Composition for treatment of periodontal disease and related method

Abstract

Disclosed is a composition suitable for treatment of periodontal disease. The composition comprises the peptide LL37 (SEQ ID NO: 1) in admixture with a gelling agent / mixture, which upon introduction into a periodontal pocket increases its viscosity. Also disclosed is an injection device comprising the composition. A method for treatment of periodontal disease is also disclosed.

Description

[0001] COMPOSITION FOR TREATMENT OF PERIODONTAL DISEASE AND RELATED METHOD

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of odontology. More specifically, the present invention provides a composition useful for treatment of periodontal disease as well as a method for such a treatment.

[0004] BACKGROUND OF THE INVENTION

[0005] Periodontitis (herein termed "periodontal disease") is a chronic inflammatory disease, which occurs when the balance between the patient's immune system and the pathogenicity of oral bacteria is upset. Its development depends on whether the immune system (neutrophils) complements and antibodies neutralize the metabolites of the bacterial plaque and its metabolites in the periodontal connective tissue. Neutrophils prevent bacteria and their metabolic products from getting into the connective tissue, but if they get there, pro- inflammatory cytokines IL-1 and PGE2 are released, which increase inflammation and thus the destruction of connective tissue (including bones). Periodontal pockets are formed, which provides even easier conditions for the development of periopathogens. Treatment of periodontal disease is characterized by different treatment modalities including surgery.

[0006] Nonsurgical treatments

[0007] If periodontitis is not too advanced, treatment may involve less invasive procedures, including :

[0008] Scaling removes tartar and bacteria from tooth surfaces and below the gumline. It may be performed using instruments, a laser or an ultrasonic device.

[0009] Root planing smooths the root surfaces. This helps prevent further buildup of tartar and bacteria. It also facilitates the ability of the gums to attach teeth again.

[0010] Topical or oral antibiotics can help control bacterial infection. Topical antibiotics can include antibiotic mouth rinses or application of gels containing an antibiotic into gum pockets. Sometimes oral antibiotics are needed to fight bacteria that cause infections. Surgical treatments

[0011] For advanced periodontal disease dental surgery may be necessary. This includes:

[0012] Flap surgery, which is a common procedure, where surgeons make small cuts in the gum and lift a section of tissue back. Then, they remove tartar and bacteria from the tooth and from under the gums. The gums are sutured back, so the tissue fits firmly around the teeth. Once healed, it will be easier to clean areas on and around the teeth and gums.

[0013] Bone grafting is a surgical procedure that replaces missing bone in order to repair bone fractures that are either extremely complex, pose a significant health risk to the patient, or fail to heal properly. Some small or acute fractures can be cured without bone grafting, but the risk is greater for large fractures like compound fractures.

[0014] Bone generally has the ability to regenerate completely but requires a very small fracture space or some sort of scaffold to do so. Most bone grafts are expected to be resorbed and replaced as the natural bone heals over a few months' time.

[0015] The principles involved in successful bone grafts include osteoconduction (guiding the reparative growth of the natural bone), osteoinduction (encouraging undifferentiated cells to become active osteoblasts), and osteogenesis (living bone cells in the graft material contribute to bone remodeling). Osteogenesis only occurs with autograft tissue and allograft cellular bone matrices.

[0016] Guided tissue regeneration involves placing a small piece of material between your bone and gum tissue to allow bone to regrow.

[0017] Soft tissue graft or gum graft is a straightforward surgical operation that will correct receding grafts by removing a piece of soft tissue from the roof of the oral cavity or creating a flap that will cover the exposed portion of the tooth.

[0018] Sometimes, surgeons apply a gel that contains special proteins to the diseased tooth root. This can encourage healthy bone and tissue growth.

[0019] Biofilm and removal

[0020] Scientifically, the term 'biofilm' is used to describe the communities of micro-organisms attached to a surface. In simple terms, Biofilm occurs when bacteria present in a watery environment such as saliva begin to adhere to surfaces. These bacteria secrete sticky substances that attract more bacteria and lead to communities of disease-causing microbes. Once they begin to multiply uncontrollably and accumulate, they become associated with cavities and gum disease such as gingivitis and periodontitis.

[0021] In the mouth, the biofilm appears as a yellowish buildup on the surfaces of teeth but underneath the tissues such as below the gum margin, physical removal of the biofilm may not be accomplished with a home care routine alone. This biofilm has now settled and become harder to form plaque and calculus. Not only is it more stubborn but it also causes infection of the gums and surrounding tissues if left untreated. The tissues become inflamed and bleed, the pocket depths continue to increase, and the pathogenic organisms become more and more harmful to the environment.

[0022] Dental biofilm is primarily composed of micro-organisms which exist within an intercellular matrix that consists of organic and inorganic materials derived from saliva, the fluid present in the gingival crevices also known as crevicular fluid and bacterial products.

[0023] If gum disease has damaged the bone surrounding your tooth root, the dentist might have to replace it with a graft. The bone graft can be made from small parts of autologous bone, a synthetic bone, or donated bone. This procedure helps prevent tooth loss and may help promote natural bone regrowth.

[0024] In spite of this variety of possible interventions, there is still a need for treatments that can help improve the result of both the surgical and non-surgical interventions.

[0025] LL37

[0026] LL37 is an antimicrobial peptide, which has been shown to have antimicrobial activity against multiple Gram-positive and Gram-negative human pathogens. LL37 belongs to the cathelicidin family and is released as a mature peptide by stimulated neutrophil granulocytes. LL37 plays an important role in the first line of defence against infections and is toxic to both bacterial and normal cells.

[0027] It has been reported that LL37 is effective against certain bacterial infections event at nanomolar concentrations. In addition to its ability to be anti-biofilm and antimicrobial, LL37 plays an important role in regulating the balance of pro- and anti-inflammatory molecules and appears to play a role in balancing inflammation with healing.

[0028] OBJECT OF THE INVENTION

[0029] It is an object of embodiments of the invention to provide improved treatment modalities for management of periodontal disease. In this connection it is also an object of embodiments of the invention to provide a composition useful in treatment of periodontal disease.

[0030] LEGENDS TO THE FIGURES

[0031] Fig. 1 : Bar graph showing development in periodontal pocked depth after treatment for 12 days with LL37 formulated in poloxamer.

[0032] Fig. 2 : Bar graph showing development in plaque index after treatment for 12 days with LL37 formulated in poloxamer.

[0033] Fig. 3 : Bar graph showing development in gingival index after treatment for 12 days with LL37 formulated in poloxamer.

[0034] Fig. 4: Bar graph showing development in periodontal disease score after treatment for 12 days with LL37 formulated in poloxamer.

[0035] Fig. 5 : Bar graph showing development in periodontal disease score after treatment for 12 days with LL37 formulated in GMO.

[0036] SUMMARY OF THE INVENTION

[0037] It has been found by the present inventor(s) that periodontal disease advantageously can be treated by controlled application of the peptide LL37 (SEQ ID NO: 1) to the affected periodontal pocked - by providing LL37 as a constituent of a controlled release gel, which has a sufficiently high viscosity to remain in a periodontal pocket for a prolonged period i.a. of time.

[0038] So, in a 1staspect the present invention relates to a composition suitable for treatment of periodontal disease by application to periodontal pockets in humans, comprising as an active ingredient peptide LL37 (SEQ ID NO: 1) in admixture with a gelling agent or gelling mixture, wherein said gelling agent or gelling mixture exhibits increased viscosity upon introduction into the environment of a periodontal pocket, wherein said increased viscosity is triggered by any one of

[0039] 1) an increase of the composition in temperature to about 37°C as present in a periodontal pocket, or

[0040] 2) by contact of the composition with water as present in a periodontal pocked, or

[0041] 3) by contact of the composition with calcium ions, and wherein said increased viscosity causes protracted release of LL37 from the composition.

[0042] A 2ndaspect of the invention relates to an injection device such as a syringe comprising a closed compartment containing a composition of the 1staspect of the invention as well as any embodiment thereof disclosed herein, as well as a outlet component dimensioned to facilitate injection of the composition into a periodontal pocket.

[0043] A 3rdaspect of the invention relates to a method for treatment, including prophylactic treatment, of periodontal disease, the method comprising injection of an effective amount of a composition comprising LL37 (SEQ ID NO: 1) into one or more periodontal pockets in the oral cavity of a subject in need thereof, wherein the composition further comprises a gelling agent / mixture and wherein the composition increases its viscosity at the time of injection from at most 1000 mPa.s to at least 4 Pa.s in the periodontal pocket to attain a semi-solid form, whereby LL37 exhibits protracted release from the composition so as to exert antimicrobial and anti-inflammatory effects in the periodontal pocket(s) for a period of at least 24 hours.

[0044] Aspects closely related to the 3rdaspect of the invention are 1) composition of the 1staspect of the invention or as defined in the 2ndaspect of the invention for use as medicament, in particular for use in a method for treatment of periodontal disease; in these closely related aspects the composition is preferably for use in the method of the 3rdaspect of the invention as well as any embodiment thereof disclosed herein.

[0045] DETAILED DISCLOSURE OF THE INVENTION

[0046] Definitions

[0047] "LL37" denotes the peptide having the sequence SEQ ID NO: 1 (LLGDFFRKSK EKIGKEFKRI VQRIKDFLRN LVPRTES). As used herein, the term LL37 includes salt forms, in particular the acetate salt of LL37. A "gelling agent" or "gelling mixture" is a part of a composition, which can confer gel properties to a composition in which the gelling agent or gelling mixture is present.

[0048] "Viscosity" is the resistance of a fluid to changes in shape. It is by nature temperature dependent and measured using standardized equipment at fixed temperatures. The unit for viscosity is Pascals x seconds, Pa.s.

[0049] "Protracted release" (or "sustained release") denotes the quality of non-immediate release of a substance from a composition, whereby an effective concentration of the substance will persist to exist in a desired location in the body. It will be understood that protracted release in the present context is defined as protracted release of a composition of the invention when it has attained a high viscosity and is present in a periodontal pocket in the oral cavity. In this situation, the protracted release is at least 24 hours, such as at least 36, at least 48, at least 60, and at least 72 hours.

[0050] Specific embodiments of the invention

[0051] 1staspect of the invention as well as embodiments thereof

[0052] The 1staspect of the invention relates to a composition suitable for treatment of periodontal disease by application to periodontal pockets in humans, comprising as an active ingredient peptide LL37 (SEQ ID NO: 1) in admixture with a gelling agent or gelling mixture, wherein said gelling agent or gelling mixture exhibits increased viscosity upon introduction into the environment of a periodontal pocket, wherein said increased viscosity is triggered by any one of 1) an increase of the composition in temperature to about 37°C as present in a periodontal pocket, or 2) by contact of the composition with water as present in a periodontal pocked, or 3) by contact of the composition with calcium ions, and wherein said increased viscosity causes protracted release of LL37 from the composition.

[0053] It is preferred that L37 is present in the form of a salt, preferably in the form of an acetate salt, which provides a higher degree of water-solubility to the LL37 molecule; generally, LL37 has a water solubility at 25°C of 50 mg / ml, whereas the acetate salt has a higher water solubility of about 100 mg / ml. Methods for producing LL37 are well-known to the skilled person - one possibility is to produce the peptide recombinantly in a suitable expression system, but due to its short length, the peptide is preferably produced synthetically, e.g. by the standard technique for solid-phase peptide synthesis originally developed by Merrifield. According to the present invention, it is preferred that LL37 or the salt thereof is present in the composition in the form of free monomers, i.e. in soluble form or in suspension, but without being part of a larger structure such as a nanoparticle.

[0054] The exact concentration of LL37 in the composition can vary, since it is the amount ultimately administered to a periodontal pocket over time that is decisive for the therapeutic effect. Previously, it has been demonstrated that a concentration of LL37 of 37 pg / ml (corresponding to « 0.0037% w / w) provides a therapeutic effect on cells, but in the present invention it is of relevance to administer a higher amount to be released over a prolonged period. For instance, it is desirable to obtain a concentration of at least 200 pg / ml, corresponding to about 0.02% w / w, but as is shown in the examples, much higher concentrations can be provided (0.1% w / w is exemplified, corresponding to approximately 1 mg / ml). Hence, the composition preferably comprises LL37 in a concentration between 0.05 (w / w) and 0.40% (w / w), such as in a range selected from 0.05%-0.30%, 0.01%-0.25%, 0.02%-0.20%, 0.03%-0.18%, 0.04%-0.16%, 0.05%-0.15%, 0.06%-0.14%, 0.07%-0.13%, 0.08%-0.12%, and 0.09%-0.11%.

[0055] The increase in viscosity of the composition after application to a dental pocket is an important feature of the present invention. Since it is necessary to introduce the composition into a periodontal pocket, typically from a manually operated syringe, the viscosity during injection must be of such a low value that the composition can be distributed freely in the periodontal pocket, but on the other hand, the viscosity should not be so low that that the composition is unable to remain until the increase in viscosity has taken place. So typically the viscosity prior to application must be somewhat higher than the viscosity of water at room temperature (i.e. higher than 1 mPa.s at 20°C , but lower than a value that can compromise the injection of the composition. Hence the increased viscosity attained after application is typically from a viscosity value at injection of at most 1,000 mPa.s (but higher than the above-indicated viscosity of water) to a post-injected value of at least 4 Pa.s whereby the composition attains a semi-solid form. Again, the increase should ensure that the composition in the dental pocket maintains a patient-compliant "soft" texture. Typically, the increased viscosity is an increase from a value of at most 500 mPa.s to a value of at least 4.5 Pa.s.

[0056] When the composition attains the increased viscosity as described in option 1 i.e. when contacted with water), the gelling agent / gelling mixture typically comprises a monoglyceride as the principal component, i.e. as the component in highest concentration. In addition, the composition will typically contain at least one triglyceride and water (the latter in very small amounts), where the triglyceride is added in order to adjust the viscosity, cf. Example 1. Water will typically be present in a concentration between 0.7% (w / w) and 1.5% (w / w), such as in a concentration between 0.8% (w / w) and 1.3% (w / w), and preferably in a concentration between 0.9% (w / w) and 1.1% (w / w). The viscosity-modifying triglyceride is typically present in a concentration between 4% (w / w) and 15%(w / w), such as between 5% (w / w) and 13% (w / w), preferably between 6% (w / w) and 11% (w / w), more preferably between 7% (w / w) and 9% (w / w).

[0057] When the composition attains the increased viscosity as described in option 2 ( / .e. when subjected to a temperature increase up to the temperature typically found in a periodontal pocket, the gelling agent / gelling mixture preferably comprises an aqueous mixture of polymers exhibiting increases in viscosity when subjected to an increase in temperature from 20°C to 37°C. Examples of such polymers are (polyethylene oxide-b-propylene oxide b- ethylene oxide) triblock co-polymers, for example Poloxamer 407 or 188. The polymers of the composition will then be present in a preferred concentration between 15% (w / w) and 30% (w / w), such as between 17% (w / w) and 27% (w / w), preferably between 19% (w / w) and 25% (w / w).

[0058] When the composition attains the increased viscosity as described in option 3 ( / .e. when contacted with calcium ions), the composition preferably comprises a polymer, which is cross-linked when contacted with calcium ions. An example of such a polymer is pectin, and according to the invention, the pectin is preferably an amidated low methoxy (LMA) pectin. In typical compositions of this sub-type, the pectin is present in an amount of between 0.5% (w / w) and 15% (w / w), preferably in a range selected from 0.6% (w / w) - 14% (w / w), 0.7% (w / w) - 13% (w / w), 0.8% (w / w) - 12% (w / w), 0.9% (w / w) - 11% (w / w), 1.0% (w / w) - 10% (w / w), 1.5% (w / w) - 9% (w / w), 1.8% (w / w) - 8% (w / w), 2.0% (w / w) - 7% (w / w), and 2.5% - 6% (w / w).

[0059] 2ndaspect of the invention and embodiments thereof

[0060] The 2ndaspect relates to an injection device such as a syringe comprising a closed compartment containing a composition according to any one of the preceding claims and an outlet component dimensioned to facilitate injection of the composition into a periodontal pocket.

[0061] This injection device can be in any of a plurality of forms - often, the device will be a traditional syringe equipped with a needle / cannula having a configuration, which makes it simple for a practitioner to introduce the composition into a periodontal pocket. But the device may also be more technical advanced and for instance include means for automated delivery of the composition via e.g. motorized action. In some versions (particularly useful when using disposable injection devices), the injection device comprises one single unit dose of the composition, but in other version, the injection device comprises a plurality of unit doses and is equipped with a component that facilitates delivery of separate unit doses to periodontal pockets (such as motor unit that step-wise can eject predefined unit doses from a larger volume of the composition).

[0062] At any rate, such unit doses to be administered will typically have a volume of 0.05-1.0 ml, such as 0.07-0.5 ml, and preferably 0.1-0.3 ml.

[0063] 3rdaspect of the invention and embodiments thereof

[0064] The 3rdaspect of the invention relates to a method for treatment, including prophylactic treatment, of periodontal disease, the method comprising injection of an effective amount of a composition comprising LL37 (SEQ ID NO: 1) into one or more periodontal pockets in the oral cavity of a subject in need thereof, wherein the composition further comprises a gelling agent / mixture and wherein the composition increases its viscosity at the time of injection from at most 1000 mPa.s to at least 4 Pa.s in the periodontal pocket to attain a semi-solid form, whereby LL37 exhibits protracted release from the composition so as to exert antimicrobial and anti-inflammatory effects in the periodontal pocket(s) for a period of at least 24 hours. In preferred embodiments, the composition comprising LL37 is a composition of the first aspect of the invention and any embodiment thereof disclosed herein.

[0065] Typically the method of the 3rdaspect is preceded by a physical of surgical periodontal disease treatment of the one or more periodontal pockets. Such physical and surgical treatments are e.g. those detailed above when discussing the background of the invention, since the present treatment conveniently can be applied as follow-up or adjuvant treatment. Thus, the physical or surgical step is preferably selected from the group consisting of scaling, root planning, flap surgery, bone grafting, guided tissue regeneration, soft tissue graft, and gum graft.

[0066] The period of at least 24 hours may conveniently be longer, such as at least 36, at least 48, at least 60, and at least 72 hours.

[0067] The injection of the composition is normally preceded or followed by a step of injection of a calcium ion containing solution when the composition is a composition that attains a higher viscosity when contacted with calcium ions, i.e. as described as option 3 in respect of the 1staspect of the invention; here, the amount of calcium ions injected are effective to increase viscosity of the composition. As described in the examples the calcium ion containing solution will typically be aqueous and and also contain a polymer such as a poloxamer in order to ensure that the calcium ion containing composition stably remains in the periodontal pocket.

[0068] The viscosity of the composition at the time of injection is preferably in the range 1-1000 mPa.s, such as 50-500 mPa.s, 60-400 mPa.s, 70-300 mPa.s, 80-250 mPa.s, and 90-220 mPa.s. It will be understood that the viscosity prior to injection can vary considerably (for instance, storage of a temperature-sensitive composition as described above and in the examples at elevated temperature will cause an increase in viscosity. This is however of less relevance as long as it is ensured that the desired lower viscosity is attained at the time of injection.

[0069] The viscosity after injection is preferably increased to at least 4.5 Pa.s, such as at least 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, and at least 13.0 Pa.s. The higher viscosity of the composition in the periodontal pocket can hence vary widely - it is merely important to ensure that the texture of the composition is patient compliant.

[0070] The injected volume in each periodontal pocket is preferably the volume of a unit dose discussed above in respect of the 2ndaspect of the invention, i.e. 0.05-1.0 ml, such as 0.07- 0.5 ml, and preferably 0.1-0.3 ml. In line with this, it is in the practice of the 3rdaspect preferred that the composition is injected with an injection device discussed under the 2ndaspect of the invention and the embodiments therof.

[0071] The method of the 3rdaspect is typically applied to human subjects. In this connection it is of special relevance to target patients that exhibit a deficiency (compared to the average population) of LL37, e.g. due to lower levels of this expression product of due to increased metabolization thereof. However, other animals also suffer from periodontal disease, in particular domesticated animals such as dogs and cats as well as animals kept in zoological gardens. Hence, the presently described method of the 3rdaspect of the invention generally finds use in treatment of periodontal disease in mammals, in particular animals that are in human care. PREAMBLE TO EXAMPLES

[0072] Formulation principles

[0073] The aim is to incorporate LL37-peptide (the peptide having SEQ ID NO: 1) into a low viscous formulation e.g. less than 1000 mPa.s in a concentration of 1 mg per ml to be applied into the gingival dental pocket environment where it will changes into a high viscous semisolid form e.g. over 5 Pa.s.

[0074] When the formulation is applied into the dental pocket of a patient it is typically administered manually through a syringe flushing needle at an appropriate force of at most 5 Kp. From the semisolid dental gel formed in the dental pocket the release of LL37 will be retarded aiming at a release time of more than 24 hours to be effective in treating periodontal disease by exerting its anti-microbial and anti-inflammatorily effect locally in the dental pocket.

[0075] It is anticipated that the volume of the dental gel injected into the dental pocket is between 0.1-0.3 ml.

[0076] The formulation principles of dental gels described below are:

[0077] Principle 1. Gel based on Glycerol monooleate + triglyceride (e.g. sesame oil), originally developed for Alpharma-Dumex with metronidazole as an active substance and marketed under the name Elyzol 25%. The gel is a liquid when injected but becomes semi-solid when in contact with water in the dental pocket causing a crystalline liquid phase to be formed.

[0078] Principle 2. Gel based on a thermo-gelling polymer, e.g. a Poloxamer mixture of types 407 and 188. The formulation has low viscosity at room temperature and forms a semi solid gel at body temp.

[0079] Principle 3. Gel based on pectin (LMA), which forms a gel by cross-linking with calcium ions. LMA is a specific type of pectin where a proportion of the methyl ester groups are converted to amide groups. This facilitates cross-binding of the polymer chains at very low calcium concentrations. It is intended first to apply a low viscous formulation containing calcium chloride into the dental pocket e.g. one droplet followed by a LMA pectin solution which then forms a semisolid pectin gel by cross-bonding with calcium ions. EXAMPLE 1

[0080] Gel according to principle 1

[0081] Reference is generally made to references 1 and 2.

[0082] Pure monoglycerides can form different liquid crystalline phases when exposed to water. When a peptide, particularly LL37, is incorporated in the lipid mixture, this mixture can be injected into a dental pocket where it takes up water and forms a liquid crystalline form. This gel has a very high viscosity and does not change behaviour in the environment (« 37°C in the gingival fluid) over several days. The incorporated LL37 peptide will consequently be released slowly as the matrix is eroded.

[0083] The melting point of pure >80% glycerol monooleat (termed "GMO" herein) is approximately 35°C. The melting point and the crystallinity / viscosity can be adjusted / reduced by adding a triglyceride, e.g. sesame oil and soyabean oil or alternatively glycerol or cholesterol.

[0084] With 8% sesame oil added, the melting point of GMO drops to around 30°C.

[0085] If the viscosity of this mixture is measured at 30°C, 35°C, and 37°C, the respective viscosities are 207, 127 and 110 mPa.s (measured at a shear rate of 168 / s by a cone-plate method).

[0086] Hence, peptides are surprisingly well shielded in the gel matrix and released slowly.

[0087] If a mixture of 92% GMO and 8% Sesame oil is stored in a ready to use container, e.g. a dental cartridge, then the viscosity of this mixture will increase over time, as the mixture becomes more and more crystalline. However, small amounts of water up to 1% surprisingly prevent crystallinity and preserve the properties that ensure an optimal injection force from the cartridge when applied into the dental pocket.

[0088] Example of composition utilizing principle 1 :

[0089] All percentages provided in the following are by weight (w / w).

[0090] Components:

[0091] 1) 91% GMO (Peceol from Gattefosse) 2) 8% Sesame oil (Ph. Eur.)

[0092] 3) 1% purified water

[0093] 4) 0.1% LL37

[0094] Procedure:

[0095] Heat to max. 60°C and mix constituents 1-3 with a spatula in a small beaker. When the composition is uniform, cool to 35°C degrees and then add LL37 by stirring.

[0096] For different viscosity performance and release times, the Sesame oil amount can be varied.

[0097] Added water can be as low as 0.7%.

[0098] During preparation and storage it is advantageous to prevent exposure to oxygen, as the GMO and Sesame oil contain unsaturated fatty acids. Also, excessive heat will degrade the mixture. Antioxidants can optionally be added to prolong the shelf-life of the composition.

[0099] For different viscosity performance and release times, the Sesame oil amount can be varied.

[0100] Added water can be as low as 0.7%.

[0101] 1) Glycerol monooleate (Cithrol) : 1155.78 g

[0102] 2) Sesame oil (Ph. Eur.) : 164.54 g

[0103] 3) purified water: 64.68 ml

[0104] 4) LL37: 3.637 g

[0105] Procedure:

[0106] GMO is melted, weighed and dispensed into a 2 litre laboratory bottle (Duran). Sesame oil is weighed and added to the GMO. Purified water is weighed and added under vigourous stirring until the composition is uniform, cooled after which LL37 is added by stirring. EXAMPLE 2

[0107] Gel according to principle 2

[0108] General reference is made of Reference 3.

[0109] Mixtures of polymers (polyethylene oxide-b-propylene oxide-b-ethylene oxide) triblock copolymers (PEG-PPO.PEO), such as Poloxamer 407 (Pluronic F127, BASF) and Poloxamer 188 (Pluronic F68, BASF) are liquids at room temperature and, depending on the composition and ratio between types, form semi-solid gels with different viscosities at body temp of 37°C.

[0110] All percentages provided in the following are by weight (w / w).

[0111] 1) Poloxamer 407 20%

[0112] 2) Purified water 80 %

[0113] 3) LL37 0.1%

[0114] Procedure 1 :

[0115] Poloxamer and water were mixed in a vial and placed at 4°C until a clear solution was obtained. LL37 was dissolved by stirring. Viscosity at 10°C = 0.03 Pa.s. Viscosity at 37°C = 4.6 Pa.s.

[0116] Poloxamer 407 20%

[0117] Poloxamer 188 10%

[0118] Purified water 70 %

[0119] LL37 0.1%

[0120] Procedure 2:

[0121] Poloxamer and water were mixed in vial and placed at 4°C until a clear solution was obtained. LL37 was dissolved by stirring. Viscosity at 10°C = 0.15 Pa.s. Viscosity 37°C = 5.5 Pa.s. Components 3:

[0122] Poloxamer 407 24%

[0123] Purified water 76 %

[0124] LL37 0.1%

[0125] Poloxamer and water were mixed in a vial and placed at 4°C until a clear solution was obtained. LL37 was dissolved by stirring. Viscosity at 10°C = 0.05 Pa.s. Viscosity 37°C = 6.15 Pa.s.

[0126] Components 4:

[0127] Poloxamer 407 24%

[0128] Poloxamer 188 10%

[0129] Purified water 66 %

[0130] LL37 0.1%

[0131] Procedure 4:

[0132] Poloxamer and water were mixed in vial and placed at 4°C until a clear solution was obtained. LL37 was dissolved by stirring. Viscosity at 10°C = 45 Pa.s. Viscosity 37°C = 11.9 Pa.S.

[0133] Further examples of compositions according to principle 2

[0134] Stock solution :

[0135] Poloxamer 407: 64.89 g

[0136] Poloxamer 188: 26.93 g

[0137] Purified water: 174.14 g

[0138] The 3 components are mixed at 5°C and left overnight in a refrigerator.

[0139] To produce two final solutions («0.02% and «0.04% LL37)for preclinical testing, LL37 is dissolved in water and mixed with the stock solution as follows:

[0140] A) 24.07 mg LL37 dissolved in 1.03 g purified water and mixed with 93.709 g stock solution,

[0141] B) 30.61 mg LL37 dissolved in 0.686 mg water and mixed with 59.266 g stock solution.

[0142] Components 6:

[0143] Poloxamer 407 24%

[0144] Poloxamer 188 10% Purified water 66 %

[0145] LL37 0.1%

[0146] EXAMPLE 3

[0147] Gel according to principle 3

[0148] General reference is made to reference 4.

[0149] Pectin polymer of the LMA quality (methylated and amidated pectin) is effectively crosslinked in the presence of calcium ions resulting in significantly increase in viscosity of the gel. The calcium concentration in saliva is low (< 1 pg total) and insufficient to cross-link pectin. The principle is based on initial injection of one droplet (50 mg) of an aqueous calcium chloride solution with a gelling agent e.g. Poloxamer 407 into the dental pocket followed by injection of an aqueous solution of LMA pectin (0.1-0.3 ml) into same dental pocket to form a semisolid gel by cross-linking.

[0150] An appropriate concentration of LMA pectin aqueous solution is in the range of 3-5% resulting in viscosity range of approx. 100-1000 mPa.s at 25°C (resulting pH=4). After crossbonding the viscosity will be increased significantly. Calcium concentration calculations below is based on maximal cross-bonding corresponding to 30 mg Ca-ions per gram pectin.

[0151] Examples of compositions

[0152] All percentages provided in the following are by weight (w / w).

[0153] Components of calcium solution 1 (applied to 3% LMA solution below) : Calcium chloride 1.5% (one droplet 0.27 mg Calcium) Poloxamer 407 25% Purified water 74%

[0154] Components of LMA pectin 3% solution:

[0155] LMA pectin 3%

[0156] Purified water 97% LL37 0.1% Procedure 1 :

[0157] Pectin is dissolved in 80°C hot water by stirring and after cooling to room temperature LL37 is dissolved.

[0158] Components of calcium solution 2 (applied to 5% LMA solution below') :

[0159] Calcium chloride 2.5% (one droplet 0.45 mg Calcium)

[0160] Poloxamer 407 25%

[0161] Purified water 74%

[0162] LMA pectin 5% solution:

[0163] LMA pectin 5%

[0164] Purified water 95%

[0165] LL37 0.1%

[0166] Procedure 2:

[0167] Pectin is dissolved in 80°C hot water by stirring and after cooling to room temperature LL37 is dissolved.

[0168] EXAMPLE 4

[0169] In vivo testing

[0170] Summary

[0171] The objective of this study was to determine the efficacy of two oral gel formulations containing LL37 as treatment for periodontal disease.

[0172] Ten (10) pure-bred Beagle dogs that had previously demonstrated suitable gingival pocket depths and severity of periodontal disease were selected for the study to assess the efficacy and safety of two test articles for periodontal disease.

[0173] Twelve (12) sequential daily applications of test articles was performed. The left side of the mouth was treated with the designated test article; the right side of the mouth was not treated and assessed as a control.

[0174] More details about the study are provided in the following : Animals

[0175] Animals were primarily group housed for the duration of the study. Following anaesthesia for dental assessments, animals were individually housed for as long as deemed necessary by the attending Veterinarian to mitigate animal welfare concerns during recovery. Animals were also placed briefly into individual metabolic cages to facilitate daily husbandry. Individual cages consisted of a slatted floor with water bowl holders affixed to the wall of the enclosure. Room and cage allocations for the dogs provided each animal with at least the minimum acceptable square footage of personal space according to the recommendations of the CCAC. Enrichment, such as beds and social interaction was provided over the course of the study. Environmental management including temperature regulation, ventilation and humidity regulation and lighting were provided, maintained and controlled according to facility SOP. A combination of commercially acceptable artificial lighting and natural light was provided for the animals. The photoperiod was approximately 12 hours (7:00 to 19:00, maintained on a timer). Temperature was electronically controlled and set to maintain the animal housing room in a temperature range of 18°C to 28°C. The housing room's ventilation was individually controlled by air exchangers and is set at a level required to maintain the environmental temperature levels and allow for fresh air to be supplied continually. Feed was provided in stainless steel bowls that were cleaned daily, and water wa provided to the animals via stainless steel bowls in each cage or housing room ad libitum via automatic water sippers. Feed was provided once daily in the form of a standard commercial diet (Purina ProPlan All Ages Sport Active 27 / 17- Chicken and Rice Formula Dry Dog Food) with tap water added. Animals were fasted for a minimum of 8 hours prior to assessments. On study days where anaesthesia was required, water was withheld for at minimum 30 minutes prior to sedation. On dosing days, water was withheld for at minimum 30 minutes post-dosing.

[0176] Treatment and examination

[0177] 6 dogs were randomly allocated to test group 01 (GMO gel) and 4 dogs to test group 02 (poloxamer gel).

[0178] On Study Day 0, animals were sedated, put under general anaesthesia (utilizing a cuffed endotracheal tube with isoflurane), underwent dental assessments, and had digital photographs of the mouth taken. Following baseline dental evaluations, animals received their respective test article (into 2-4 designated gingival pockets per animal) while under anaesthesia. On Days 1 to 11, application of the test articles to the gumline was performed on conscious animals. Dental assessment and digital photographs was repeated under anaesthesia on Day 12. Administration was performed using a 20-gauge cannula or blunt needle attached to the syringe pre-filled with 0.5 ml of the test article. Following dental assessments on Day 0, the assigned treatment of either test article was administered into 2-4 of the designated gingival pockets.

[0179] A small amount of product was expressed into the pocket to ensure the pocket was filled approximately to the gingival margin according to the following procedure:

[0180] Drying the surface of the tooth to be treated.

[0181] Drying the pocket to be treated.

[0182] Gently inserting the cannula / blunt needle into pocket, taking care to keep it parallel to the tooth.

[0183] Pressing the syringe slowly to completely fill the pocket Withdrawing the cannula.

[0184] The remaining volume of the dose was applied to the gingival margin in the same side of the mouth designated for treatment.

[0185] For the remaining treatment period, the daily dose of the assigned test article was applied to the gingival margin in the same side of the mouth designated for treatment, paying specific attention to cover the gumline of previously treated teeth with gingival pockets. The dogs received twelve (12) doses of their assigned treatment (Days 0 to 11).

[0186] Digital photographs of all four quadrants of the mouth were taken following anaesthetic induction prior to dental assessments and test article administration on Day 0. Further, each dog underwent pocket depth assessment, while maintained under general anaesthesia. The assessment included examination of pocket depth (measurement via probing the sulcus; a normal gingival sulcus (probing) depth is 0.5-3 mm in dogs). Probing was performed on six sites of the tooth : mesiobuccal (MB), mid-buccal (B) distobuccal (DB), mesiolingual / palatal (ML), mid-lingual / palatal (L) and the distolingual / palatal (DL).

[0187] In addition to probe depth measurements, animals underwent a general periodontal assessment to establish their plaque index, gingival index, furcation index, mobility index and the degree of bleeding on probing.

[0188] These parameters were scored based on the following criteria:

[0189] Plaque Index (PI) - Whole Mouth Score 0 = No plaque

[0190] 1 = Thin film along gingival margin covering < 1 / 3 of buccal tooth surface 2 = Moderate accumulation of plaque covering 1 / 3 to 2 / 3 of buccal tooth surface

[0191] 3 = Abundant soft plaque covering > 2 / 3 buccal tooth surface

[0192] Gingival Index (GI) - Whole Mouth Score

[0193] 0 = Normal gingiva

[0194] 1 = Marginal gingivitis, mild swelling, some colour change, no BOP 2 = Moderate swelling and inflammation of gingiva, BOP

[0195] 3 = Marked swelling and inflammation, spontaneous bleeding

[0196] Furcation Index (F) - Scored on Teeth with Designated Gingival Pockets for Test Article Administration

[0197] Fl = Probe goes into furcation and up to 1 / 3 buccolingual crown width of multirooted tooth

[0198] F2 = Probe goes up to 2 / 3 buccolingual crown width of multirooted tooth F3 = Probe goes all the way through buccolingual crown width of multirooted tooth

[0199] Mobility Index (M) - Scored on Teeth with Designated Gingival Pockets for Test Article Administration

[0200] Ml = Slight mobility > 0.2 mm, less than 0.5 mm

[0201] M2 = Moderate mobility, > 0.5, less than 1 mm in any lateral direction M3 = Severe mobility > 1 mm or intruded into socket or can be extruded out of socket

[0202] Animal observations were conducted and recorded twice daily while animals are on study for any signs that would not be expected in normal dogs relating to general appearance, behaviour, and attitude to identify and note any abnormality or undesirable concern occurring to an animal prior to or during the administration of the investigational product. This allowed that appropriate intervention could be initiated if necessary.

[0203] Results and discussion

[0204] At the conclusion of the study, it turned out that all 4 dogs receiving the gel according to principle 2 (the poloxamer formulation) consistently exhibited an improvement in their periodontal status compared to baseline. Combined results are presented in Figs. 1-4, where Fig. 1 shows the improvement in pocket depth, Fig. 2 shows the improvement in plaque index, Fig. 3 shows the improvement in gingival index, and Fig. 4 shows the improvement in periodontal disease overall.

[0205] The experiment with GMO-based gels generally did not show any improvement in pocket depth, plaque index or gingival index, but a general improvement in periodontal disease was found (Fig. 5).

[0206] It is noted that the experiment due to the limited number of dogs and the short experimental period was not powered to provide statistically significant data; but in view of the fact that all dogs in the poloxamer group exhibited an improvement in their periodontal scores, it appears safe to conclude that the method is effective over a longer time span. Also the general improvement in periodontal disease observed in the GMO treated dogs suggest that that treatment is effective.

[0207] LIST OF REFERENCES

[0208] 1. Norling T. et al. Formulation of a drug delivery system based on a mixture of monoglycerides and triglycerides for use in the treatment of periodontal disease. Drug Delivery System. 1992, 687-692.

[0209] 2. Hansen J. Use of fatty acid esters as bioadhesive substances. US 6,228,383.

[0210] 3. Zhang K. et al. Poloxamer in situ hydrogels for controlled delivery of hydrophilic macromolecules after intramuscular injection in rats. Drug Delivery. 2015;22(3), 375-383.

[0211] 4. Genu®Pectin handbook. Kelco.

Claims

CLAIMS1. A composition suitable for treatment of periodontal disease by application to periodontal pockets in humans, comprising as an active ingredient peptide LL37 (SEQ ID NO:1) in admixture with a gelling agent or gelling mixture, wherein said gelling agent or gelling mixture exhibits increased viscosity upon introduction into the environment of a periodontal pocket, wherein said increased viscosity is triggered by any one of1) an increase of the composition in temperature to about 37°C as present in a periodontal pocket, or2) by contact of the composition with water as present in a periodontal pocked, or3) by contact of the composition with calcium ions, and wherein said increased viscosity causes protracted release of LL37 from the composition.

2. The composition according to claim 1, wherein LL37 is in the form of an acetate salt.

3. The method according to claim 1 or 2, wherein LL37 or the salt thereof is in admixture with the gelling agent or gelling mixture as free LL37 monomers.

4. The composition according to any one of the preceding claims, wherein LL37 is present in a concentration between 0.05 (w / w) and 0.40% (w / w), such as in a range selected from 0.05%-0.30%, 0.01%-0.25%, 0.02%-0.20%, 0.03%-0.18%, 0.04%-0.16%, 0.05%- 0.15%, 0.06%-0.14%, 0.07%-0.13%, 0.08%-0.12%, and 0.09%-0.11%.

5. The composition according to any one of the preceding claims, wherein the increased viscosity is an increase from a value of at most 1,000 mPa.s to a value of at least 4 Pa.s whereby the composition attains a semi-solid form6. The composition according to claim 5, wherein the increase is from a value of at most 500 mPa.s to a value of at least 4.5 Pa.s.

7. The composition according to option 1 in any one of the preceding claims, wherein the gelling agent / gelling mixture comprises a monoglyceride as the principal component.

8. The composition according to claim 7, which further comprises at least one triglyceride and water.

9. The composition according to claim 8, wherein the water is present in a concentration between 0.7% (w / w) and 1.5% (w / w).

10. The composition according to claim 9, wherein the water is present in a concentration between 0.8% (w / w) and 1.3% (w / w)11. The composition according to claim 9, wherein the water is present in a concentration between 0.9% (w / w) and 1.1% (w / w)12. The composition according to any one of claims 8-11, wherein the triglyceride is present in a concentration between 4% (w / w) and 15%(w / w)13. The composition according to any one of claims 8-11, wherein the triglyceride is present in a concentration between 5% (w / w) and 13% (w / w)14. The composition according to any one of claims 8-11, wherein the triglyceride is present in a concentration between 6% (w / w) and 11% (w / w)15. The composition according to any one of claims 8-11, wherein the triglyceride is present in a concentration between 7% (w / w) and 9% (w / w).

16. The composition according to option 2 in any one of claims 1-6, wherein the gelling agent / gelling mixture comprises an aqueous mixture of polymers exhibiting increases in viscosity when subjected to an increase in temperature from 20°C to 37°C17. The composition according to claim 16, wherein the polymers are (polyethylene oxide- b-propylene oxide b-ethylene oxide) triblock co-polymers18. The composition according to claim, wherein the polymers are Poloxamer 407 or Poloxamer 18819. The composition according to any one of claims 16-18, wherein the polymers are present in a concentration between 15% (w / w) and 30% (w / w)20. The composition according to any one of claims 16-18, wherein the polymers are present in a concentration 17% (w / w) and 27% (w / w)21. The composition according to any one of claims 16-18, wherein the polymers are present in a concentration between 19% (w / w) and 25% (w / w).

22. The composition according to option 3 in any one of claims 1-6, which comprises a polymer, which is cross-linked when contacted with calcium ions.

23. The composition according to claim 22, wherein the polymer is pectin24. The composition according to claim 23, wherein the pectin is an amidated low methoxy (LMA) pectin25. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 0.5% (w / w) and 15% (w / w)26. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 0.6% (w / w) and 14% (w / w)27. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 0.7% (w / w) and 13% (w / w)28. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 0.8% (w / w) and 12% (w / w)29. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 0.9% (w / w) and 11% (w / w)30. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 1.0% (w / w) and 10% (w / w)31. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 1.5% (w / w) and 9% (w / w)32. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 1.8% (w / w) and 8% (w / w)33. The composition according to claims 23 or 24, wherein pectin is present in an amount of between 2.0% (w / w) and 7% (w / w)34. The composition according to claims 23 or 24, wherein pectin is present in an amount of between and 2.5% and 6% (w / w).

35. An injection device comprising a closed compartment containing a composition according to any one of the preceding claims and comprising an outlet component dimensioned to facilitate injection of the composition into a periodontal pocket.

36. The injection device according to claim 35, which is in the form of a syringe.

37. The injection device according to claim 35 or 36 wherein the injection device comprises 1) one single unit dose of the composition or 2) a plurality of unit doses and a component that facilitates delivery of separate unit doses to periodontal pockets.

38. The injection device according to any one of claims 35-37, wherein the unit dose has a volume of 0.05-1.0 ml.

39. The injection device according to any one of claims 35-37, wherein the unit dose has a volume of 0.07-0.5 ml.

40. The injection device according to any one of claims 35-37, wherein the unit dose has a volume of 0.1-0.3 ml.

41. A method for treatment, including prophylactic treatment, of periodontal disease, the method comprising injection of an effective amount of a composition comprising LL37 (SEQ ID NO: 1) into one or more periodontal pockets in the oral cavity of a subject in need thereof, wherein the composition further comprises a gelling agent / mixture and wherein the composition increases its viscosity at the time of injection from a value of at most 1000 mPa.s to a value of at least 4 Pa.s in the periodontal pocket to attain a semi-solid form, whereby LL37 exhibits protracted release from the composition so as to exert antimicrobial and anti-inflammatory effects in the periodontal pocket(s) for a period of at least 24 hours, wherein the composition comprising LL37 is a composition according to any one of claims 1- 34.

42. The method according to claim 41, wherein the injection of the composition is preceded or followed by a step of injection of a calcium ion containing solution when the composition is a composition according to option 3 in claim 1, wherein the amount of calcium ions injected are effective to increase viscosity of the composition.

43. The method according to claim 41 or 42, wherein the viscosity at the time of injection is in the range 1-1000 mPa.s.

44. The method according to claim 41 or 42, wherein the viscosity at the time of injection is in the range 50-500 mPa.s.

45. The method according to claim 41 or 42, wherein the viscosity at the time of injection is in the range 60-400 mPa.s.

46. The method according to claim 41 or 42, wherein the viscosity at the time of injection is in the range 70-300 mPa.s.

47. The method according to claim 41 or 42, wherein the viscosity at the time of injection is in the range 80-250 mPa.s.

48. The method according to claim 41 or 42, wherein the viscosity at the time of injection is in the range and 90-220 mPa.s.

49. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 4.5 Pa.s.

50. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 5.0 Pa.s.

51. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 5.5 Pa.s.

52. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 6.0 Pa.s.

53. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 6.5 Pa.s.

54. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 7.0 Pa.s.

55. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 7.5 Pa.s.

56. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 8.0 Pa.s.

57. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 8.5 Pa.s.

58. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 9.0 Pa.s.

59. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 9.5 Pa.s.

60. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 10.0 Pa.s61. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 10.5 Pa.s.

62. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 11.0 Pa.s.

63. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 11.5 Pa.s.

64. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 12.0 Pa.s.

65. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 12.5 Pa.s.

66. The method according to any one of claims 41-48, wherein the viscosity after injection is increased to at least 13.0 Pa.s.

67. The method according to any one of claims 41-66, wherein the injected amount has the volume defined for a unit dose in any one of claims 38-40.

68. The method according to any one of claims 41-67, which is preceded by a physical of surgical step periodontal disease treatment of the one or more periodontal pockets.

69. The method according to claim 68, wherein the physical or surgical step is selected from the group consisting of scaling, root planning, flap surgery, bone grafting, guided tissue regeneration, soft tissue graft, and gum graft.

70. The method according to any one of claims 41-69, wherein the composition is injected with the injection device defined in any one of claims 35-40.

71. The method according to any one of claims 41-70, wherein the subject is a human subject.

72. The method according to claim 71, wherein the human subject exhibits a deficiency of LL37.

73. A composition according to any one of claims 1-22 or as defined in claim 41 for use as medicament.

74. A composition according to any one of claims 1-22 or as defined in claim 41 for use in a method for treatment of periodontal disease.

75. The composition for the use according to claim 74, wherein the method is as defined in any one of claims 41-72.

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