsurth of the original HAP suspension which has been washed with three milliliters of water, The remaining 1.5 mL in test tubes labeled D1--Dn were centrifuged for ten minutes, the test tubes removed, and the liquid layer decanted. Three milliliters of water were . .
added to these test tubes and the HAP solids resuspended/washed using disposable pi pettes. ` ~ ;
The tubes were centrifuged for ten minutes, the tubes removed and the liquid layer decanted.
20 An additional three milliliters of water were added to these tubes and the H~P solids resuspended/washeci by pipette. These tubes were again placed ;n the centrifuge and spun for ; ~ ~;
ten minutes. The tubes were removed, the liquid layer decanted and 1.5 mL of water added to : .
each tube. The HAP solids were resuspended to the original 100 mglmL cuncentration and a ~ ~ .
0.5 mL sample removed and placed i n each of several S mL polystyrene test tube labeied B1--Bn.
25 This sample B contained 50 mg HAP solids which had been treated with test solution and then :
washed with a total of eleven milliliters of water. ;;, . ~ ~
The procedure given above was repeated a third and fourth time to create a ; ~
seriesoftesttubeslabeledC~ nand D1-Dn.~TheCsamplescontained HAPsolidswhichhad : .
30 been treated with test solution and then washed with a total of 18.5 mL of water. The D
samples contained HAP solids which had been treated with the test solution and then washed ~ ;
with atotal of 2~5.5 mLwater.

J4 glycolysis pH test was then performed as described above by adding 0.75 mL ..
aliquots of the saliva/sucrose/bacterial mixture to~the test tubes labeled At--Dn, each containing 0.5 mL of the treated washed HAP suspension. ;~
', ..
~; . .

~ ~ -37 W093/11741 PCT/US92/10~
212~i76 Using the e~perimental procedure described above, the following compounds were tested for HAP substantivity: water (control); CPC; chlorhexidine (CH~; DOTMP; and DOTMP/CPC. All compounds being tested present as 0.0015 M aqueous solutions. The results from this trial are shown in Table Vil.
s The solution of DOTMP/CPC shows markedly better antirnicrobial actiYity after a series of washes than the other solutions tested.

TABLE VII
pH AS A FUNCTION OF HAP WASHINGS
.

Wa.~h (mL) ~ater CPC1 CH2 DoTMP3 CPC1 ? 3 . ___ , ~__ 3 5.0 7.17~08 4.977.08 . . = . . _ 11 4.94 5.62 5.0 5.0 7.1 .
18.5 4.g8 4.975.01 4.97 7.1 25.5 5.2 5.125.43 5.1 6.0 1CPC = cetylpyridinium chloride 2CH= chlorhexidine 3DoTMP = 1,4,7,10--~etr~azacyclododecane-1,4,7,10--tetramethylenepho~phonic acid ~, .
ExamPle 9 Usingthe experimental proceduredes~ribed in Example 8,thefollowing compounds were tested for HAP substantivity: water Icontrol); cetylpyridinium chloride (CPC);
25 DOTMP/CPC; and PCTMP/~PC The compounds were tested as 0.0015 M aqueous solutions.

The results frQm this trial are shown in Table Vll 1. The HAP suspension treatedwith 0.0015 M CPC showed antimicrobial activity after being washed with 3 mL of water, however the level of CPC dropped considerably after being washed with 1 1 mL of water. The HAP suspension treated with a one to~one solution of 0.û015 M DOTMP plus CPC formulation 30 remained an~imitrobial even after 25 mL of water washing. Likewise, the HAP suspension treated with a o,ne-to-one PCTMP~ plus CPC formulation remained antimicrobial through 25 mL
of water washing.

.
.
:.

W093/11741 PCT/US92/~0897 ~
2125~6 ~

TABLE VIII
pH AS A FUNCTION OF HAP WASHINGS
I _ i' s Volume of ~ DOTMP/ PCTNP
Wa~h (mL) Water CPC CPC~ /CPC~
~ _ _ _ . _ _ , _ 3 5~16 7.50 7.45 7.39 ::
_ _ _ 11 5.17 7.55 7.40 7.45 ~.
_ "
18.5 5~20 5.76 7.47 7~30 _ _ _ _ ..
25.5 5.24 5.64 7.31 6.g3 ~
*CPC = cetylpryidinium chloride; :
DOTMP - 1,4,7,10-tetraazacyclododecane~
- -1,4,7,10-tetramethylene-pho3phonic acid;
PCTMP = 3,6,g,15-tetrabicyclo--[9.3.1]-tetradeca -1S15),11,13-triene :
3,6,9-trimethylenephosphonic acid :
,:, .
ExamPle 10 .

Using the experimen~al procedure described in Example 8, the following ~.
compounds were tested for ~IAP substantivity: water (control); cetylpyridinium chloride (CPC); .. ~ :
DOTMP/CPC; Calcium-DOTMP (Ca-DOTMP); and Ca-DOTMP/CPC. The compounds being tested .
present as 0.0015 M aqueous solutions and the Ca present as a .0014 M ac~ueous solution.

The results from this trial are shown in Table IX The results show that . ~ ~
DOTMP/CPC and Ca-DOTMP/CPC retained substantially more an~imicrobial activity after a ~ `
series of washes than ttle other solutions tested, indicating that the presence of calcium does .:
not interfere with the CPC substantivity enhancing ability of DOTMP. ; `

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WO 93/11741 212 5 5 7 6 P~/US92/10897 :
. : .
Exampl e 1 1 ! .: `
Usingthe experimental procedure described in Example 8, the following . ~
compounds were tested for HAP substantivity: water (control); cetyipryidinium chloride (CPC); . .~;;
Sn-DOTMP; Sn-DC)TMP/CPC; Zn-DOTMP; and Zn-DOTMP/CPC. The compounds being tested ::
presentas0.0015Maqueoussolutionsandth~metalspresentas00014Maqueoussolutic)ns. ;;

The results from this trial are shown in Table X, Bo~h the Sn-DOTMP/CPC and Zn-DOTMPlCPC solutions displayed substantivity through a series of washes as measured by the ;;
retention of antimicrobial activity, with the Sn-DOTMP/CPC solution showing the greatest 10 Substantivity, lS ~
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Example 12 212 5 5 7 6 Usi ng the experimental procedure descri bed i n Example 8, the fol l owi ng ' compounds were tested for HAP substantivity: water (control); cetylpyridinium chloride (CPC);
Sr-DOTMP; Sr-DOTMP/CPC; and Mg-DOTMP/CPC. The compounds being tested present as 0,0015MaqueoussolutionsandthemetalspresentasO.0014Maqueoussolutions, , . , The results from this trial are shown in Table Xl, Both the Sr-DOTMP/CPC and ; ,~,, Mg-DOTMP/CPC sol uti ons showed substantivity through a series of washes as measured by the '~ "
retention of antimicrobial activity, :
~

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i W0 93JI 1741 212 5 5 7 6 PCJ'/U592/10897 Example 13 Using the experimental procedure described in Example 8, the following `:
compounds were tested for HAP substantivity: water (control~; cetylpyridinium chloride (CPC);
Ca-DOTMP/CPC; Sn-DOTMP/CPC; Zn-DOTMP/CPC; Sr-DOTMP/CPC; Mg-DC)TMP/CPC;
S Fe-DOTMP; and Cu-DOTMP. The compounds being tested present as 0.0015 M aqueous solutions and the metals present as 0.0014 M aqueous solutions. : ' ... .
,: . .
The results from this trial are shown in Xl1, Of the metal complexes tested withDOTMP and CPC, Sn has the best slJbstantivity as measured by the retention of antimicrobial :
activity, '.'',, :' . ~; . .
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' : ` .- ,, iwo 93/11741 212 5 5 7 ~ i P~/US92/10897 ExamPle 14 i.
An in-vivo study was conducted to determine the ability of the test formulationscontaining phytic acid to inhibit the experimental forrnation of gingivitis in Beagle dogs.

Purebred female Beagle dogs, 2-3 years old, with naturally occurring gingivitis were randomly divided into groups of four animals each. After 14 days of adaptation, the teeth of the dags were scaled to remove supragingival calculus and polished. One week ~ ;
foliowing the prophylaxes, during which time oral care was maintained by brushin~, a baseline gingivitis index was obtained as measured by the procedure of Loe, ~. Periodontal., 38, 610 10 (1967~ and Loe and Silness, Acta OdontScand, 21, 533 (1963). After the initial gingîvitis index reading, the teeth of each group were sprayed twice daily, five days per week, with approximately 10 mL of one o~ the following mouth rinses: ;

(A) cetylpyridium chloride (CPC);
(B) tin/DOTMPl~PC (Sn-DOTMP/CPC);
(C) calcium/DOTMP (Ca-DOTMP);
(D) DOTMP/CPC

The cetylpryidinium chloride and DOTMP were present at a concentration of approximately 1.5 millimolar (mM) and the metai ions, calcium and tin, at approximately ;1 20 1.4 mM.

The CPC solution was prepared by dissolving 1.096 g CPC in 30 mL of water. The solution was transfe~rred to a 2 0 liter volumetric flask and diluted to the mark with water. The ;
pH of the final solution was approximately S.9.

The Sn-DOTMPlCPC solution was prépared~by initially making separ~te solutions - ;
o~ Sn-DOTMP (0.447 9 stannous fluoride and 1.674 9 DOTMP in one liter) and CPC (1.096 9 in ~ ~
one liter). The DOTMP was suspended in 50 mL of water and~ brought into solution with the ~.
addition of 50 percent by weight todium hydroxide to bring the pH to about 7 prior to addi ng ~
tothe stannousfluoride solution. The Sn-DOTMP~and CPC solutionwere then combined to ; ~;
give approximately 1.4 mM Sn, 1.5 mM DOTMP and 1.5mM CPC.

TheCa-DOTMPsolutionwaspreparedby~issolvingO.419gcalciumchloride ;
dihyrate in 50 mL of water and transferring to a two liter volumetric flask. The DOTMP was prepared by~slJspending 1.674 g DOTMP in 50 mLof water and then adding 5~ percent by 35~ weight sodium hydroxide to b~ring the DOTMP Into solution. The DOTMP solution was then added to the two liter flask and diluted to the mark wlth water. The final pH was : :
approximately 7.4.

. .
.

~ -WO 93/11741 2 1 2 5 5 7 6 PCT/US92/10~

The DOTMP/CPC solution containç!d 1.096 g CPC and 1.674 9 DOTMP. The DOTMP
was prepared separately as described above, added to a two liter volumetric flask containing CPC dissolved in 30 mL of water, and then diluting to the mark with water. The final pH was approximately 7.66.

After four weeks of treatment, the gingival index was again measured. The results given in Table Xlll, showing the change in gingival index, show that the DOTMP/CPC
composition was the most effective composition of those ~ested for inhibiting deterioration of the gingival health. The Ca-DOTMP and Sn-DOTMP/CPC compositions gave a lower increase in the gingival index when compared to the cetylpyridinium chloride alone.

TABLE XIII
,., Formulation1 Change in Gingival Index o~er ~ week~ ~:
,,, , j CPC 0.528 _ Sn-DOTMP/CPC 0.312 _ _ , . ~ ,.
Ca-DOTMP 0.300 :.
,. ..
DOTMP/CPC 0.124 ~
:, 1CPC = cetylpyridinium chloride;
DOTMP = 1,4,7,10-tetraazadod~¢ane~
-1,4,7~10-tetramethylenephogphonic acid;
Sn = tin; and Ca = calcium , 25 ExamPle15 CompatibilityofCakiumwithDOTMPandCPC ;-To determine the compatibili~y of metal ions with DOTMP and CPC in solution, ;~15 pL inaemental amounts of a 0.01 M CaCI2 solution were added to 100 ~L of a sample cQntaining DOTMP, CaCI2 and CPC, each component at a concentration of 1.5 mM (pH 5.~5).
The solutions were mixed and observed for precip;tation. The results shown in Table XIV ~ ;:
indicate that at least five calcium ions are~ compatible with DOTMP/CPC mixtures at these concentrations with no vis;ble precipitate.

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-48~ ;, ~wo g3,l,7~l 2 1 2 S ~ 7 ~ PCT/US92/1~97 ' TABLE XIV
_ ,. .~", .

. Amount of Molar Ratio Ob~ervation CaC ~ DOTMP:Ca:CPC*

5O (initial 1:1:1 clear, ~olu~ion) water white _ _ .
1:2:1 clear, water white :~
1:3:1 clear~ .
water white .
1 n _.
,u ~5 1:4:1 clear, wat~r white _ , 1:5:1 clear, :
water white *DOTMP - 1,4,7 9 10 tetraazacyclo-d~deoane-1~4,7,10-tetra- .
methyl~nepho phoni~ aoid;
Ca = Calci~m;
CPC = cetylpyridinuum ¢hloride Exam~le 16 Using the above pH stat procedure as described for crystai growth inhib;tion determination under General Experiments, two inhibi~ors, ethylenediamine- ;
"
tetramethylenephosphonic acid (EDT~AP~ and 1,4,7,1~tetraazacyclodocecane -1 ,4,7,1~tetramethylenephosphonic acid (DOTMP) were each evaluated at various leveis ~nd compared to a control. Both inhibitors were prepared as 0.09 M solutions o~ the ammonium salts. Levels of EDTMP used were 10, 15, 20, and 30 ~uL. Levç!ls of DOTMP used were 4, 1 û, 15, ~ -20 ~L (20 ~L in 54 mL equals 3.3 X 10 5 M, or on ~ wei~ht basis, 14.5 pprn of EDT~P and ~ -18.3 ppm of DOTMP). As can b~ seen from the Figures 1 and 2, 4 IIL of DOTMP is equivalent to 10 ~uL of EDTMP.
~ .
Example 17 ! ~ Usin9theaboYeptlstatpro~edure,si~inhibitors,allat3,3X'lOsM(20~Lofa Q.09 M soiution in 54 mL) were ~ested againstwater as a control. They are, in order of ~ -increasing efficacy, HEt)P, NDATMP, EDTMP, DCDATMP, DTPMP and DOtMP As can be seen from Figures 2 and 3, DOTMP stands out as the best HAP scaie inhibitor.
~ -: 49 ,-, ~.

WO 93/11741 PCI/US92/10~. ~

Example 18 Using the above pH stat procedure, an extended run (4 hours) was rnade wi~h DOTMP at the 3.3 X 10 5 M level (20 ~lL of a 0.09 M solution in 54 rnL). As can be seen from Figure 4, there is no appreciable HAP formation even at 4 hours.

ExamPle 19 Using the above pl I stat procedure, the inhibitors EDTMP, PCTMP and NOTMP al I
at ~.71 x 10 5 M, were tested against water as a control. The results of this trial are shown in 10 Fi~ure 5.
Exarnple 20 :
Using the above pH stat procedure, the effect of adding an equal molar concentration of cetylpyridinium chloride (CPC)to the inhibitor DOTMP was determ;ned. As 15 can be seen from Figure 6, the addition of an equal molar amount of CPC to DC)TMP (both at 2.73 x 10 5 M~ did not substantially inhibit the ability of DOTMP (2 73 x 10 5 M) to prevent hydroxyapatite formation.

Example 21 Using the above pH stat procedur~, adding an equal molar amount of m~gnesium orcalcium iontoa3.3x 105 Msolutionof DOTMPdid notinhibittheabil;ltyof DOTMPto ;;
prevent hydroxyapatite formation.
, .
Example 22 ' -~5 An in-vivo study was conducted to determine the ability of formulations containing DOTMPto inhibitformation of experimental dental calculus in ~eagle dogs.
.:
Purebred femaie Beagle dogs, 2~3 years old, with naturally occurring gingivitis were rands:~mly divided into groups of four animals each. After 14 days of adaptation, the teeth of the dogs were scaled to remove supraglngival calculus and polished. One week fQllowing the prophylaxis, during which time oral care was rnaintained by brushing, a baseli ne calculus index measurement as measured by the procedure of H. Loe, J. PeriQdontal., 38, 610 (1967)wastaken. Afterthe initial caiculusindexreading,theteeth of eachgroupwere :~
sprayed twice daily, five d~ys perweek, with approximately 10 mL of one of the following mouth rinses: ;

. ~;
: ,:,.
-50~
"

YYO 93/11741 2 1 2 5 5 7 6 pcr/uss2/1o~97 (A) 0.0015 M cetylpyridinium chloride (CPC);
tB) 0 0015 M DOTMP, 0.015 M CPC (DOTMP/CPC);
(C,~ 0.0015 M stannous, 0.0l 5 M DOTMP (Sn-DOTMP~; or (D) 0.0015 M calcium, 0.015 M DOTMP ~Ca-DOTMP).

The CPC solution was prepared by dissolving 1.096 g of ce~ylpyridinium chloride in 2.0 liters of water.

The DOTMP/CPC solution was prepared by adding 1.674 g of DOTMP to 50 mL of water and dissolving with the addition of S0 percent by weight sodium hydroxide to rais~ the 10 pl I to abolJt 7Ø This solution was then added to 1.096 g of cetylpyridinium chlorid~ dissolved in 30 mL of water. This solution was then dilutQd to 2.0 lit~rs with the final pH being about 7.66.

To prepare the solutions containing th~ stannous or calcium, DOTMP was 15 prepared as above and add~d to 30 rnL of water containing 0.419 9 of calcium chloride dîhydrate or 0.477 9 stannous fluoride. The solu~ionswere then diluted to a total volume of 2 liters.

After four weeks of treatment, the calculus index was again measured. The results given in Table 1, show that the Ca-DOTMP composition was th2 most effective composition of those tested for preventing an increase in calculus index.
TABLE I
~ _ ~ :
. *Increase in Calculu~
FormulatlonIndesc over 4 week3 2s ( A ) CPC ~ ~0 . 295 __ _ _ ( B ) DOTMP ~CPC O . ~09 _ :
( C) Sn-DOTMP~ ~ 0:~.158 ___ ~ D ~ C--007 ~ ~ . 1 Z 7 .
; I *CP~ etylpyridinium chloride;
DOTMP ~ 4:, 7, 1 0-tetraazadodeane-4,7, 10-tetrame'ch~rlene phosphonie acid; Sn _ tin;: :Ca - oa~cium ~ : ~

::

WO 93/11741 21 2 ~ 5 7 6 P~/US92/ltO

Other embodiments of the invention wilt be apparent to those skilled in the art from a consicderation of this specification or practice of the invention disclosed herei n. It is intended that the specification and examples be consiclered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

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Claims (18)

WHAT IS CLAIMED IS:

1. An oral composition comprising an orally acceptable vehicle containing therein an effective amount of, as an antiplaque agent or an anticalculus agent, a cyclic alkylamine or a cyclic amine selected from one or more of the compounds represented by the Formulae I to IV:
(I) (II) (III) (IV) wherein each R is independently hydrogen, C1-C8 hydrocarbon radical, C2-C6 hydroxyalkyl or wherein Z is independently -PO3H2, -COOH, -H, or C1-C18 alkyl or a physiologically acceptable salts of the acid radicals; and X and Y are independently-H or C1-C3 hydrocarbon radicals, with the proviso that each cyclic alkylamine or cyclic amine contains at least two Z
groups which are -PO3H2 or a physiologically acceptable salt thereof;
each n is independently 2 or 3;
m is 3 to 6 inclusive; and with the proviso that n and m are selected such that the total number of carbon and nitrogen atoms in the cyclic amine ring does not exceed 18.

2. The oral composition of Claim 1 wherein the anticalculus or antiplaque agent is a cyclic alkylamine represented by Formula I.

3. The oral composition of Claim 1 wherein the anticalculus or antiplaque agent is a cyclic alkylamine represented by Formula II.

4. The oral composition of Claim 1 wherein the anticalculus or antiplaque agent is a cyclic amine represented by Formula III.

5. The oral composition of Claim 1 wherein the anticalculus or antiplaque agent is a cyclic amine represented by Formula IV.

6. The oral composition according to any of Claims 1 to 5 wherein each R is a methylenephosphonic acid or a physiologically acceptable salt thereof.

7. The oral composition according to Claim 1 or 4 wherein the cyclic amine is 1,4,7,10-tetraaza-cyclododecane 1,4,7,10-tetramethylenephosphonic acid or a physiologically acceptable salt thereof.

8. The oral composition according to any one of Claims t to 7 wherein the anticalculus or antiplaque agent is present in a concentration from 0.5 millimolar to 20 millimolar.

9. The oral composition of Claim 8 containing an anticalculus or an antiplaque agent wherein the oral composition further contains from 0.001 to 15 percent by weight of one or more cationic antimicrobial compounds.

10. The oral composition of Claim 9 wherein the cationic antimicrobial compound is chlorhexidine, sanguinarine, a quaternary ammonium compound represented by one or more of Formulae V or VI:
(V), (VI), WO 93/11741 PCT/US92/10?

or a mixture thereof;
wherein R1 is a C8-C20 alkyl;
R2 is benzyl or C1-C12 alkyl;
R3 and R4 are independently a C1-C7 alkyl or -(CH2-CHOH-CH2-O)nH
wherein n is an integer from 1 to 6 inclusive;
R5 is -H, a C1-C7 alkyl or -(CH2-CHOH-CH2-O)nH wherein n is an integer from 1 to 6 inclusive; and X- is chloride, bromide, iodide or fluoride ion.

11. The oral composition of claim 10 wherein the quaternary ammonium compound is cetylpyridinium chloride.

12. The oral composition of according to any one of Claims 1 to 8 containing an anticalculus or antiplaque agent wherein the oral composition further contains a metal ion;
wherein the metal ion is Sr2+, Mg2+, Sn2+, Zn2+, Ca2+ or mixtures thereof and the ratio of the metal ion to anticalculus or antiplaque agent is from 5:1 to 1:5.

13. The oral composition of according to any one of Claims 9, 10 or 11 containing (a) an anticalculus or antiplaque agent; (b) an antimicrobial compound; and (c) a metal ion, wherein the metal ion is Sr2+, Mg2+, Sn2+, Zn2+, Ca2+ or mixtures thereof and the ratio of the metal ion to anticalculus or antiplaque agent is from 5:1 to 1:5.

14. A method for inhibiting the formation of dental plaque comprising administering to mammalian teeth a plaque inhibiting amount of an oral composition containing an antiplaque agent according to any one of Claims 1 to 13.

15. A method for inhibiting the formation of dental calculus comprising administering to mammalian teeth a calculus inhibiting amount of an oral composition containing an anticalculus agent according to any one of Claims 1 to 13.

16. The use of a cyclic alkylamine or a cyclic amine represented by one or more of the compounds of Formulae I to IV as defined in Claim 1 for the manufacture of an oral composition for the treatment of dental calculus or dental plaque.

17. A kit for inhibiting the formation of dental calculus or dental plaque comprising in a first compartment an orally acceptable vehicle containing one or more cyclic alkylamines or cyclic amines as claimed in any one of Claims 1 to 8 and in a second compartment an orally acceptable vehicle containing one or more cationic antimicrobial compounds as claimed in any one of Claims 8 to 11.

18. A commercial package comprising as an active ingredient one or more anticalculus or antiplaque agents as claimed in any one of Claims 1 to 8, together with instructions for the use thereof for inhibiting the formation of calculus or plaque in an oral cavity.