TECHNICAL FIELD

[0001] The present invention relates to an oral delivery composition, more particularly a solid dosage form, for delivering a therapeutic agent, e.g., an antibacterial agent, to a soft tissue in the oral cavity such as a periodontal pocket so as to treat an oral cavity related disease such as periodontal disease; and to a method of use.

BACKGROUND ART

[0002] Periodontal diseases are common ailment which affect high proportion of the population especially at advanced age. Gingivitis, often caused by inadequate oral hygiene, is the mildest form of a periodontal disease that causes the gingiva (or gums) to become red, swollen, and bleed easily. While gingivitis can be reversible with professional treatment and good oral home care, untreated gingivitis can advance to periodontitis. With time, plaque can spread and grow below the gum line. Toxins produced by the bacteria in plaque irritate the gums, and stimulate a chronic inflammatory response following which the tissues and bone supporting the teeth are broken down and destroyed. Consequently, gums separate from the teeth, forming pockets (spaces between the teeth and gums) that become infected. As the disease progresses, those pockets deepen and more gum tissue and bone are destroyed. Often, this destructive process has very mild symptoms. Eventually, teeth may become loose and might have to be removed.

[0003] In contrast to the tooth' s carries that might be rather effectively treated, periodontal diseases are more difficult to treat, inter alia, due to the markedly different environments of the oral and periodontal cavities. In particular, whereas the oral cavity is essentially an aerobic environment constantly perfused by saliva, the periodontal microenvironment is more anaerobic and perfused by a plasma filtrate known as the "crevicular fluid" . The growth of microorganisms within the periodontal microenvironment may cause periodontal disease, and as the periodontal disease becomes more established, said microenvironment becomes more anaerobic and the flow of crevicular fluid increases. The increased outward flow of said fluid prevents therapeutic agents placed within the oral cavity from entering the periodontal pocket, and has the effect of diluting and removing therapeutic agents placed within the periodontal crevice. Thus, antimicrobial agents introduced to the oral cavity are generally ineffective. [0004] Indeed, antibacterial agents such as chlorhexidine and quaternary ammonium salts, in the form of mouth rinses, have proved to be successful in preventing periodontal disease (Loe et ah, 1970). Yet, when administered in the form of mouth rinses, these agents have no effect on the subgingival flora as they do not penetrate into the pockets resulting from the disease. Moreover, oral usage of chlorhexidine, even for short periods of time, may have side effects such as tooth discoloration.

[0005] Oral systemic administration of antibiotics has been shown to be a useful method of controlling the subgingival flora (Listgarten et ah, 1978); however, while long-term therapy has the potential dangers associated with the development of resistant strains and super-imposed infections, discontinuation of therapy is often associated with the return of the potential pathogens to the pockets. In fact, due to side effects such as those of the digestive system, oral systematic administration has had only limited short term use, and variable success in treating periodontal disease (Genco, 1981).

[0006] Trying to overcome these difficulties, use of sustained release of drugs directly into the periodontal pocket has been tested, trying to achieve local drug concentration higher than that achieved by systematic administration or achievable by mouth rinse.

[0007] Goodson et al. (1979 and 1985) proposed the use of a device that could be placed within the periodontal pocket and provide a sustained release of an antibacterial agent, e.g., for a period of up to 10 days. Particular such devices comprise a drug incorporated into a polymeric matrix, which is then shaped into a convenient form and implanted into the periodontal cavity. US 4,175,326 describes the use of a drug-filled polymer hollow fiber. This delivery system is tied around a tooth and gently pressed below the margin of the gingiva such that it resides in the periodontal pocket, and is capable of delivering a dose of 2.5 micrograms tetracycline per day for a period of a week or more. Although these nonbiodegradable devices are capable of dispensing an appropriate ingredient for a time span of a week or more, they are inappropriate for widespread use as they are difficult to apply and in certain cases must be dislodged in an operative procedure carried out by a dentist.

[0008] Degradable polymers and copolymers that have been substantially investigated as potential implant compositions include poly(lactic acid) (Kulkarni et ah, 1966), poly(glygolic acid) (US 2,676,945), and poly(lactic acid)poly(glycolic acid) copolymer (US 3,397,033); and the properties and uses of such polymers and/or copolymers have been disclosed (FR 2,059,690 and FR 2,059,691; JP 72-43,220; US 3,642,003). For example, the biodegradation of poly(lactic acid) and poly(glycolic acid) may require three to five months (FR 1,478,694; Darkik, 1971), and it may thus not be advisable to employ implants composed of such polymers where a more rapid biodegradation is desired.

[0009] Absorbable periodontal implants have been described by Noguchi et al. (1984), which used a hydroxypropylcellulose polymer. US 4,569,837 discloses the use of water- soluble polymeric substances (such as methylcellulose, gelatin, etc.) as a polymeric matrix for a periodontal implant. Pharmaceutical compositions containing gelatin are disclosed in US 2,961,374, US 4,344,967 and US 5,002,769, as well as in WO 2011/001425.

[0010] An acrylic strip made of polyethylmethacrylic impregnated with metronidazole, chlorhexidine acetate, or tetracycline is described in Addy et al. (1982). A similar strip made of ethyl cellulose, for drug release, is disclosed in US 4,568,538. Ethylcellulose matrix polymer as periodontal implant was also described, e.g., in Friedman et al. (1982). Other non-biodegradable polymers tested for drug release for periodontal pockets include polyethylmethacrylate or biocompatible ethylene vinyl acetate (Goodson et al., 1983).

SUMMARY OF INVENTION

[0011] In one aspect, the present invention provides an oral delivery composition, said composition being in a solid dosage form comprising: (i) a matrix comprising a water insoluble biodegradable or bioerodible pharmaceutically acceptable polymer, low molecular weight hyaluronic acid or a salt thereof, and a cross-linking agent cross linking said polymer and said hyaluronic acid or salt thereof; (ii) a plasticizer; and (iii) a therapeutic agent, more specifically an antibacterial agent, wherein upon contact with a fluid, e.g., an aqueous fluid, said composition adsorbs said fluid and consequently swells, and then degrades and releases said therapeutic agent in a sustained release manner. In particular embodiments, said solid dosage form is an essentially two-dimensional (or practically flat three-dimensional) solid implant adapted for implantation in a periodontal pocket, e.g., a solid implant that is from about 3 to about 10 mm in length, from about 1 to about 5 mm in width, and from about 0.01 to 0.5 mm in thickness.

[0012] In another aspect, the present invention relates to a method for treatment of periodontal disease in a subject in need thereof comprising the step of implantation of an oral delivery composition as defined above (e.g., such a composition comprising an antibacterial agent), i.e., a solid dosage form, that is an essentially two-dimensional (or practically flat three-dimensional) solid implant adapted for implantation in a periodontal pocket of said subject, to thereby release said therapeutic agent in said periodontal pocket in a sustained release manner.

[0013] In a further aspect, the present invention relates to an oral delivery composition as defined above (e.g., such a composition comprising an antibacterial agent), i.e., a solid dosage form, that is an essentially two-dimensional (or practically flat three-dimensional) solid implant adapted for implantation in a periodontal pocket, for use in the treatment of periodntal disease.

BRIEF DESCRIPTION OF DRAWINGS

[0014] Fig. 1 shows the swelling of the commercial product (Periochip) vs. that of the Periodontal film shown in Study 1 , indicting that the initial swelling of the Periochip is faster, reaching more than 400 percent; however, it falls down within half an hour, whereas the swelling of the Periodontal film is around 240 percent and it stays constant.

[0015] Fig. 2 shows the changes (%) in the X dimension of the Periodontal film shown in Study 1 vs. Periochip in water, as measured at room temperature after incubation at 37°C, at different time points over 60 minutes in total. Each experiment was conducted in duplicates.

[0016] Fig. 3 shows the changes (%) in the Y dimension of the Periodontal film shown in Study 1 vs. Periochip in water, as measured at room temperature after incubation at 37°C, at different time points over 60 minutes in total. Each experiment was conducted in duplicates.

[0017] Fig. 4 shows the changes (%) in the Z dimension of the Periodontal film shown in Study 1 vs. Periochip in water, as measured at room temperature after incubation at 37°C, at different time points over 60 minutes in total. Each experiment was conducted in duplicates.

[0018] Fig. 5 schematically illustrates the swelling of the Periodontal film shown in Study 1 (middle and left side) vs. that of Periochip™ (right side), showing that Periodontal film swells in the z direction (top-bottom direction) substantially more than in the planar x and y (horizontal) directions, as opposed to the commercial product (Periochip) wherein swelling is substantially higher in the x-y horizontal directions. Left scheme represents a slice cut from the Periodontal film prepared (an essentially two-dimensional solid implant).

[0019] Fig. 6 schematically depicts a Periodontal film inside a periodontal pocket.

[0020] Figs. 7A-7B show AFM images (top tiew) of the composite film shown in Study 1 (7A, 400-700 nm features) and of the commercial product, Periochip (7B, 200-400 nm features). Both images cover area of 20x20 μηι.

[0021] Figs. 8A-8B are three-dimensional images showing deeper and wider clefts in the composite film shown in Study 1 (8A) compared to Periochip (8B). Both images cover area of 20x20 μηι. The larger features (400-700 nm) of the surface shown in the image of 9A are probably due to the different surface composite (hyaluronan enriched) of the periodontal film. The imaging was performed under ambient conditions.

[0022] Fig. 9 shows a three-dimensional image taken from the cut side (thickness) of the composite film shown in Study 1. Image area is of 20x20 μιη. Loss of detail was observed when scanning this sample site, probably due to the increased interaction between the instrument tip and the sample surface, which resulted from higher water content inside the film sample than on its surface.

[0023] Fig. 10 shows the total cumulative octenidine release (%) at different time points from the Periodontal film shown in Study 1 in a combined saliva mix, as measured at room temperature, after incubation at 37°C, over 24 hours in total. Two experiments (each in two duplicates) were conducted, each with a saliva mix obtained from several donors. As known from the literature, certain parameters of the saliva, e.g., viscosity, are different from individual to individual, and the work with different saliva mixtures led to a variance in the results measured reflected in the apparent decrease in the cumulative octenidine release shown. In addition, during the last measurements conducted it could be observed that the saliva mixtures were not homogeneous and were characterized by air bubbles and/or mucos- like bodies. Due to the presence of those, it was difficult to take samples for measurements having accurate and uniform volume, and the samples taken at the last measurement points thus varied in volume and had smaller volume than that of the samples taken at the first measurement points.

[0024] Fig. 11 shows the changes (%) in the X, Y and Z dimensions of <10kDa HA- and ~48kDa HA-based Periodontal films vs. Periochip™, in water, as measured at room temperature after 15-minute incubation period at 37°C. The results relating to the <10kDa HA-based Periodontal film and Periochip™ were taken as an average from Figs. 2-4, and the results relating to the ~48kDa HA-based Periodontal film are the average of a duplicate experiment with error bars representing the standard deviation.

[0025] Fig. 12 shows the total cumulative octenidine release (%) from the ~48kDa hyaluronic acid (HA)-based Periodontal film vs. the <10kDa HA-based Periodontal film, in a combined saliva mix, at different time points, as measured after incubation at 37°C over 24 hours in total. The results relating to the <10kDa HA-based Periodontal film are the average of those shown in Fig. 10, and the results relating to the ~48kDa HA-based Periodontal film are the average of a duplicate experiment. DETAILED DESCRIPTION

[0026] It has now been found, in accordance with the present invention, that a solid dosage form (referred to herein as an "oral delivary composition") comprising a gelatin matrix cross- linked via glutaraldehyde to a low molecular weight hyaluronan, e.g., hyaluronic acid having a molecular weight that is lower than 50000 Da, and a therapeutic agent, e.g., an antiseptic agent such as octenidine, upon contact with an aqueous fluid, adsorbs said fluid and swells, and consequently biodegrades and releases said therapeutic agent in a sustained release manner. As postulated by the inventors, the low molecular weight hyaluronan chains actually contribute to a particular and unexpected swelling pattern of the solid dosage form, which seems to render said dosage form highly advantageous when used in treatment of periodontal disease.

[0027] Such solid dosage forms can be cut as an essentially two-dimensional film for implantation into a periodontal pocket, wherein upon insertion into a periodontal pocket, said film adsorbs fluids in the inflated pocket and swells, and consequently biodegrades and releases the therapeutic agent in a sustained release manner to thereby treat the periodontal disease; and upon degradation of the polymeric matrix, said low molecular weight hyaluronan contribute to the healing process of the inflamed tissue of the dental pocket. The terms "periodontal pocket", "periodontal crevice", "gingival pocket", "gingival crevice", and "dental pocket", used herein interchangeably, refer to an abnormal space between the cervical enamel of a tooth and the overlying unattached gingiva, resulting from a chronic inflammatory response associated with untreated gingivitis or periodontitis, which leads to destruction and fracture of the tissues and bone supporting said tooth.

[0028] In one aspect, the present invention thus provides an oral delivery composition, said composition being in a solid dosage form comprising: (i) a matrix comprising a water insoluble biodegradable or bioerodible pharmaceutically acceptable polymer, low molecular weight hyaluronic acid (also called hyaluronan or hyaluronate) or a salt thereof, and a cross- linking agent cross linking said polymer and said hyaluronic acid or salt thereof (i.e., crosslinked to said polymer and said hyaluronic acid or salt thereof); (ii) a plasticizer; and (iii) a therapeutic agent, wherein upon contact with a fluid, e.g., an aqueous fluid, said composition adsorbs said fluid and consequently swells, and then degrades and releases said therapeutic agent and said hyaluronic acid in a sustained release manner. In a particular such aspect, the therapeutic agent comprised within the oral delivery composition of the invention is an antibacterial agent, also referred to herein as "antiseptic agent" or "disinfecting agent". [0029] The terms "sustained-release", "extended release" and "controlled release", used herein interchangeably, refers to the release of an active agent from a composition comprising it at predetermined intervals or gradually, in such a manner as to make the contained active agent available over an extended period of time, e.g., hours (e.g., up to 6, 12, 18, 24, 36, or 48 hours) or days. The release profile of the therapeutic agent from the oral delivery composition of the present invention depends on various parameters such as the particular biodegradable or bioerodible pharmaceutically acceptable polymer used, and its amount in the composition; the ratio (by weight) between said polymer and the hyaluronan; the extent to which said polymer is cross-linked; and the biodegradation or bioerosion rate of said composition. For example, a certain amount of the therapeutic agent (e.g., about 30%, 35%, 40%, 45%, 50%, 55% or 60%, by weight) might be released from the composition within the first several hours after swelling, e.g., within the first 4, 5, 6, 8, 10 or 12 hours after swelling, while the remainder of the therapeutic agent is slowly released afterwards due to the biodegradation or bioerosion of the composition, e.g., in a constant rate.

[0030] In order to provide a biodegradable or bioerodible polymeric matrix for the sustained release of the antibacterial agent, it is preferable to employ a polymeric matrix composed of cross- linked matrix polymer.

[0031] In certain embodiments, the water insoluble biodegradable or bioerodible pharmaceutically acceptable polymer comprised within the oral delivery composition of the invention is selected from polylactide (PLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), chitosan oligosaccharide (Mahima et ah, 2015), dextran, starch, alginic acid, carrageenan, heparin, hydroxyethylcellulose, or a combination thereof.

[0032] In other embodiments, the water insoluble biodegradable or bioerodible pharmaceutically acceptable polymer comprised within the oral delivery composition of the invention is a protein, more specifically a structural protein. Partiuclar such proteins include, without being limited to, proteins derived from connective tissue such as collagen and gelatin; albumin proteins such as serum albumin, milk albumin, or soy albumin; enzymes such as papain, or chymotrypsin; serum proteins such as fibrinogen. In particular such embodiments, the water insoluble biodegradable pharmaceutically acceptable polymer comprised within the oral delivery composition of the invention is gelatin, preferably partially hydrolyzed gelatin.

[0033] The present invention does not require the use of proteins having a specific level of purity, and thus proteins of any grade of purity may be employed. It is, however, preferable to employ proteins having a high degree of purity, and especially a defined (i.e., specifiable) composition, since the use of such proteins increases the degree with which the release of the antibacterial agent may be controlled. Thus, it is more preferable to employ a degradation product of proteins such as gelatin (a degradation product of collagen), and degradation product of hyalarunan such as short chain (low molecular weight) hyaluronan or more precisely oligomeric hyaluronan. A preferred protein for use in the oral delivery composition of the invention is gelatin which has been partially hydrolyzed by enzymatic action to molecular weight of, e.g., 1000-12000 Daltons (Da). Byco ^R proteins (Croda Colloids, Ltd.), and particularly Byco ^R C, A, and O, having molecular weights in the range of about 8000 Da to about 30000 Da, have been found to be the preferred proteins for use in the polymeric matrix of the oral delivery composition.

[0034] The water insoluble biodegradable or bioerodible pharmaceutically acceptable polymer comprised within the oral delivery composition of the present invention is cross- linked to an extent that is sufficient to render said polymer insoluble but insufficient to prevent the release of said therapeutic agent from the composition, provided that it is capable of being degraded at the treatment site.

[0035] According to the present invention, the biodegradable or bioerodible pharmaceutically acceptable polymer composing the matrix of the oral delivery composition is present at a concentration that is sufficient to provide said composition with structural stability, but insufficient to render said composition incapable of degradation, or incapable of permitting the release of said antibacterial agent. For example, in certain embodiments, the biodegradable or bioerodible pharmaceutically acceptable polymer is present in said composition in an amount of from about 10% to about 90%, preferably from about 20% to about 70%, or from about 30% to about 50%, more preferably from about 35% to about 45%, e.g., in an amount of about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45%.

[0036] The matrix composing the oral delivery composition of the present invention comprises a biodegradable or bioerodible pharmaceutically acceptable polymer as defined above, e.g., a protein such as gelatin, and hyaluronic acid or a salt thereof, wherein said polymer and hyaluronic acid or salt thereof are cross-linked via a cross-linking agent. The mixture of said polymer and hyaluronan or salt thereof in fact improves the bio-compatibility of the composition.

[0037] Hylauronic acid is an anionic, nonsulfated glycosaminoglycan (GAG) widely distributed throughout connective tissues of vertebrates, being the most abundant glycosaminoglycan of higher molecular weight in the extracellular matrix of soft periodontal tissues.

[0038] Hyaluronan has important hygroscopic, rheological and viscoelastic properties that fluctuate with changes in temperature, pH, ionic environment, and binding partners. However, these properties are also highly dependent on chain length. Hyaluronan can reach over 10 ⁷ Da in molecular mass, but also exists in multiple smaller forms, referred to as low molecular weight hyaluronan or oligomeric hyaluronan.

[0039] As disclosed in West and Kumar (1989), recombinant hyaluronan exerts varied bacteriostatic effects on all the bacterial strains tested, depending on its molecular weight and concentration, wherein high concentrations of medium molecular weight hyaluronan had the greatest bacteriostatic effect, particularly on the Actinobacillus actinomycetemcomitans, Prevotella oris, Staphylococcus aureus, and Propionibacterium acnes strains.

[0040] Hyaluronan has been found to be effective in treatment of inflammatory processes in medical areas such as orthopedics, dermatology and ophthalmology, and it has been further found to be anti-inflammatory and antibacterial in gingivitis and periodontitis therapy. Due to its tissue healing properties, it could be used as an adjunct to mechanical therapy in the treatment of periodontitis (Sukumar and Drizhal, 2007).

[0041] As previously shown, high molecular weight hyaluronan are immunosuppressive, antiangiogenic and anti-inflammatory, and were shown to protect against lymphocyte- mediated cytolysis (McBride and Bard, 1979), suppress septic responses to lipopolysaccharides, maintain immune tolerance, induce production of immunosuppressive macrophages, and reduce expression of inflammatory cytokines. Such hyaloronan was further found to have antiaging and anticancer effects; and are known to cause cell cycle arrest, mediated by transmembrane association between cluster of differentiation 44 (CD44) and the intracellular protein merlin (Morrison et ah, 2001), and to protect against apoptosis by a mechanism mediated by nuclear factor kappa-B (NF-κΒ) (Jiang et ah, 2011).

[0042] By contrast, short or low molecular weight hyaluronan are highly angiogenic, and immunostimulatory. Even the smallest fragment (the tetrasaccharide) has specific functions, with an ability to induce heat shock proteins and suppress apoptosis. The very smallest fragments apparently have the ability to ameliorate the intensity of the reactions induced by the small to intermediate- size fragments of hyaluronan. These small tetrameric to hexameric polysaccharides identify tissue injury through Toll-like receptors (TLRs) (Taylor et al, 2007). They also have the ability to inhibit the growth of tumor cells. It was shown that human umbilical cord hyaluronan that was hydrolyzed with hyaluronidase to molecular weight range of 1350-4500 Da 3-10 disaccharide units was angiogenetic, and when applied on skin of pig, can penetrate up to 800 μιη and increase the number of blood vessels in the skin. In a study directed to the bacteriostatic effects of hyaluronic acid, it has been suggested that hyaloronan having molecular weight of 1300 kD may prove beneficial in minimizing bacterial contamination of surgical wounds when used in guided tissue regeneration surgery (Pirnazar et al, 1999).

[0043] Hyaluronan affects endothelial cell proliferation and monolayer integrity. In addition, oligosaccharides were found to stimulate angiogenesis in vivo and endothelial cells proliferation in vitro.

[0044] In certain embodiment, the hyaluronic acid or salt thereof comprised within the oral delivery composition of the invention has a molecular weight that is lower than 50000 Da, e.g., from about 4000 Da to about 30,000 Da, 33000 Da, 36000 Da, 39000 Da, 42000 Da, 45000 Da or 48000 Da, preferably from about 4000 Da to about 48000 Da, from about 5000 Da to about 25000 Da, or from about 6000 Da to about 12500 Da, more preferably from about 6500 Da to about 8000 Da. Hyaluronan having molecular weight of below 10000 Da is an important component of the extracellular matrix and has been used as a viscoelastic biomaterial for medical purposes, in cosmetics and as a drug delivery system.

[0045] In certain embodiments, the hyaluronic acid or salt thereof comprised with the oral delivery composition of the present invention is present in an amount of from about 0.001% to about 20%, preferably from about 0.01% to about 10%, or from about 0.1% to about 3%, more preferably from about 0.4% to about 2.5%, e.g., in an amount of 0.4-0.6%, 0.6-0.8%, 0.8-1.0%, 1.0-1.2%, 1.2-1.4%, 1.4-1.6%, 1.6-1.8%, 1.8-2.0%, 2.0-2.2%, or 2.2-2.5%.

[0046] Examples of hyaluronan salts include, without limiting, alkaline metal or alkaline earth metal salts of hyaluronan, e.g., hyaluronan sodium salt and hyaluronan potassium salt.

[0047] Combination of collagen and hyaluronate were tested as suitable matrix for wound treatment (Kirk et ah, 2013); and the combination of collagen fibers and hyaluronan has shown advantages such as enhancement of cell migration and division compared with either material alone.

[0048] In certain embodiments, the cross-linking agent comprised within the matrix of the oral delivery composition of the present invention is an aldehyde such as glutaraldehyde or formaldehyde, aluminum, chromium, titanium zirconium, bisdiazobenzidine, phenol 2,4- disulfonyl chloride, l,5-difluoro-2,4-dinitrobenzene, urea, 3,6-bis(mercurimethyl)-dioxane urea, dimethyl adipimidate, or N,N'-ethylene-bis-(iodo-acetamide). In particular such embodiments, said cross-linking agent is an aldehyde, preferably glutaraldehyde. In any case, it should be noted that the cross-linking agent is present in the oral delivery composition in its crosslinked form, i.e., as cross-linking agent residues linked to the polymer molecules and to the hyaluronan, rather than in its free form.

[0049] According to the present invention, the cross-linking agent comprised within the matrix of the oral delivery composition is present in an amount that is sufficient to render said polymer insoluble but insufficient to prevent the release of said therapeutic agent from the composition. For example, in certain embodiments, said crosslinked cross-linking agent is present in an amount of from about 0.1% to about 20%, preferably from about 0.5% to about 10%, or from about 1% to about 5%, more preferably from about 2% to about 4%, e.g., in an amount of about 2.0%, about 2.1%, about 2.3%, about 2.5%, about 2.7%, about 2.9%, about 3.1%, about 3.3%, about 3.5%, about 3.7%, about 3.9%, or about 4.0%.

[0050] Hyaluronic acid can be cross-linked via various functional groups, e.g., via the acetyl group (CH ₃ C(0)-) after deacetylation, via the carboxylic acid group, or via one of the hydroxyl groups. Hyaluronic acid can be cross-linked with glutaraldehyde via hemiacetal formation. Hyaluronic acid was chemically cross-linked with glutaraldehyde, using uncross- linked hyaluronan films in acetone- water mixtures, to produce water- insoluble hyaluronan films having low water content (as low as 60 wt%) when brought into contact with phosphate-buffered saline of pH 7.4 at 37°C. Infrared spectra of the cross-linked films led to the conclusion that intermolecular formation of hemiacetal bonds with glutaraldehyde between the hydroxyl groups belonging to different hyaluronan molecules led to crosslinking (Tomihata and Ikada, 1997).

[0051] In certain embodiment, the plasticizer comprised within the oral delivery composition of the present invention is a phthalate ester (also termed phthalate), i.e., an ester of phthalic acid, a phosphate ester (also termed organophosphate), i.e., an ester of phosphoric acid, glycerin, or sorbitol. In particular such embodiments, the plasticizer is glycerin.

[0052] In certain embodiments, the plasticizer comprised within the oral delivery composition of the present invention is present at a concentration of from about 0.01% to about 25%, preferably from about 0.1% to about 20%, from about 1% to about 20%, or from about 3% to about 15%, more preferably from about 5% to about 10%, e.g., at a concentration of about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0053] The oral delivery composition disclosed herein is aimed at releasing a therapeutic agent, e.g., an antiseptic agent, in a periodontal pocket in a sustained release manner, i.e., in a predetermined rate aimed at maintaining a constant drug concentration for a specific period of time, e.g., hours or days, with minimum side effects.

[0054] The term "therapeutic agent" as used herein refers to any agent having a therapeutic effect that might be beneficial in treatment of periodontal disease, e.g., an antimicrobial agent; an antibacterial agent, also referred to herein as "antiseptic agent" or "disinfecting agent, more particularly a topical antiseptic; antifungal agent, also referred to herein as fungicide or fungistatic; an anti-inflammatory agent, e.g., a non-steroidal anti-inflammatory drug; and an anti-pain agent.

[0055] Examples of antifungal agents include, without being limited to, fluconazole, itraconazole, amphotericin B, voriconazole, nystatin, clotrimazole, econazole nitrate, miconazole, terbinafine, ketoconazole, enilconazole, boric acid, and miconazole.

[0056] The term "non-steroidal anti-inflammatory drug" (NSAID) as used herein refers to any non-steroidal anti-inflammatory drug/agent/analgesic/medicine, and relates to both cyclooxygenase (COX)-2 selective inhibitors such as celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib and lumiracoxib, as well as to COX-2 non-selective inhibitors such as etodolac, aspirin, naproxen, ibuprofen, indomethacin, piroxicam and nabumetone.

[0057] Examples of anti-pain agents include, without being limited to, lidocaine, benzocaine, dibucaine, tetracaine, and proparacaine.

[0058] Topical antiseptics in general are less likely to induce resistance compared with antibiotics, owing to their unspecific mode of action and the high concentrations in which they can be used. In certain embodiments, the antibacterial agent comprised within the oral delivery composition of the present invention is a topical antibacterial agent such as a quaternary ammonium compound (QAC) or a pharmaceutically acceptable salt thereof, e.g., benzalkonium chloride, or cetylpyridinium chloride; a guanidine compound or a pharmaceutically acceptable salt thereof, e.g., chlorhexidine, alexidine, or polyhexamethylene biguanide (PHMB); hexetidine, triclosan; liniment (Glossary of common dental product ingredients. Version one: May 2011. British dental health foundation); stannous fluoride; eucalyptol; menthol; methyl slisylate; thymol; peppermint oil; amyloglucosidase; or glucose oxidase (Silje Storehagen, Nanna Ose og Shilpi Midha. Dentifrices and mouthwashes ingredients and their use. Thesis for degree Candidata Odontologiae (DDS) . Faculty of dentistry, University of Oslo, 2003).

[0059] In other embodiments, the topical antiseptic agent comprised within the oral delivery composition of the present invention is the bispyridinamine octenidine (1,Γ,4,4'- tetrahydro-N,N'-dioctyl- 1 , 1 '-decametylenedi-(4-pyridylideneamine)), an antimicrobial agent capable of inhibiting dental plaque (Bailey et ah, 1984), or a pharmaceutically acceptable salt thereof such as octenidine dihydrochloride (CAS -number 70775-75-6) that is exemplified herein. Octenidine dihydrochloride is a cationic, surface active antimicrobial compound, which differs from QACs such as benzalkonium chloride, and guanidines such as chlorhexidine, by the lack of an amide- and ester structure in its molecule, which results in lower toxicity due to possible metabolites. Octenidine can be sterilized by steam up to 130°C in aqueous solutions and stored at room temperature, making it suitable for long storage. The stability of octenidine makes it a useful robust molecule that is insensitive to interactions with functional groups that might be present in the composition (e.g., during preparation and/or storage of the composition). Moreover, octenidine is stable and maintains its antimicrobial activity at an extremely broad pH range (1.6-12.2), which is particularly important in wound healing during which the wound pH changes. The octenidine mode of action involves destabilization of the microorganism membrane, and this ensures that microorganisms exposed to said agent cannot develop drug resistance in a straightforward way, as the entire cellular structure, rather than a specific molecular target, is affected. Since octenidine is a hydrophobic compound, it requires an organic solvent such as ethanol or phenoxyethanol in order to be effectively administered. However, such organic solvents may cause substantial irritation, particularly when applied to inflamed tissue, and the drug should thus be introduced in a suitable manner, e.g., in a controlled release manner precisely at the site of microbial contamination (e.g., the periodontal pocket).

[0060] In particular embodiments, the topical antiseptic agent comprised within the oral delivery composition of the invention is octenidine, or a pharmaceutically acceptable salt thereof such as octenidine dihydrochloride. Examples of other pharmaceutically acceptable salts of octenidine include, without limiting, the dimesylate salt, the diesylate salt, the ditosylate salt, the disulfate salt, the disulfonate salt, the diphosphate salt, the dicarboxylate salt, the dimaleate salt, the difumarate salt, the ditartrate salt, the dibenzoate salt, the diacetate salt, the dihydrobromide salt, and the digluconate salt.

[0061] In certain embodiments, the antibacterial agent comprised within the oral delivery composition of the present invention is present at a concentration of from about 0.001% to about 55%, preferably from about 0.1% to about 45%, from about 1% to about 40%, or from about 10% to about 35%, more preferably from about 20% to about 30%, e.g., at a concentration of about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.

[0062] Use of high molecular weight hyaluronan (molecular weight of lxlO ⁶ and 2.5xl0 ⁶ g/mol) with octenidine is disclosed in DE 202016102375 Ul.

[0063] In certain embodiments, the oral delivery composition of the present invention, upon contact with said fluid, adsorbs said fluid and consequently swells to up to about 115%, about 120%, or about 125%, in length; up to about 115%, about 120%, or 125% in width; and to about 120-150% in thickness, of its sizes before adsorbing said fluid, i.e., before swelling.

[0064] In certain embodiments, the oral delivery composition of the present invention has a dissolution profile in saliva, at 37°C, whereby about 30% to about 60%, or about 40% to about 50%, of said therapeutic agent is released over the first five hours.

[0065] In certain embodiments, the present invention provides an oral delivery composition as defined in any one of the embodiments above, wherein the biodegradable or bioerodible pharmaceutically acceptable polymer is gelatin, preferably partially hydrolyzed gelatin; said hyaluronic acid has a molecular weight that is lower than 50000 Da, e.g., from about 4000 Da to about 30,000 Da, 33000 Da, 36000 Da, 39000 Da, 42000 Da, 45000 Da or 48000 Da, from about 5000 Da to about 25000 Da, from about 6000 Da to about 12500 Da, or from about 6500 Da to about 8000 Da, optionally hydrolyzed or partially hydrolyzed; said cross-linking agent is glutaraldehyde; said plasticizer is glycerin; and said therapeutic agent is an antibacterial agent such as octenidine or a pharmaceutically acceptable salt thereof, e.g., octenidine dihydrochloride. In particular such embodiments, (i) said polymer is present in said composition in an amount of from about 20% to about 70%, or from about 30% to about 50%; (ii) said hyaluronic acid or salt thereof is present in said composition in an amount of from about 0.01% to about 10%, or from about 0.1% to about 3%; (iii) said crosslinked glutaraldehyde is present in said composition in an amount of from about 1% to about 4%; (iv) said plasticizer is present at a concentration of from about 3% to about 15%, or from about 5% to about 10%; and (v) said antibacterial agent is present at a concentration of from about 10% to about 40%, or from about 20% to about 30%. More particular such embodiments, are those wherein (i) said polymer is present in said composition in an amount of from about 35% to about 45%; (ii) said hyaluronic acid or salt thereof is present in said composition in an amount of from about 0.4% to about 2.5%; (iii) said crosslinked glutaraldehyde is present in said composition in an amount of from about 1% to about 4%; (iv) said plasticizer is present at a concentration of from about 6% to about 8%; and (v) said antibacterial agent is present at a concentration of from about 20% to about 30%. Such oral delivery compositions may have a dissolution profile in saliva, at 37°C, whereby about 30% to about 60%, or about 40% to about 50%, of said antibacterial agent is released over the first five hours.

[0066] Oral delivery sustained release compositions as disclosed herein are formed through the solidification of a liquid solution, and may be prepared according to any procedure and utilizing any technique available, e.g., according to the process described in detail in the Examples section hereinafter. Partiuclar such octenidine-containing compositions as exemplified herein can be prepared by (i) dissolving a hydrolyzed gelatin (e.g., Byco C) in water, while stirring, to obtain a solution of said hydrolyzed gelatin; (ii) adding a first amount of an alcoholic solution of a pharmaceutically acceptable octenidine salt, e.g., octenidine dihydrochloride, to said solution, and immediately thereafter adding glycerin to said solution, while stirring; (iii) adding aqueous glutaraldehyde to the solution obtained in step (ii), while stirring; (iv) adding a second amount of said alcoholic solution of pharmaceutically acceptable octenidine salt to the solution obtained in step (iii), and immediately thereafter adding an aqueous solution of a hyaluronic acid to said solution, while stirring; and (v) pouring the solution obtained in step (iv) into a suitable vessel to thereby obtain, upon drying, said oral delivery composition.

[0067] The oral delivery composition as defined in any one of the embodiments above may further comprise one or more additional therapeutic agents, e.g., one or more antiinflammatory agents in addition to the hyaluronan that has an anti-inflammatory activity.

[0068] The oral delivery composition of the present invention is in fact a solid dosage form, e.g., in the form of a film. In certain embodiments, the said solid dosage form is an essentially two- (or practically flat three-) dimensional solid implant adapted for implantation in a periodontal pocket. Such a solid implant may have different shapes and may be, e.g., from about 3 to about 10 mm in length; and/or from about 1 to about 5 mm in width; and/or from about 0.01 to 0.5 mm in thickness.

[0069] In another aspect, the present invention thus relates to a method for treatment of periodontal disease in a subject in need thereof comprising the step of implantation of an oral delivery composition as defined in any one of the embodiments above (e.g., such a composition comprising an antibacterial agent), that is an essentially two-dimensional solid implant adapted for implantation in a periodontal pocket of said subject, to thereby release said therapeutic agent and said hyaluronic acid in said periodontal pocket in a sustained release manner.

[0070] The term "subject" as used herein refers to any mammal, e.g., a human, non-human primate, horse, ferret, dog, cat, cow, and goat. In a preferred embodiment, the term "subject" denotes a human, i.e., an individual.

[0071] In certain embodiments, the solid dosage form implanted in a periodontal pocket of the subject treated comprises a matrix, a plasticizer, and a therapeutic agent, each as defined above, wherein the biodegradable or bioerodible pharmaceutically acceptable polymer composing said matrix is gelatin, preferably partially hydrolyzed gelatin; the hyaluronic acid or salt thereof composing said matrix has a molecular weight that is lower than 50000 Da, e.g., from about 4000 Da to about 30,000 Da, 33000 Da, 36000 Da, 39000 Da, 42000 Da, 45000 Da or 48000 Da, from about 5000 Da to about 25000 Da, from about 6000 Da to about 12500 Da, or from about 6500 Da to about 8000 Da, optionally hydrolyzed or partially hydrolyzed; the cross-linking agent cross-linking said polymer and said hyaluronic acid is glutaraldehyde; the plasticizer is glycerin; and the therapeutic agent is an antibacterial agent such as octenidine or a pharmaceutically acceptable salt thereof, e.g., octenidine dihydrochloride. In particular such embodiments, (i) said polymer is present in said solid dosage form in an amount of from about 35% to about 45%; (ii) said hyaluronic acid or salt thereof is present in said solid dosage form in an amount of from about 0.4% to about 2.5%; (iii) said crosslinked glutaraldehyde is present in said solid dosage form in an amount of from about 1% to about 4%; (iv) said plasticizer is present at a concentration of from about 6% to about 8%; and (v) said antibacterial agent is present at a concentration of from about 20% to about 30%.

[0072] In a further aspect, the present invention relates to an oral delivery composition as defined in any one of the embodiments above (e.g., such a composition comprising an antibacterial agent), that is an essentially two-dimensional solid implant adapted for implantation in a periodontal pocket, for use in the treatment of periodntal disease.

[0073] Unless otherwise indicated, all numbers expressing quantities of ingredients and so forth used in the present description and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this description and attached claims are approximations that may vary by up to plus or minus 10% depending upon the desired properties sought to be obtained by the present invention.

[0074] The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Study 1. Preparation of <10kDa hyaluronic acid-based Periodontal film and structural analysis thereof

Preparation of the composition

[0075] Gelatin powder Byco ^R C (Croda Colloids, Ltd) molecular weight 19-30 kDA was added to pure water and dissolved under mixing. Sixty parts from the octenidine 20% in absolute ethyl alcohol were mixed with glycerol, used as a plasticizer for the final product, and the homogenous solution was added into the gelatin solution. Crosslinking of the composition was performed using glutaraldehyde at about half the amount used to prepare the commercially available chip (PerioChip). Glutaraldehyde (10% solution in water) was divided into 5 portions which were added every 5 minutes with mixing after each addition. The left 40 parts of the octenidine alcoholic solution was then added into the gelatin crosslinking solution. The last step of the product preparation was the addition of hyaluronic acid (Bloomage Freda Biopharm Co., Ltd., Oligo Hyaluronic Acid [HA-Oligo], <10kDa, htt :// w w .bloomagefreda.coin/proen/id 16. html ; 10%), before full crosslinking is achieved, while mixing for 2 minutes. The yellow solution thus obtained was poured into suitable drying flat (polystyrene) dishes or some other suitable material, at 360 mg/cm ² , and the drying process was performed at ambient temperature. The films formed were left to dry for few days up to one week, and the dry films obtained were peeled dry from the flat dishes and cut into chips having a suitable size. Chips were packed in sealed blisters, so as to keep them under controlled humidity level, which keeps them hard enough, and not too flexible, for easy insertion under the gum line inside the gingival pocket (excess drying of the chips may result in brittle products that might be broken in use or hurt the gingival pocket tissue during insertion).

Swelling in water (Periodontal film vs. Periochip™)

[0076] A Periodontal film was weighed in order to determine its initial weight, using electronic analytical balance (FA2004C series), and was then inserted into an Eppendorf containing 0.2 mL of purified water and placed in an oven preheated to 37°C in order to swell. The Periodontal film was removed from the Eppendorf at pre-determined times of 15, 30 and 60 minutes. Excess of water was removed by absorbing it with a paper tissue and the chip was weighed. The swelling percentage was calculated using the formula I:

Swelling % = ^ x 100 I where Wf is the weight at each measuring point and Wi is the initial weight. As found, the initial swelling of the Periochip is faster, reaching more than 400 percent; however, it falls down within half an hour, whereas the swelling of the Periodontal film is around 240 percent and it stays constant (Fig. 1).

Changes in the XYZ dimensions (Periodontal film vs. Periochip™)

[0077] The X and Y dimensions of the chip (length and width, respectively; referring to the direction of the plate used to lay the chip during the casting step) were measured using a ruler in order to determine its initial dimensions, and the Z dimension (hight; referring to the height of the casted film) was measured using a calibrated caliper (Digital caliper, "Metric", IP67 model). The chip was then inserted into an Eppendorf containing 0.2 mL of purified water and placed in an oven preheated to 37°C in order to swell. The chip was removed from the Eppendorf at pre-determined times of 15, 30 and 60 minutes. Excess of water was removed by absorbing it with a paper tissue and the chip was measured again. The change in each dimension was calculated in percentages using the formula II:

Change in X, Y, Z % = ^ x 100 II where Df is the dimension in millimeters at each measuring point and Di is the initial dimension in millimeters.

[0078] The dimensions of the commercial product tested were x=5 mm (length), y=4 mm (width), and z=0.35 mm (height), i.e., the sizes of said product in the x and y axes were more than one order of magnitude bigger than the its size in the z axis.

[0079] Surprisingly, the Periodontal film was found to behave different than expected when contacted with water, and swelled absolutely different than the commercially available product. In particular, the swelling of the Periodontal film is rather low in both the X-axis and Y-axis compared with that of Periochip; however, significantly higher than that of Periochip in the Z-axis. In particular, the swelling of the Periodontal film in the X-axis reaches about 125% in 15 min, while the swelling of Periochip in that axis reaches more than 160% in 15 min and then falls down (Fig. 2); the swelling of the Periodontal film in the Y- axis reaches about 115% in 15 min, while the swelling of Periochip in that axis reaches more than 150% in 15 min and then falls down (Fig. 3); and in contrary, the swelling of the Periodontal film in the Z-axis reaches about 120-150% in 15 min, while Periochip almost does not swell at all in this axis (Fig. 4).

[0080] In other words, whereas the Periodontal film bulk swelling is about half of the Periochip bulk swelling, the swelling of the Periodontal film is surprisingly significantly higher compared to that of the Periochip in the Z-axis, and much lower compared to the Periochip in the X and Y axes. Considering that the chip dimensions for gingival pocket treatment have to be high in the X and Y axes, and low (thin) in the Z axis, these unexpected findings suggest that the Periodontal film will have better stability inside the gingival pocket, upon insertion, as it will have absolute change (length and width) that is much lower than that of the commercial chip.

[0081] During the preparation of the composition, the wet film matrix undergoes final crosslinking by the crosslinking agent(s) used, while at the same time, drying of the wet film is proceeding. It is postulated that due to the anisotropic nature of the drying process, the crosslinking becomes anisotropic as well. More specifically, while during early stages of the casting and drying phases, the film is in a fluid state with some freedom of the polymer chains constituting the composition matrix to arrange and interact with one another, as well as with the molecules of the crosslinking agent(s), the crosslinking process increases the viscosity of the film and consequently restricts the movement of the film constituents. Surprisingly, the introduction of the hyaluronic acid into the octenidine-containing composition resulted in a film where the swelling became smaller in the x and y directions albeit less crosslinking agent (glutaraldehyde) was used and hyaluronic acid was added. Hyaluronic acid is a highly hydrophilic biopolymer known as being capable of binding water (and thus as a natural humectant), and as being capable of substantially swelling in water. It seems that the swelling properties of hyaluronic acid are exhibited in this anisotropic composition prepared, by a high degree of swelling in the z direction rather than in the z and y directions.

[0082] It is assessed that during preparation, while the outer part of the film formed is enriched with hyaluronic acid, crosslinking is proceeding in the gelatin-enriched core of the film, which consequently becomes more rigid, and less free and sensitive to hydration with water. In other words, the addition of hyaluronic acid brings more water molecule to associate with the hyaluronic acid. In the absence of hyaluronic acid, these water molecules associate with the hydrophilic motifs (hydrophilic amino acids) of the gelatin, and as a consequence, during crosslinking of the polymer matrix the gelatin chains are crosslinked where they retain much of the ability to swell while in contact with water and water solution. The hyaluronic acid molecules added compete with the gelatin molecules on the water molecules available. The gelatin hydrophilic motifs associate with one another rather than with water molecules, and the hydrophobic character of gelatin is thus more pronounced. The crosslinking of the gelatin under these conditions results in less swelling in water as compared to that of gelatin crosslinked in the absence of hyaluronic acid.

[0083] It is hypothesized that while the gelatin is crosslinked with a higher degree of hydrophobicity, the hyaluronic acid with the water molecules bound thereto is being excluded from the gelatin-enriched crosslinked core, probably creating a hydrophilic envelop (shell) comprising mainly hyaluronic acid and surrounding the less hydrophilic gelatin core.

[0084] It is assumed that some of the hydrophilic motifs of the gelatin left on the outer part of the gelatin core are bound to the hydrophilic hyaluronic molecules, which become crosslinked with the gelatin molecules while the crosslinking process proceeds. Hence, upon completion of said process, the film produced is actually a layer consisting of a gelatin enriched core and an hyaluronan enriched outer layer overcoating said core. Consequently, and as schematically illustrated in Fig. 5, upon contacting of the composite film with water, the swelling in the x and y directions is relatively minor due to the enhanced hydrophobic nature of the gelatin core (or even absent, due to the fact that the chip is cut from a continuous flat crosslinked film); however, the swelling in the z direction (height) is remarkably higher due to the relatively high hyaluronan concentration in the outer hydrophilic coat.

[0085] It may be envisioned that the low molecular weight hyaluronan chain are gradually expelled out of the core formed during the crosslinking of gelatin, and these expelled hyaluronan molecules undergo gradual crosslinking with the glutaraldehyde. This way, the matrix formed is gradually enriched with hyaluronan going from inside the film towards its outer part. Glutaraldehyde crosslinks both gelatin and hyaluronan chains, where crosslinking with the nucleophilic amino groups-containing gelatin chains is preferred over the slow crosslinking reaction with the hyaluronan side chains.

[0086] The minor swelling in the x-y direction of the product disclosed herein significantly reduces the probability that such a product will swell out of the dental pocket into which it is inserted. Swelling outside the dental pocket often results in higher probability of dislodgement of said product during use. Moreover, the minor swelling in the x-y direction of the product disclosed herein, compred with that of the commercial product, is expected to significantly reduce the pain (toothache and gum ache) caused by the pressure developed inside the dental pocket as a result of the swelling in the x-y direction, which is one of the complains when using the commercial product and appears in more than 50% of the cases.

[0087] The composite film disclosed herein swells significantly less than the commercial product in the x-y direction and hence substantially reduces both the probability of dislodgment after insertion into the pocket and the pain associated with the expansion (swelling) of the product inside the pocket. On the other hand, the fact that said composite film swells substantially more than the commercial product in the z axis is advantageous, as in this case, a better contact is secured between the inserted product and the inflamed walls of the gingival pocket, and the amount of drug released from the inserted composite film that may leak outside the treatment site is significantly reduced (Fig. 6). Thus, the product disclosed herein can be positioned in an optimal structure hence fill the dental pocket and therefore prevent the re-infestation of the pocket by pathogenic bacteria.

Structural studies of the Periodontal film

[0088] To further understand the behavior of the composite film disclosed herein, the surface of said composite film was scanned with an atomic force microscope (AFM).

[0089] As shown in Fig. 7, the surface of the x-y plain of the composite film was found to have a roughness of 400-700 nm, whereas the surface of the commercial product, Periochip, tested under similar conditions, has shown roughness of 200-400 nm. Three-dimensional imaging of the surface of the two products shows deeper and wider cleft in the composite film disclosed herein in comparison with the commercial Periochip where structure feature are smaller and shallower (Fig. 8).

[0090] Based on these structural data, it is assumed that during the preparation of the composite film disclosed herein, more particularly after pouring the liquid formulation onto the flat bath in a shallow wet film, the crosslinking process between the chains of the abundant gelatin, where most of the chains are crosslinked between themselves, proceeds, while the hyaluronan chains are expelled outside from the gelatin core formed and are shown as the non-uniform 400-700 nm size structures on the surface of the film in the AFM image. The hyaluronan chains expelled from the gelatin core are probably also crosslinked albeit at lower extent compared to the gelatin chains, and the relatively low-level of crosslinkage of those outer chains, mainly composed of hyaluronan, is responsible for the high level of swelling in the z axis schematically depicted in Fig. 5.

Total cumulative octenidine release in saliva

[0091] A Periodontal film was inserted into an Eppendorf containing 0.2 mL of saliva, obtained from several donors and mixed, and the Eppendorf was then placed in an oven preheated to 37°C to release octenidine into the saliva sample. 20 μΐ _^ of saliva were removed at pre-determined times of 0.5, 1, 4, 8 and 24 hours, and diluted. Total octenidine release at each point was measured using a spectrophotometer (Biomate 3, Thermo Spectronic, USA), at a wavelength of 281 nm, in a cumulative manner, using a calibration curve that was determined on pure octenidine solution according to the formula III:

Mrotal Oct. Rel % = 100 III

"Oct.

where MoctRei. is the cumulative octenidine release ^g), Moct. is the octenidine amount (dosage) per Periodontal film ( .g), and Mrotai octrei. is the total octenidine release (%).

[0092] As found, the intitial burst release of the antiseptic agent was about 40-50% during the first 1-5 hours, reaching almost plateau and leaving about 50% of the octenidine content to be slowly released during several days, upon bio-degradation of the chip structural matrix (gelatin and hyaluronan) by proteinase and hyaluronase enzymes present in the tissue sourounding the treated gingival pocket (Fig. 10).

Study 2. Preparation of ~48kDa hyaluronic acid-based Periodontal film and structural analysis thereof

[0093] A ~48kDa HA-based Periodontal film was prepared following the exact procedure described in Study 1, using a particular batch of hyaluronic acid having a molecular weight of ~48kDa (Bloomage Freda Biopharm Co., Ltd., Hyaluronic Acid [HA-TLM], htip ://www . bloomagefreda.com/proen/id 1.6. html ) . In particular, gelatin powder Byco ^R C (Croda Colloids, Ltd) molecular weight 19-30 kDa was added to pure water and dissolved under mixing. Sixty parts out of octenidine 20% in absolute ethyl alcohol were mixed with glycerol, used as a plasticizer for the final product, and the homogenous solution was added into the gelatin solution. Crosslinking of the composition was performed using glutaraldehyde at about half the amount used to prepare the commercially available chip (PerioChip™). Glutaraldehyde (10% solution in water) was divided into 5 portions which were added every 5 minutes with mixing after each addition. The left 40 parts of the octenidine alcoholic solution was then added into the gelatin crosslinking solution. The last step of the product preparation was the addition of hyaluronic acid ~48kDa (10%), before full crosslinking is achieved, while mixing for 2 minutes. The yellow solution thus obtained was poured into suitable drying flat (polystyrene) dishes or some other suitable material, at 360 mg/cm ² , and the drying process was performed at 35°C. The films formed were left to dry for 48 hours, and the dry films obtained were peeled dry from the flat dishes and cut into chips having a suitable size. Chips were packed in sealed blisters, so as to keep them under controlled humidity level.

Changes in the XYZ dimensions (Periodontal films vs. Periochip™)

[0094] The changes in the XYZ dimensions of the ~48kDa HA-based Periodontal film were measured following the exact procedure described in Study 1, and compared with those measured for the <10kDa HA-based film and for the commercially available product Periochip™. Aftre measuring the initial X, Y and X dimensions, the film was inserted into an Eppendorf containing 0.2 mL of purified water and placed in an oven preheated to 37°C in order to swell, and was removed from the Eppendorf after an incubation period of 15 minutes, a time point at which (as previously determined) the XYZ dimensions reached their highest values. Excess of water was removed by absorbing it with a paper tissue and the film was measured again. The change in each dimension was calculated in percentages.

[0095] The -48 kDa HA-based Periodontal film has shown a behavior very similar to that of the <10kDa HA-based Periodontal film shown in Study 1, and definitely different than that of the commercially available Periochip™. The swelling of the chip was anisotropic, more specifically rather low in both the X and Y axes compared with that of the Periochip™; however, remarkably higher than that of Periochip™ in the Z-axis. The swelling of the chip in each one of the X and Y axes reached about 120-140%, while the swelling in the Z axis was higher than that in the X and Y axes reaching about 150%. As shown in Study 1, the swelling of the commercially available Periochip™, under the same conditions, in the X and Y axes is much higher than that in the Z axis that is practically negligible (Table 1; Fig. 11).

Table 1. Changes in XYZ axes after 15-minute incubation period in water at 37°C - <10kDa HA- and ~48kDa HA-based Periodontal chip vs. Periochip™ [0096] The behavior of the ~48kDa HA-based Periodontal film, in terms of swelling in the various axes following incubation in water at 37°C, is thus very similar to that of the <10kDa HA-based Periodontal film shown in Study 1, wherein both Periodontal films show a completely different behavior, and particularly a remarkably higher swelling in the Z axis, than that of the commercial product Periochip™ (to -150% compared to practically none).

[0097] The increase measured in the swelling of the ~48kDa HA-based chip in the Z axis is greater than that in the <10kDa HA-based chip (to -150% vs. to -130% compared to the dry size, respectively); however, it is still beneficial in enhancing holding of the chip inside a periodontal pocket. Yet, a further increase in swelling in the Z axis with further increase in the molecular weight of the hyaluronic acid used might be too excessive and thus counterproductive as it may cause pain.

Total cumulative octenidine release in saliva

[0098] The total cumulative octenidine release in saliva, from the ~48kDa HA-based Periodontal film, was measured as described in Study 1 and compared with that measured for the <10kDa HA-based film. As found, replacing the <10kDa hyaluronic acid with the same weight concentration of ~48kDa hyaluronic acid resulted in only slight change in the rather fast initial release of the octenidine from the Periodontal chip (Fig. 12).

Study 3. The efficacy of the Periodontal film as anti infective therapeutic device

[0099] In the present study, the efficacy of a Periodontal film prepared as shown in Study 1 in treating patients suffering from chronic periodontitis or aggressive periodontitis was tested. The number of patients participated in the study was eleven (both females and males), all having periodontal pockets of various depths.

[00100] Prior to the treatment, all patients were evaluated by clinical and radiological examinations by means of full mouth periapical x-rays and diagnosed. Patients under study at initial stage suffered from chronic periodontitis or aggressive periodontitis. The initial medical status was based on the principles of American Society of Anesthesiologists (ASA) physical status classification system. Smoking as a factor that enhanced periodontal diseases and prevent repair was taken in consideration.

[00101] Periodontal measurements status was evaluated at the first control by a charting. This evaluation included (i) periodontal pockets depth (PPD) at 6 sites of each tooth which is more than 4 mm; (ii) recession (REC) level at each tooth in the buccal and lingual/palatal side; (iii) clinical attachment level (AL) of every tooth which enters the study for the distance between cement-enamel junction (CEJ) and the bottom of the pocket level (attachment loss was calculated according to the formula: PPD - (MG-marginal gingiva - CEJ to bottom of pocket); and (iv) tooth mobility and furcations.

[00102] Periodontal film devices were provided randomly into the deepest pockets at the teeth (test group), while contra laterally teeth were served as control (control group). Periodontal pockets that received the Periodontal film together with scaling and root planning in periodontal pockets at initial therapy (test group) were compared to the control group of pockets that received scaling and root planning alone.

[00103] Preliminary measurement charts were performed after 10, 18, and 25 days. The preliminary results in the test group were significantly better in all the clinical aspects compared to the control group. Accordingly, PPD in the test group was reduced by 4-5 mm, while in the control group it was reduced by 2-3 mm; PPD at the adjacent tooth in the test group was reduced by 3-4 mm probably due to diffusion of the active agents, while in the control group it was reduced by 1-2 mm; clinical attachment level in the test group was 3-4 mm, while in the control group it was 1-2 mm; and recessions in the control group were significantly deeper. These results clearly show the ability of the Periodontal film of the invention as a valuable anti infective therapeutic device in diverse treatments and surgical procedures.

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