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United States Patent (19) 11) Patent Number: 4,767,615 Geho et al. 45 Date of Patent: "Aug. 30, 1988

54; DENTAL THERAPY BY VESICLEDELIVERY 4,603,044 7/1986 Geho ...... 514/3 (75) Inventors: W. Blair Geho; Joseph Jacob; John R. OTHER PUBLICATIONS Lau, all of Wooster, Ohio Accepted Dental Therapeutics, 38th Ed., American Den 73) Assignee: Technology Unlimited, Inc., Wooster, tal Association, Chicago, Ill. 1979, p. 343. Ohio Jones et al. in Chemical Week, McGraw-Hill Inc., Jul. *) Notice: The portion of the term of this patent 30, 1986, pp. 426-5129, "Liposome Research: New Path subsequent to Jul. 29, 2003 has been for '. disclaimed. Primary Examiner-Ronald W. Griffin (21) Appl. No.: 877,862 Assistant Examiner-F. T. Moezie (22 Filed: Jun. 24, 1986 Attorney, Agent, or Firm-Frijouf, Rust & Pyle 57 ABSTRACT Related U.S. Application Data A known procedure is used to prepare liposomes of 63 Continuation-in-part of Ser. No. 606,714, May 3, 1984, bipolar membrane which are permeable and hence Pat. No. 4,603,044. "leak' their contents at a rate which is variable by 51) Int. Cl...... A61K 7/16 choice. The liposome is supplied with or 52 U.S. Cl...... 424/57; 424/49; cosmetic material for the oral cavity, or specifically for 424/54 the teeth and gums. The liposome is then attached to a 58) Field of Search ...... 424/19, 21, 22, 27, molecule that has affinity for the hydroxyapatite. Thus, 424/28, 38, 49, 54, 57 the liposome will bind to oral cavity hydroxyapatite and (56) References Cited bathe the surrounding support surface with its contents for extended hours of service. U.S. PATENT DOCUMENTS 4,483,929 11/1984 Szaka ...... 435/7 2 Claims, 5 Drawing Sheets U.S. Patent Aug. 30, 1988 Sheet 1 of 5 4,767,615

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U.S. Patent Aug. 30, 1988 Sheet 4 of 5 4,767,615

Wash out of Vesicles Bound to Hydroxyapatite

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seLosen go LuIOg upM WH go u009 unrog esee easoonte-Owl log quino eAkaelruno LeoL 4,767,615 1 2 frequency sound waves, to give a dispersion of closed DENTAL THERAPY BY WESCLE DELIVERY liposomes that are quite uniform in size. There are other methods of forming such liposomes, and one specific CROSS REFERENCE TO RELATED recommended procedure is set forth in the specification APPLICATION hereinafter. This invention is a continuation-in-part of application Molecules, such as sodium fluoride for dental ther Ser. No. 606,714, filed May 3, 1984, now issued as U.S. apy, can be trapped in the aqueous compartment of Pat. No. 4,603,044. liposomes by forming them in the presence of these substances. For example, if liposomes as small as 500 A BACKGROUND OF THE INVENTION O in diameter are formed in a 0.1M glycine , 1. Field of the Invention Stryer states that about 2000 molecules of glycine will A chemically-structured delivery system for target be trapped in each inner aqueous compartment. This ing liposomes containing medication to the tooth struc manner of packaging oral cavity enhancement chemi ture of the oral cavity. cals is the first step of the present invention. 2. Description of Prior Art 15 The of the polyphosphoinositides and A general background for understanding the chemi the diphosphonates as noted in the scientific literature cal process steps that go into making vesicles and lipo demonstrates that these molecules are capable of partic somes is set forth clearly in a publication "Biochemis ipating in chemical reactions that result in the formation try” by Lubert Stryer, published by W. H. Freeman and of exceptionally strong coordination complexes with Company, San Francisco, Calif., U.S.A., copyright 20 the calcium ions of the hydroxyapatite crystal over a 1981. very broad pH range. The repertoire of membrane is extensive, and Stryer states they may even be bewildering, but they do SUMMARY OF THE INVENTION possess a critical common structural theme in that mem brane lipids contain both a hydrophilic and hydropho 25 Lipid vesicles, otherwise known as liposomes, are bic moiety. envelopes having, in part, a lipophilic membrane. Basi A space-filling model of a typical lipid has a general cally, the vesicle walls are composed of bipolar mole shape roughly rectangular with two fatty acid chains cules having a lipophilic end and a hydrophilic end. approximately parallel to one another and a hydrophilic These molecules are intertwined with the hydrophilic moiety pointing in the opposite direction. 30 ends forming inner and outer walls with the lipophilic It is common practice to use a short hand illustration ends sandwiched therebetween. which has been adopted to represent these membrane This invention employes vesicles whose membrane is lipids. The hydrophilic unit called the polar head group permeable and contain entrapped chemicals useful for is represented by a circle and the hydrocarbon tails are oral cavity enhancement, such as fluorides, antiplaque represented by lines which may be straight or wavy. 35 materials and breath fresheners. Permeability is usually The polar head groups have affinity for water and the accomplished by the use of lysolecithin as a wall mem hydrocarbon tails avoid water and seek lipid media. A brane component. A full teaching of liposomal mem bi-molecular sheet, known also as a lipid bi-layer, is the branes containing lysolecithin is contained in the 1976 favored structure for most and glycolip addition of the Journal of Biochemistry wherein the ids in aqueous media. work of Takayuki Kitagawi, Keizo Inoue and Shoshichi The structure of a bi-molecular sheet is inherent in Nojima, department of chemistry, National Institute of the structure of lipid molecules. Their formation is a Health, Kamiosaki, Shinagawa-Ku, Tokyo, Japan, de rapid and spontaneous process in water. Hydrophobic scribing liposomes which have been prepared with lyso interaction is the major driving force for the formation , lecithin, dicetyl phosphate and cholesterol. of lipid bi-layers. It is important to the final construction 45 This report states that generally, lysolecithin incorpora of a targeted liposome that there are van der Waals tion decreases the effectiveness of the membranes as a attactive forces between the hydrocarbon tails. These barrier to glucose and made the membranes more 'os van der Waals forces favor close packing of the hydro motically fragile'. This terminology simply means that carbon tails, and also will accept the hydrocarbon moi by including lysolecithin a fault dislocation is produced ety of target molecules from an . 50 in the membrane wall, allowing the contents to leak Clustering of bipolar lipids is favored by the van der from the vesicle. The amount of the lysolecithin incor Waals attractive forces with the significant biological poration will decidedly influence the rate at which the consequence that they will tend to close on themselves vesicles will leak the contents. Relatively low concen so that there are no ends with exposed hydrocarbon trations of lysolecithin cause an increase in the permea chains and therefore result in the formation of a com 55 bility of the liposomes, this report states. These studies partment which is normally self sealing because a hole suggested that the induction of a change in the molecu in a bi-layer is energetically unfavorable. lar organization by lysolecithin molecules may cause However, if one of the lipid components of such a the permeability change. closed compartment has one R-group missing, there Since the work published by the National Institute of will be a fault dislocation which defeats the self sealing 60 Health in Tokyo, the manufacture of vesicles from to behavior and allows the contents of the liposome to leak tally non-leaking structure to those which quickly lose from the inner aqueous compartment. their contacts, is now fully developed and well known Therefore, as explained in the prior art and particu prior art. larly in the Stryer publication supra, liposomes are This invention provides a means whereby the perme aqueous compartments enclosed by a lipid bi-layer. 65 able liposome, with its cargo of oral cavity enhance They can be formed by suspending a suitable lipid, such ment material, anchored to tooth structure of the oral as phosphatidyl choline in an aqueous medium. This cavity in order that eating, drinking and normal saliva mixture is then sonicated, which is an agitation by high wash will not dislodge the vesicle. Keeping it in place 4,767,615 3 4. until the contents are fully expanded is the touchstone application of fluoride and , or by of this invention. incorporation such medication into and A long chain target molecule is composed having one . end lipophilic and the other end characterized by the The effectiveness of such procedures is the product ability to chemisorb with the surface of hydroxyapatite of concentration and time in contact with the treated crystals. The lipophilic end is caused to penetrate the areas. The dentist uses a very high concentration of the hydrophilic wall of the liposome and form weak van fluoride medication in the liquid or and washes the der Waals bonds characterized as a transient attraction, area free of excess material. The dentist thereafter ob with the lipophilic membrane. The hydrophilic end of tains a high degree of effectiveness without danger of the target molecule will then project from the liposome. 10 adverse effect of ingesting the strong solution. The use The resultant composition when exposed to tooth or of dentifrice is a low concentration application repeated bone hydroxyapatite will cause an attempt by the hy often and is therefore safe for home application. drophilic end of the target molecule to form strong This invention addresses the growing needs of ex bident metalligands with the hydroxyapatite in a chem tended application time of the medical and cosmetic ical bond. 15 materials in contact with hydroxyapatite and provides a The normal chemical relationship of the chelating means of effectively treating peridontal disorders as hydrophilic end would be to form a chelate ring with well as those attendant to dental plaque and dental calcium, but because the calcium of tooth structure is a carries, with the goal of providing better dental health component of the hydroxyapatite, the attraction which care. The invention also addresses the problem of social anchors the hydrophilic end of the target molecule is 20 acceptance by eliminating breath and mouth odors. better characterized as chemisorption. The first step in the discovery of this invention was to The net result of this invention is that a permeable recognize the capability of incorporating the medical or liposome, having a core volume of an oral cavity en cosmetic material into a liposome, which liposome is hancement chemical, is attached to the tooth structure permeable to allow the material contained in the core by exposing the tooth to a wash or other carrier con 25 volume to leak slowly from the vesicle and provide a taining the targeted structure of this invention. continuous supply for an extended period of time. The manufacture and use of liposomes is now well DEFINITIONS known by organic chemists and researchers. Basically, a Vesicle-Substantially spherical thin walled bladder, liposome is created by sonication of polar lipid material. usually in a range of about 250A to 1500 A. 30 The liposome will trap a core volume of a water base Liposome-A larger spherical bladder, often of lay environment, or will carry lipid materials in the lipo ered walls, ranging from about 1000 A to several mi some membrane. CO. Generally, the components of the liposomes are mate For the purpose of this teaching, a target molecule rials such as L-distearoyl lecithin and cholesterol. Soni may be a chemical structure directly connected to a 35 cation causes the lipids to form into spheroidal configu liposome and having a hydrophilic moiety capable of ration. chemisorptive bonding to hydroxyapatite, or it may be The literature contains much teaching of the actual a composite (conjugate) molecule with two separate and proposed uses of liposomes. One structure germane molecules joined by a bridge, thereby establishing a to this present invention is a "leaky" membrane made lipophilic moiety and a hydrophilic moiety. by introducing reagents which cause fault dislocation. The work of Kitagawa; Inoue, and Nojima, "Properties DESCRIPTION OF THE DRAWINGS of Liposomal Membranes Containing Lysolecithin', J. FIG. 1 is a structural representation of a unilamellar Biochem, 79: 1123-1133 (1976), is an example. In this liposome carrying a core volume of radioactive trace prior work, liposomes were prepared with lysolecithin, material for delivery to tooth hydroxyapatite, and a 45 , dicetyl phosphate, and cholesterol. The target anchoring molecule linking the liposome to the ability to function as a barrier to the of glucose surface of a tooth. marker and the sensitivities of the liposomes to hypo FIG. 2 is a list of the sample codes, lipid constituents, tonic treatment and other reagents which modifies the weights in mg., sonication and annealing times and permeability were examined. Generally, lysolecithin temperatures and conditions under which the various 50 incorporation decreased the effectiveness of the mem vesicle preparations are made. branes as a barrier to glucose and made the membranes FIG. 3 illustrates the results of an experiment de more "osmotically fragile", i.e. permeable. Cholesterol signed to study liposome binding to hydroxyapatite. incorporation counteracted the effect of incorporated FIG. 4 depicts the results of an experiment designed lysolecithin. The more cholesterol incorporated into to wash away free or loosely held liposomes on the 55 liposomes, the more lysolecithin could be incorporated hydroxyapatite surface, and into the membrane without loss of function as a barrier. FIG. 5 illustrates that events that occur when lipo Therefor, it is known how to capture water soluble somes are bound to hydroxyapatite and allowed to leak substances within a leaky faulted liposome. This inven their core volume contents over time into the external tion is directed to attaching a leaky, or sustained release media. liposomes to the hydroxyapatite. See Kitagawa, Inoue and Nojima, supra. DESCRIPTION OF THE PREFERRED Using this type of vesicle it has been observed objec EMBODIMENT tively that the treatment materials adhered to the hy The primary object of this invention is to provide a droxyapatite for a period of time longer that could be sustained release mechanism for medical and cosmetic 65 expected of, for example, a deodorant. materials in the oral cavity of a warm-blooded animal. The present invention was conceived wherein the It is well known that treatment of dental carries and properties inherent in the unique molecular structure of peridontal problems is carried out in the dental office by phosphate compounds that belong to the classes of the 4,767,615 5 6 polyphosphoinositols and diphosphonates could be em such as the connector shown, can bond directly to the ployed to bind the vesicle to the hydroxyapatite for tooth surface, it is capable of producing unwanted lev increased time of exposure. els of tooth etching. Accordingly, a targeted vesicle delivery system has Accordingly, those who are skilled in the chemical been developed wherein selected phosphate com arts, having this teaching before them, may select from pounds and their derivatives are attached at one end to a class consisting of the diphosphonates, the class con the lipid vesicle membrane and the other end is avail sisting of the polyphosphoinositides, and the class con able to form strong bidentate metal ligands which result sisting carboxylic acids, as the preferred general classes in the formation of coordination complexes with the of compounds, those which have a moiety which is calcium of the hydroxyapatite lattice of bones and teeth. 10 lipophilic and a moiety which has affinity for the hy This attraction is known as chemisorption binding. droxyapatite. In this selection, those skilled in the art One of the important considerations related to the will be able to select various combinations having the preparation of the delivery system takes into account required characteristics, and join them by a chemical the fact that the hydroxyapatite of tooth enamel is ex bridge if desired as taught by the FIG. 1. Otherwise, posed in the oral cavity to the external environment and 15 direct binding is acceptable although in some instances thus facilitates the use of a topical vesicle drug delivery not as desirable as the combination shown in FIG. 1. system. To join the moiety by a chromium bridge, the lipid According to this invention, the polyphosphoinosi vesicles were collected and then, with respect to the tides, the diphosphonates and their derivatives have a initial concentration of vesicle connector molecules, moiety held to the lipid membrane of a liposome for 20 were reacted with a five-fold molar excess of CrCl3. targeting and subsequent binding of the liposomes to the The vesicles were then rechromatographed using the hydroxyapatite of tooth enamel. same buffer to remove unreacted CrCl3. The collected The vesicle delivery system utilizing, for example, vesicles were then reacted with a five-fold molar excess the membrane constituent L-aphosphatidyl inositol of connector molecules. Following this step the vesicles 4,5-diphosphate as a chelating agent for chemisorption 25 were then rechromatographed using the same buffer binding to hydroxyapatite, suggests a variety of new system to remove unreacted connector molecules. Fol therapeutic uses for this dental delivery system. lowing the final chromatography, the vesicles were Since the polyphosphoinositides are naturally occur stored under nitrogen in the refrigerator at 5° C. ring phospholipids with hydrophilic phospoinositol Because there is no known practical means of measur head groups and hydrophobic fatty acid tail groups, 30 ing the extent to which the present invention effectively they are uniquely suited for the incorporation into vesi delivers and anchors vesicles to the appetite of the oral cle membranes. cavity in vivo, applicant devised a means for establish FIG. 1 of the drawings illustrates what is considered ing the extent of the effectiveness of the present inven to be the preferred embodiment of this invention. tion. That is, the experiment will demonstrate the affin A general definition of the invention is the discovery 35 ity of the delivery system for lipids to the hydroxyapa that an osmotically fragile, i.e. permeable, liposome may tite. be attached to a tooth surface by provision of a mole cule having a moiety which is lipophilic and therefore DENTAL DELIVERY SYSTEM (DDS) held by van der Waals forces in the lipophilic mem PREPARATION brane of the liposome, and a moiety which is hydro The synthetic procedure for the preparation of the philic and has an affinity for the hydroxyapatite of a targeted dental and drug delivery system is described as tooth surface. Such a structure will bind to the tooth follows: surface for an extended period of time and thereby 28.96 mg of Ladistearoyl lecithin and 1.67 mg of permit the contents of the liposome to bathe the tooth cholesterol, for the formation of a bipolar vesicle, plus surface much more efficiently than any available tech 45 1.40 mg of L-aphosphatidylinositol-4,5-diphosphate, nique known prior to this invention. the target molecule, are solubilized in CHCl3. MeOH In the FIG. 1, a complex molecule is shown as the (2:1 v/v) and dried under house vacuum for 15 minutes preferred means to target the liposome to the tooth at 60° C.-0.5 C. to form a lipid crust. Following the surface. The connector is bi-polor with one moiety held drying procedure, 2.0 ml of 40 mM. KH2PO4-K2H by van der Waals attraction in the liposome lipid mem 50 PO4, pH 7.4, was added to the lipid crust. The lipid brane and the other end terminating in oxygen ions. constituents were then sonicated in the cuphorn at 60 Note, then, that the portion of the molecule labeled C.--0.5 C. for 15 minutes at setting #4 on the sonica "target' also terminates in oxygen ions which are tor. The sample was then annealed with slow turning at shown (...) attracted or bonded to the calcium ion of 60+0.5 C. for 15 minutes. Following the annealing the hydroxyapatite by chemisorption. The target also 55 step, the sample was centrifuged in the Triac Clinical has oxygen ions which are connected by bonding forces Centrifuge on the bloodsetting mode at ambient temper to a chromium bridge. The chromium bridge connects ature for 15 minutes. The supernatant containing the the oxygen ions of the connector and the target and lipid was chromatographed over a 1.5 therefor completes the structure. cmx25 cm Sephadex G-100-120 column that had been It is important to note that the connector L-aphos 60 equilibrated with 40 mM phosphate buffer, pH 7.4. The phatidylinositol 4,5-diphosphate could be connected by pooled vesicle fractions collected after chromatogra bonding directly to the hydroxyapatite without the phy comprised 4.6 ml and were designated as batch necessity of the target and bridge illustrated. As stated 250-E. herein above, the FIG. 1 is the preferred ideal structure This step by step procedure causes on inherent plac and the reason is that the selected target N,N,N',N', 65 ing the lipophilic part of the target substituent in the ethylene diamine tetra (methylene phosphoric acid), lipophilic membranes of the liposome with the hydro known as Editempa or Dequest produces a minimum philic head orientated in three-dimensional space ex etching of tooth surfaces. Although other molecules, tended away from the membrane surface. 4,767,615 7 8 CONTROL MATERIAL A control preparation, referred to hereinafter as Tube if 1 Tube i2 Tube i3 Tube #4 250C, was prepared as described for the DDS, except Clear St. Cloudy SAFE that no target material was supplied, i.e., material such 5 DDS control vesicles as the polyphosphoinositides or diphosphonates. TEST PROCEDURE The data of tubes 1-4 are interpreted as showing a complete binding of the DDS-250E to HA as evidence This test procedure was chosen to demonstrate the 10 by the clear supernatant in Tube #1. The lack of settling ability of the DDS to bind to hydroxyapatite. The vesi in Tube #2 indicates that the DDS 250E, which did not cle contents will leak out the medication, breath fresh have hydroxyapatite solution to bind to, is a stable col ener, of other content as taught by the prior art, and by loid that does not spontaneously settle. Tube #2 is a fixing these vesicles in place on the tooth surface, will control for Tube #1. be effective in bathing the tooth surfaces and gum tis 15 Tube #3 had a cloudy supernatant, indicating that the sues for any desired time period. Usually a 24-hour time control vesicles of batch 250C (the vesicles without period will be selected because a fresh supply will nor target molecules) did not bind efficiently to the HA. mally be presented through tooth brushing of mouth Close examination indicated, however, a weak binding wash at least once in each 24-hour period. of some vesicles. The partial settling of Tube #4 indi Although the DDS will have for its purpose to bind 20 cates that the vesicles without the target molecule is a to dental enamel in the mouth, the laboratory demon less stable colloid than the complete DDS 250E. stration of the ability of the DDS to bind hydroxyapa The conclusion is that the DDS-250E by virtue of the tite (HA) utilizes the binding of DDS to a fine aqueous L-aphosphatidylinositol-4,5-diphosphate target mole suspension of HA purchased from Sigma Chemical cule does efficiently bind the HA, which is the mineral Company, St. Louis, Miss. HA is the mineral compo 25 component of dental enamel. The important conclusion to be made from these nent of dental enamel. The HA suspension alone will observations is that a binding profile can be depicted settle out upon standing and leave a perfectly clear extending from the weak interaction of the control supernatant. The DDS preparations (250E and 250C) vesicles to the strong interaction with hydroxyapatite as are bipolar lipid vesicles that form colloidal suspen 30 evidenced by the experimental sample. The fact that sions. This experiment utilizes both of these attributes: there is weak vesicle adherence by hydroxyapatite, as clear, supernatant for HA alone and cloudy supernatant well as strong adherence, introduces the option of man for DDS alone. ufacturing vesicles that either bind weakly or strongly If the HA and DDS are combined the resultant super to hydroxyapatite, depending upon the type of vesicle natant, after permitting settling, can be used to indicate 35 that is needed. This binding is predicated on the number whether or not the DDS became bound to the HA. The and character of the functional target groups on the two resultant possibilities are: vesicle surface. 1. If the resultant supernatant is cloudy, then the HA The synthetic processes employed in the manufacture did not bind the DDS in a significant way. of targeted vesicles for dental drug delivery systems 2. If the resultant supernatant is clear, then the HA have been expanded hereafter to include all other pro bound all of the DDS. cedural variations that offer an array of targeting mech The experiment was designated to demonstrate the anisms which will selectively seek and bind to crystal enhanced DDS binding to HA when the L-aphos line hydroxyapatite surfaces such as are found in tooth phatidylinositol-4,5-diphosphate was used as a DDS enamel and bone. 45 These methods produce dental delivery systems with target molecule (preparation 250E) compared to a vesi maximal, as well as nominal, hydroxyapatite binding cle with no target molecule (250C). affinities. The experiment test was carried out as follows: The table, FIG. 2, is a list of the sample codes, lipid Four test tubes numbered 1-4 were used with num constituents, weights in mg., sonication and annealing bers 1 and 2 for DDS 250E. Tube numbers 3 and 4 were SO times and temperatures and conditions under which the used for the control vesicles labelled 250C. The addi various vesicle preparations are made. tions were according to the following table: Each of the vesicle preparations outlined in FIG. 2 is treated as follows to produce the final vesicle product. The lipid constituents are first solubilized in a solu DDS-250E Vesicles-250C 55 tion of Chloroform-methanol (2:1 v/v) and then dried 2 3 4. with slow rotation using a Buchi rotoevaporator and 1.0 ml buffer X X X X 2 drops HA X X accompanying waterbath at 60 C.E0.5' C. The lipids 2 drops buffer X X are dried under pump vacuum for 15 minutes before 0.5 ml 250E X X being transferred to a vacuum desiccator and further 0.5 ml 250C X X 60 dried for one hour at ambient temperature. Following the drying period each lipid crust is reconstituted with either an aqueous solution of glucose in water at a con The tubes were covered and stirred with small mag centration of 1 mg/ml or 10 mM phosphate buffer at netic stirring bars for 70 hours at room temperature (25 7.4. C.) to achieve binding equilibrium. When stirring 65 The lipid constituents are then sonicated in a cuphorn ceased, the tubes were then allowed to stand overnight sonicator powered by a Heat Systems Model W 200R to permit the HA to settle. The supernatants were then amplifier. The sonication and annealing procedures described: then proceed according to the schedule outlined in 4,767,615 10 Table I. After the annealing procedure the samples are phatidyl inositol-4,5-diphosphate appears to be interme centrifuged in a Triac Clinical Centrifuge on the blood diate between chromium-Dequest and phosphatidyl setting mode for 15 minutes at ambient temperature. glycerol in its binding affinity for hydroxyapatite. The supernatant is then chromatographed over a FIG. 4 depicts the results of an experiment designed freshly prepared 1.5 cmx25 cm Sepharose CL-2B-300 to wash away any free or loosely held vesicles on the column that has been equilibrated with 10 mM phos hydroxyapatite surface. Once again, the percent of 14C phate buffer at pH 7.4. Vesicle fractions are then evalu DSL that is bound is measured in the same manner as ated for their lipid concentration based on ultraviolet observed for the experiment illustrated in FIG. 3. light scattering and radiochemical analysis. Ultraviolet The vesicles with the L-aphosphatidyl inositol-4,5- light is not absorbed by lipid vesicle but it is scattered. 10 diphosphate groups on the vesicle surface bind to hy This refracted light shows up on a ultraviolet monitor as droxyapatite very well and near 100% capacity, even a light scattered signal which is subsequently recorded. after three consecutive equal volume washes with 10 The extent to which light is scattered is proportional to mM potassium phosphate buffer, pH 7.4. However, the the peak height on the recorder. In FIG. 1, the core vesicles with simply phosphatidyl glycerol on their volume is shown as carbon 14. This material is not to be 15 surface are washed off the hydroxyapatite rather rap included in commercial product, but is used and illus idly, as shown with the washout curve in FIG. 4. Only trated for test purposes. 9% of the original vesicles remain after three consecu RESULTS tive washes of the hydroxyapatite. FIG. 5 demonstrates the events that occur when The vesicle sample codes and their lipid constituents 20 are listed in FIG. 2 for easy reference to the following vesicles which contain L-aphosphatidyl inositol-4,5- figures: diphosphate are bound to hydroxyapatite and allowed FIG. 3 illustrates the results of an experiment that to leak their soluble C core volume contents over time was designed to study vesicle binding to hydroxyapatite into the external media. (H.A.) (Type III, Sigma). Along the abscissa of the 25 FIG. 3 shows that vesicles with the phosphatidyl graph, increasing levels of hydroxyapatite are used to inositol-4,5-diphosphate moiety bind convincingly to generate a hydroxyapatite crystal sink that is capable of hydroxyapatite. Thus, for the experiment shown in being saturated with a given vesicle preparation. The FIG. 5, it can be assumed that the phosphatidyl inositol degree of vesicle binding and subsequent hydroxyapa 4,5-diphosphate vesicles are bound substantially to the tite saturation is measured by incorporating a radiola 30 hydroxyapatite. The physical event which is concomi beled C-DSL constituent into the vesicle membrane at tantly observed after binding is the continual and cumu the time of synthesis and then comparing the amount of lative leakage of C-glucose from the core volume as a radiolabel bound to hydroxyapatite versus the amount function of time. of radiolabel that is free in the supernatant following the In a separate experiment, Sample Code #4, with centrifugation of hydroxyapatite crystals. The results 35 “C-glucose-DSL-CHOL L-aphosphatedyl inoselot graphed in FIG. 3 are expressed as a percentage of 14C --4,5-diphosphate was observed to bind to a single bound relative to the hydroxyapatite concentration. human tooth which was immersed in a vesicle suspen FIG. 3 shows that Sample Code #5, which contains sion for 15 minutes at ambient temperature. In this pre chromium and Dequest in addition to the L-aphosphati liminary experiment, 18.1% of the available vesicles dyl inositol-4,5-diphosphate group, has a greater bind bound to the crystalline surface of the tooth in 10 mM ing affinity at any given concentration of hydroxyapa phosphate buffer, pH 7.4. tite than does the phosphatidyl inositol-4,5-diphosphate Experimentally 50 pull of the stock vesicle preparation or the phosphatidylglycerol moiety. from Sample Code 1904 was added to 650 ul of 10 mM Phosphatidyl glycerol (PG) is also inserted in the potassium phosphate buffer, pH 7.4, to form the incuba vesicle membrane at the time of sonication, even though 45 tion medium. At the concentration of lipid vesicles used (PG) does not in this particular circumstance function in this experiment, it is likely that a vesicle as a connector molecule. However, it occupies the same was chemisorbed to the tooth, signaling that a maxi spatial or three-dimensional position as a connector mum level of vesicle saturation was achieved within the molecule. Phosphatidyl glycerol is an example of a parameters of the experiment. molecule that shows weak binding affinity to hydroxy 50 In summary, it can be concluded that maximal bind apatite at all concentrations of hydroxyapatite tested. It ing to hydroxyapatite is achieved with the Dequest can be concluded that Sample Code #1 with phosphati binding molecule, and that by altering the mole ratio of dylglycerol present in the vesicle membrane is a good lipid constituents in the vesicle membrane the core vol control vesicle with insignificant hydroxyapatite bind ume contents can be made to leak at designated and ing affinity. 55 variable rates. Intermediate between Sample Code #5 (Chromium What is claimed is: Dequest) and Sample Code #1 (Phosphatidylglycerol) 1. A composition of matter for prolonged oral admin is Sample Code #2 with L-aphosphatidyl inositol-4,5- istration to a warm blooded animal of dental therapy diphosphate present as the functional binding molecule and oral cavity breath freshener chemicals, comprising: occupying the connector molecule position. L-aphos 60 a first component which is a tooth structure protec phatidyl inositol-4,5-diphosphate has two opposed tive and therapeutic chemical or cosmetic breath phosphated groups in positions #4 and #5 on the inosi freshener, said first component being encapsulated tol ring structure that serve to bind to the crystalline in or associated with; lattice of hydroxyapatite. These phosphate groups can a second component which comprises lipid mem also bind to chromium ions. Furthermore, phosphatidyl 65 brane structures in the form of vesicles; and inositol-4,5-diphosphate is not capable of more than a third component which is a molecule having a fatty 86% binding capacity as defined by the parameters of substituent attached to the vesicle wall and a target the experimental results shown in FIG. 2. Thus, phos substituent selected from the class consisting of 4,767,615 11 12 chemicals which are classed biologically as having affinity for hydroxyapatite. atite attracted being selected from the class consisting of 2. The composition of matter as claimed in claim 1 diphosphonates, polyphosphoinositides and carboxylic wherein the third component target substituents are acids. chemicals which are classed biologically as hydroxyap 5

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