Formulations comprising ENPP1 inhibitors

Abstract

This invention generally relates to formulations comprising an ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor, kit comprising the same and methods of use comprising an ENPP1 inhibitor or a formulation comprising the same.

Description

FORMULATIONSCOMPRISING ENPP1 INHIBITORSCROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 693,651, filed September 11, 2024, the contents of which are hereby incorporated herein by reference in their entireties.FIELD OF THE INVENTION[0001 J This invention generally relates to formulations comprising an ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor, kits comprising the same and methods of use comprising an ENPP1 inhibitor or a formulation comprising the same.

[0002] BACKGROUND OF THE INVENTION

[0003] Periodontal disease (generally known as gum disease) is a disease involving the periodotium, which is a complex structure composed of both hard (alveolar bone and cememtum) and soft (gingiva or gum tissue and periodontal ligament which are connective tissue fibers that run between the alveolar bone and cememtum) tissues that surround and support the teeth. Early stage of periodontal disease is gingivitis where the gums become red and swollen due to inflammation and infection of the gums. Gingivitis is generally reversible upon improvement in oral hygiene. If left untreated, however, gingivitis progresses to mild periodontitis where bacteria seep beneath the gums, affecting the supporting bone. The gums may pull away from the affected tooth, creating pockets around them where plague and bacteria hide, and brushing and floss cannot reach. Left untreated, mild periodontitis progresses to moderate periodontitis where bacteria begin to erode the ligaments, soft tissues and bone that support the affected tooth. As periodontitis advances, bone loss continues and the affected tooth becomes loose and may eventually fall out. Periodontitis is chronic, destructive and generally irreversible if advanced too far.

[0004] Treatment of mild gum disease generally involves dental cleaning, including scaling and root planning which cleans deeper beneath the gums to remove plague and bacteria where toothbrushing and floss cannot reach. Moderate to advanced gum disease such as periodontitis may require pocket reduction surgery which involves an incision in the gums to reach the root and removal of plaque and bacteria from the root surfaces and smoothing, reshaping and regeneration of the bone around the affected tooth if necessary,which includes replacement of membranes and dental bone grafts (e.g., bone from donor bank or lab-made dental bone substitute such as hydroxyapatite).

[0005] Due to the challenges in penetrating periodontal pockets, there is a need for localized delivery of therapeutic agents to the periodontal pocket of the affected tooth.SUMMARY OF THE INVENTION

[0006] It is an object of the invention to provide formulations comprising an ENPP1 inhibitor, e.g., useful for the treatment or prophylaxis of an ENPP1 related disease or disorder, for example, for the treatment or prophylaxis of periodontal disease, particularly, periodontitis. Therefore, in the first aspect, the invention provides a composition (Composition I) comprising (i) an ENPP1 inhibitor having a structure defined by Formula I et seq., as disclosed herein, for example:Formula Iin free or pharmaceutically acceptable salt form, wherein:T is a heteroaryl (e.g., quinolinyl, quinazolinyl, isoquinolinyl) optionally substituted with one or more hydroxy, C1-6alkoxy (e.g., methoxy) and / or cyano;Li is absent (i.e., a bond connecting T and B), -OC1-6alkylene (e.g., -OCH2-) or -N(R5)-C1-6alkylene wherein R5 is H or C1-6alkyl (for example, L1 is -N(H)-CH2-);B is aryl (e.g., phenyl), 5-10 membered heterocycloalkyl, a 6-10 membered bridged heterocycloalkyl or 6-10 membered bridged heterocycloalkenyl;L2 is absent (i.e., a bond connecting T and B) or a C1-6alkylene; orL2 has the structure:d and di are points of attachment to B and HG, respectively, each Ra, Rb, Rc, and Rd are independently hydrogen, C1-6alkyl (e.g., ethyl, n-propyl, n-butyl), haloC1-6alkyl (trifluoropropyl) or Ra, Rb, and the carbon atom to which they are attachedtogether form a Cs-Cecycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclopropyl or cyclobutyl);m and n are independently 0 or 1, preferably both m and n are 1;HG is phosphonate or boronic acid,in admixture with (ii) a polymer system comprising a polylactide (PLA) polymer and N-methyl-2-pyrrolidone (NMP) present in a ratio ranging from 20:80 to 50:50, for example, the system is a solution comprising polylactide (PLA) polymer and N-methyl-2-pyrrolidone (NMP) present in a ratio of 20:80 to 40:60.

[0007] In another aspect, the disclosure provides a method for the treatment or prophylaxis of an ENPP1 related disease or disorder, e.g., for the treatment or prophylaxis of periodontal disease, particularly, periodontitis, in a subject (e.g., a human or non-human animal), comprising administering an effective amount of Formula I et seq. or Composition I et seq. as disclosed herein, to a subject in need thereof (Method I). In still another aspect, the disclosure provides Formula I et seq. or Composition I as disclosed herein, for the treatment or prophylaxis of an ENPP1 related disease or disorder, e.g., for the treatment or prophylaxis of periodontal disease, particularly, periodontitis, as described in Method I et seq. (Use 1). In still another aspect, the disclosure provides a kit comprising Composition I et seq. as disclosed herein, and instructions for using the composition in Method I et seq. as disclosed herein, e.g., for the treatment or prophylaxis of an ENPP1 related disease or disorder as disclosed herein, e.g., for the treatment or prophylaxis of periodontal disease, particularly, periodontitis (Kit 1). In another embodiment, the kit further comprises one or more syringes, preferably a two syringe mixing system comprising Composition I et seq. (e.g., wherein one syringe comprises an effective amount of an ENPP1 inhibitor as disclosed herein optionally in a solvent such as N-methyl-2-pyrrolidone (NMP), and a second syringe comprising a solution comprising polylactide (PLA) and NMP present in a ratio of 20:80 to 40:60, preferably 35:65), optionally with a needle (e.g., with a 14-25 Gauge needle, for example 25 Gauge, 23 Gauge, 16 Gauge or 14 Gauge needle).BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure la-c shows dissolution profile of three samples of 15% loading of PTGN-19cb HC1 in 25:75 PLA / NMP over time.

[0009] Figure Id shows dissolution profile of control (25:75 PLA: NMP without active) over time.

[0010] Figure 2a-b shows dissolution profile of 15% and 10% loading of PTGN-19cb HC1 in 25:75 PLA: NMP over time.

[0011] Figure 3 shows dissolution profile of 10% of loading of PTGN-19cb HC1 in 35:65 PLA: NMP over time.

[0012] Figure 4 shows the change in pocket depth after 42 days based on the administration of one dose, two doses and three doses of the composition comprising PTGN-19cb compared to cleaning alone.DETAILED DESCRIPTION OF THE INVENTION Compounds of Formula I et seq. (Formula I)

[0013] The present invention provides ENPP1 inhibitors of Formula I et seq., in free or pharmaceutically acceptable salt form, as disclosed herein (Formula I), in a formulation or composition as disclosed herein (in Composition I et seq.) for use in a method as disclosed herein (in Method I et seq.). ENPP1 inhibitors of Formula I et seq., useful for the compositions and methods of the present disclosure include the following:1.1 A compound having the structure:Formula Iin free or pharmaceutically acceptable salt form, wherein:T is a heteroaryl (e.g., quinolinyl, quinazolinyl, isoquinolinyl) optionally substituted with one or more hydroxy, C1-6alkoxy (e.g., methoxy) and / or cyano;Li is absent (i.e., a bond connecting T and B), -OCi ^alkylene (e.g., -OCH2-) or - N(R5)-C1-6alkylene wherein R5 is H or C1-6alkyl (for example, Li is -N(H)- CH2-);B is aryl (e.g., phenyl), 5-10 membered heterocycloalkyl, a 6-10 membered bridged heterocycloalkyl or 6-10 membered bridged heterocycloalkenyl; L2 is absent (i.e., a bond connecting T and B) or a C1-6alkylene; orL2 has the structure:d and di are points of attachment to B and HG, respectively, each Ra, Rb, Rc, and Rd are independently hydrogen, C1-6alkyl (e.g., ethyl, n-propyl, n-butyl), haloC1-6alkyl (trifluoropropyl) or Ra, Rb, and the carbon atom to which they are attached together form a C3-C6cycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclopropyl or cyclobutyl); m and n are independently 0 or 1, preferably both m and n are 1;HG is phosphonate or boronic acid.Formula 1.1, wherein T is selected from:Formula 1.1 or 1.2, wherein T is:OCH3Formula 1.1 or 1.2, wherein T is:Formula 1.1 or 1.2, wherein T is:^vw>IFormula 1.1 or 1.2, wherein T is:Formula 1.1 or 1.2, wherein T is:Any of formulae 1.1-1.7, where L1 is absent (i.e., a bond connecting T and B), -OC1-6alkylene (e.g., -OCH2-) or -N(R5)-C1-6alkylene wherein R5 is H or C1-6alkyl (for example, L1 is -N(H)-CH2-);Any of formulae 1.1-1.8, wherein L1 is absent (i.e., a bond connecting T and B); Any of formulae 1.1-1.8, wherein L1 is -OC1-6alkylene (e.g., -OCH2-) or -N(R5)-C1-6alkylene wherein R5 is H or C1-6alkyl (e.g., methyl), for example, L1 is -N(H)-CH2-);Any of formulae 1.1-1.10, wherein B is aryl (e.g., phenyl), 5-10 membered heterocycloalkyl, a 6-10 membered bridged heterocycloalkyl or 6-10 membered bridged heterocycloalkenyl;Any of formulae 1.1-1.11, wherein B is aryl (e.g., phenyl);Any of formulae 1.1-1.11, wherein B is 5-10 membered heterocycloalkyl (e.g., piperidin-4-yl);Any of formulae 1.1-1.11, wherein B is a 6- 10 membered bridged heterocycloalkyl or a 6-10 membered bridged heterocycloalkenyl (e.g., 8-azabicyclo[3.2.1]octan-3-yl, 3-azabicyclo[3.2.1]octan-8-yl, 2-azabicyclo[2.2.2]octan-5-yl or 8-azabicyclo[3.2.1]oct-2-en-3-yl);Any of formulae 1.1-1.11, wherein B is 8-azabicyclo[3.2.1]oct-2-en-3-yl;Any of formulae 1.1-1.15, wherein HG is a phosphonate or boronic acid;Any of formulae 1.1-1.16, wherein HG is a phosphonate;Any of formulae 1.1-1.17, wherein HG is a boronic acid;Any of formulae 1.1-1.18, wherein:T is a heteroaryl (e.g., quinazolinyl) optionally substituted with one or more Ci- ealkoxy (e.g., methoxy);Li is absent (i.e., a bond connecting T and B);B is a 6-10 membered bridged heterocycloalkenyl (e.g., 8-azabicyclo[3.2.1]oct-2-en-8yl);L2 has the structure:d and di are points of attachment to B and HG, respectively, each Rc, and Rd are hydrogen, and Ra, Rb, and the carbon atom to which they are attached together form a C3-C6cycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclopropyl or cyclobutyl); m and n are 1; andHG is a phosphonate.Any of the foregoing formulae, wherein the ENPP1 inhibitor is a compound selected from:. 0... N. - "f1' ' A ■> OH / ?? OH O P SM 6 OHBO-P:: Q HO- P»O MO-P::O OH OH OHOH OH H? ■' 6 Qin free or pharmaceutically acceptable salt form.Any of the foregoing formulae, wherein the ENPP1 inhibitor is a compound selected from:in free or pharmaceutically acceptable salt form.Any of the foregoing formulae, wherein the ENPP1 inhibitor is selected from the following:OCH3in free or pharmaceutically acceptable salt form.Any of the foregoing formulae, wherein the ENPP1 inhibitor is the compound (PTGN-19cb):OCH3in free or pharmaceutically acceptable salt form.Any of the foregoing formulae, wherein the ENPP1 inhibitor is in a pharmaceutically acceptable salt form (e.g., hydrochloride salt form).Any of the foregoing formulae, wherein the ENPP1 inhibitor is a compound of Formula I, in free or pharmaceutically acceptable salt form, wherein:T is a heteroaryl (e.g., quinolinyl, quinazolinyl, isoquinolinyl) optionally substituted with one or more hydroxy, Ci-6alkoxy (e.g., methoxy) and / or cyano;Li is absent (i.e., a bond connecting T and B);B is a a 6-10 membered bridged heterocycloalkyl or a 6-10 membered bridged heterocycloalkenyl, preferably 6-10 membered bridged heterocycloalkenyl;L2 has the structure:wherein:Ra, Rb, and the carbon atom to which they are attached together form a C3- Cecycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclobutyl);Rc, and Rd are independently hydrogen or Ci-6alkyl, preferably Rc, and Rd are both hydrogen; andm and n are 1; andHG is a -P(=O)(OH)2.mula 1.25, wherein T is selected from:mula 1.25 or 1.26, wherein T is:OCH31.28 Formula 1.25, 1.26 or 1.27, wherein B is 8-azabicyclo[3.2.1]oct-2-en-3-yl.Compositions

[0014] The compositions disclosed herein (Composition I et seq.) are useful as a controlled release composition, e.g., depot formulation, e.g., for the treatment or prophylaxis of an ENPP1 related disease or disorder. In some embodiments, the compositions disclosed herein are useful as a controlled release implant for subgingival administration, e.g., for the treatment of periodontal disease, particularly, periodontitis. Therefore, the compositions disclosed herein can be a depot formulation. In some embodiments, the compositions disclosed herein can be a flowable liquid or a gel, suitable for injection and / or implantation in a subject (e.g., human or other animal). As used herein, “flowable” refers to the ability of the composition to be injected through a medium (e.g., syringe, e.g., with a 14-25 Gauge needle, for example 25 Gauge, 23 Gauge, 16 Gauge or 14 Gauge needle) into the periodontal pocket of the subject. For example, the composition can be injected, with the use of a syringe, beneath the gum of a subject. The ability of the composition to be injected into a patient will typically depend upon the viscosity of the composition. The compositions disclosed herein therefore have a suitable viscosity, such that the composition can be forced through the medium (e.g., syringe) into the periodontal pocket of a patient, which upon exposure to an aqueous medium, such as physiological fluids or water, the composition exhibits an increase in viscosity to form a semi-solid or solid material. Therefore, present disclosure provides Composition I as follows:1.29 Composition I, wherein the PLA to NMP ratio is 25:75 or 22.5:67.5.1.30 Composition I, wherein the PLA to NMP ratio is 35:65.1.31 Composition I or any of formulae 1.1-1.30, wherein the percentage of ENPP1 inhibitor loading is 10-25 wt% of the total weight of the composition.1.32 Composition I or any of formulae 1.1-1.31, wherein the percentage of ENPP1 inhibitor loading is 15 wt%, of the total weight of the composition.Composition I or any of formulae 1.1-1.31, wherein the percentage of ENPP1 inhibitor loading is 10 wt%, of the total weight of the composition.Composition I or any of formulae 1.1-1.33, wherein: (i) the ENPP1 inhibitor or a pharmaceutically acceptable salt thereof is present in an amount equal to 10 wt% to 15 wt%, preferably 10 wt% of the total weight of the composition and (ii) PLA present in the PLA / NMP admixture in an amount between 25 wt% to 35 wt%, preferably 35 wt%, of the total weight of the PLA / NMP admixture and / or (iii) NMP present in the PLA / NMP admixture in an amount between 65 wt% to 75 wt%, preferably 65 wt%, of the total weight of the PLA / NMP admixture.Composition I or any of formulae 1.1-1.33, wherein: (i) the ENPP1 inhibitor or a pharmaceutically acceptable salt thereof is present in an amount equal to 10 wt% to 15 wt%, preferably 10 wt%, (ii) PLA is present in an amount between 25 wt% to 35 wt%, preferably 31 wt% to 32 wt% (e.g., 31.5 wt%), and / or (iii) NMP is present in an amount between 55 wt% to 65 wt%, preferably 58 wt% to 59 wt% (e.g., 58.5 wt%), of the total weight of the composition.Composition I or any of formulae 1.1-1.34, wherein the PLA has a molecular weight of 75,000-120,000.Composition I or any of formulae 1.1-1.36, wherein the composition provides controlled release of the active over one to seven days, e.g., one to two days, for example over 40 hours.Composition I or any of formulae 1.1-1.37, wherein the ENPP1 inhibitor and the PLA: NMP solution are mixed shortly before administration, preferably in a two syringe mixing system comprising the composition disclosed herein, (e.g., wherein one syringe comprises an effective amount of an ENPP1 inhibitor as disclosed herein, and a second syringe comprising a solution comprising polylactide (PLA) and N-methyl-2-pyrrolidone (NMP) disclosed herein).Formula 1.38, wherein the composition is in a flowable liquid or gel form, e.g., for application (injection) via a syringe.Formula 1.38 or 1.39, wherein the composition may be administered via a syringe, preferably the composition is provided in a two syringe mixing system comprising Composition I et seq. (e.g., wherein one syringe comprises an effective amount of an ENPP1 inhibitor as disclosed herein, and a second syringe comprising a solution comprising polylactide (PLA) and N-methyl-2-pyrrolidone(NMP) as disclosed herein), optionally with a needle e.g., a 14-25 Gauge needle, for example 25 Gauge, 23 Gauge, 16 Gauge or 14 Gauge needle.Any of formulae 1.29-1.40, wherein the composition is in a dose of about 1-10 mg, preferably 4-5 mg (e.g., per periodontal pocket).Any of formulae 1.29-1.41, wherein upon contact with an aqueous medium, such as physiological fluids or water, the composition exhibits an increase in viscosity to form a semi-solid or solid material (e.g., pellet).Any of formulae 1.29-1.42, wherein the ENPP1 inhibitor is a compound having a structure defined by Formula I et seq. as described herein (e.g., any of 1.1-1.24). Any of formulae 1.29-1.43, wherein the PLA / NMP admixture is present in about 75 wt% to 90 wt%, preferably 85 wt% to 90 wt%, preferably 90 wt%, of the total weight of the composition.Composition I or any of the foregoing, wherein the ENPP1 inhibitor is the compound of any formula 1.23 or 1.25-1.28, in free or pharmaceutically acceptable salt form.Composition I or any of the foregoing, wherein the ENPP1 inhibitor is released over a period of 30-50 hours, such as 40 hours.Composition I or any of 1.29-1.46, wherein the percentage loading of ENPP1 inhibitor is 1-25 wt% of the total weight of the composition.Composition I or any of formulae 1.29-1.30 or 1.36-1.46, wherein the percentage loading of ENPP1 inhibitor is 1-9 wt%, of the total weight of the composition, e.g., 5% of the total weight of the composition.Any of formulae 1.36-1.43, wherein the PLA / NMP admixture is present in about 75 wt% to 95 wt%, preferably 85 wt% to 90 wt%, preferably 90 wt%, of the total weight of the composition.Composition I or any of the foregoing, wherein the PLA to NMP ratio is 50:50. Composition I or any of the foregoing, wherein the PLA is a 75 / 25 poly (DL-lactide-co-glycolide.Composition I or any of the foregoing, wherein the ENPP1 inhibitor is a compound of Formula I, in free or pharmaceutically acceptable salt form, wherein:T is a heteroaryl (e.g., quinolinyl, quinazolinyl, isoquinolinyl) optionally substituted with one or more hydroxy, Ci-6alkoxy (e.g., methoxy) and / or cyano;Li is absent (i.e., a bond connecting T and B);B is a a 6-10 membered bridged heterocycloalkyl or a 6-10 membered bridged heterocycloalkenyl, preferably 6-10 membered bridged heterocycloalkenyl;L2 has the structure:wherein:Ra, Rb, and the carbon atom to which they are attached together form a C3- Cecycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclobutyl);Rc, and Rd are independently hydrogen or Ci-6alkyl, preferably Rc, and Rd are both hydrogen; andm and n are 1; andHG is a -P(=O)(OH)2.mula 1.52, wherein T is selected from:mula 1.52 or 1.53, wherein T is:OCH31.55 Formula 1.52 or 1.53 or 1.54, wherein B is 8-azabicyclo[3.2.1]oct-2-en-3-yl.Methods of Using

[0015] ENPP1 is a type II transmembrane glycoprotein containing two identical disulfide-bonded subunits, and possesses nucleotide pyrophosphatase and phosphodiesterase enzymatic activities. ENPP1 cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 may also hydrolyze nucleoside 5' triphosphates to their corresponding monophosphates and may also hydrolyze diadenosine polyphosphates. Further, ENPP1 is widely expressed in several tissues and is critical for purinergic signaling involved in platelet aggregation, muscle contraction, cell proliferation, migration, differentiation and apoptosis and therefore, plays a role in cancers as well as in cardiovascular, neurological, immunological, musculoskeletal (e.g., periodontal), hormonal, and hematological functions in mammals (Onyedibe, et al., Molecules 2019, 24, 4192). ENPP1 is also the major hydrolase of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) that activates the Stimulator of Interferon Genes (STING) pathway, important in anti-cancer innate immunity. Also, ENPP1 can function as a molecular switch to turn cold tumors hot; demonstrating that ENPP1 levels can be used as a biomarker for patient stratification (Wang et al. PNAS, 120(52): e2313693120 (2023). Therefore, inhibiting ENPP1 can enhance treatment of cancers. Recently, loss of function mutations that knock out Enppl and other genes (e.g., ANK) have been performed and showed promotion of cementogenesis in Enppl knockouts compared to control (Nagasaki, et al., J. Dent. Res. 2021, 100(6): 639-647). Therefore, inhibiting ENPP1 can enhance treatment of periodontal diseases that involve cementum loss. Further, modulation of ENPP1 activity, such as its nucleotide pyrophosphatase and / or phosphodiesterase enzymatic activities, can be utilized to treat musculoskeletal disorders, such as bone loss.

[0016] Therefore, the compounds of Formula I (e.g., any of formulae 1.1-1.24 above), the disclosed compositions (Composition I et seq.), and the disclosed kits (Kit 1) are usefulfor the treatment or prophylaxis of diseases or disorders associated with tissues that express ENPP1, where the disease or disorder involves ENPP1 activity. The diseases or disorders associated with tissues that express ENPP1, where the disease or disorder involves ENPP1 activity include without limitation, diseases or disorders associated with activation of an ENPP1 pathway. In some embodiments, the diseases or disorders associated with ENPP1 include without limitation bone loss, reduced bone mineralization, or both. In other embodiments, the diseases or disorders associated ENPP1 are selected from periodontal disease (periodontitis); autoimmune disorders; inflammatory disorders; metabolic disorders; digestive and gastrointestinal disorders; side effects from medical procedures; cancer; hematologic / blood disorders; neurological / nervous system disorders; bone marrow disorders; endocrine disorders; bone disorders associated with ageing; and combinations thereof.Specific examples of these diseases or disorders include, but are not limited to, periodontal disease (periodontitis), rheumatoid arthritis; lupus; multiple sclerosis; ankylosing spondylitis; celiac disease; inflammatory bowel disease; side effects from weight loss surgery, gastrectomy, and gastrointestinal bypass procedures; cancer; leukemia; lymphoma; multiple myeloma; sickle cell disease; stroke; Parkinson’s disease; multiple sclerosis; vertebral column injuries; thalasemia; diabetes; hyperparathyroidism; hyperthyroidism; Cushing’s syndrome; thyrotoxicosis; irregular periods; premature menopause; low levels of testosterone and estrogen in men; and combinations thereof. As such, the disclosed compositions are useful in promoting bone growth and / or bone mineralization incidental to these diseases or disorders. The compositions can also be used to promote bone growth and / or bone mineralization due to ageing. In some forms, compositions can be used to promote bone growth and / or bone mineralization in a subject suffering from a periodontal disease. In some the compositions disclosed herein can be used to promote bone mineralization, bone growth, or both, of an alveolar bone.

[0017] In another embodiment, the compounds, compositions, and kits of the present invention are useful for the treatment or prophylaxis of periodontal disease, particularly periodontitis, in a subject (e.g., a human or a non-human animal), comprising administering to the subject in need thereof, an effective amount of Formula I (e.g., any of formulae 1.1-1.24 above) or Composition I et seq. as disclosed herein.

[0018] In a particular embodiment, the present disclosure provides Method I or Use I as follows:2.1. Method I or Use I, wherein the ENPP1 related disease or disorder is periodontal disease.Method I or Use I, wherein the ENPP1 related disease or disorder is gingivitis or periodontitis.Method I or Use I, wherein the ENPP1 related disease or disorder is mild periodontitis.Method I or Use I, wherein the ENPP1 related disease or disorder is moderate periodontitis.Method 1 or Use 1, wherein the ENPP1 related disease or disorder is severe periodontitis.Method I or Use I, wherein the ENPP1 related disease or disorder is chronic periodontitis (e.g., chronic adult periodontitis).Any of the foregoing formulae, wherein the ENPP1 inhibitor is administered via subgingival application.Any of the foregoing formulae, wherein the ENPP1 inhibitor is administered to the periodontal pocket of the affected tooth.Any of the foregoing formulae, wherein Composition I et seq. is administered via a syringe, optionally with a needle e.g., a 14-25 Gauge needle, for example 25 Gauge, 23 Gauge, 16 Gauge or 14 Gauge needle.Any of the foregoing formulae, wherein Composition I et seq. may be provided in a two syringe mixing system comprising Composition I et seq. (e.g., wherein one syringe comprises an effective amount of an ENPP1 inhibitor as disclosed herein optionally in N-methyl-2-pyrrolidone (NMP), and a second syringe comprising a solution comprising polylactide (PLA) and N-methyl-2-pyrrolidone (NMP) as disclosed herein).Any of formulae 2.1-2.10, further comprising administering water (e.g., mist of water) following the application of the composition.Any of the foregoing formulae, wherein the ENPP1 inhibitor is administered once every six hours, 12 hours, 18 hours, or 24 hours; once every 2, 3, 4, 5, 6, or 7 days; once every two weeks, once every three weeks, once a month, twice a month or once every two months, once every three, four, six, nine or twelve months, as needed.Any of the foregoing formulae, wherein the method / use promotes production of the cememtum, alveolar bone or dentin relative to baseline, e.g., as measured by pocket depth reduction and / or clinical attachment levels gain (mesio-buccal, disto-buccal, mesio- lingual and disto-lingual), e.g., via dental imaging such as CT scan or C-arm scan, as well as bleeding on probing.Any of the foregoing formulae, further comprising administering one or more other active agents, e.g., antimicrobial agent or antibiotic such as doxycycline or doxycycline hyclate (e.g., sequentially or simultaneously, separately or in the same composition as the ENPP1 inhibitor).Any of the foregoing formulae, wherein the ENPP1 inhibitor or compositions comprising the same as disclosed herein are administered in combination (sequentially or simultaneously) with one or more other therapies such as scaling and root planning.Method I or Use I, or any of the foregoing formulae, wherein the ENPP1 inhibitor is administered in once a month, twice a month or three times a month.Method I or Use I, or any of the foregoing formulae, wherein the ENPP1 inhibitor is administered once every 14 days.Method I or Use I, or any of the foregoing formulae, wherein the ENPP1 inhibitor is administered once every 14 days for 28 days.Method I or Use I, or any of the foregoing formulae, wherein the method provides a mean pocket depth change of -30% to -40%, for example, a mean pocket depth change of at least -0.5mm, e.g., at least -1mm, at least -2mm, e.g., in a starting mean pocket depth of 5mm.Method I or Use I, wherein the ENPP1 related disease or disorder is bone loss, reduced bone mineralization, or both.Method I or Use I, wherein the ENPP1 related disease or disorder is selected from autoimmune disorders; inflammatory disorders; metabolic disorders; digestive and gastrointestinal disorders; side effects from medical procedures; cancer; hematologic / blood disorders; neurological / nervous system disorders; bone marrow disorders; endocrine disorders; ageing; and combinations thereof.Method I or Use I, wherein the ENPP1 related disease or disorder is periodontal disease (periodontitis), rheumatoid arthritis; lupus; multiple sclerosis; ankylosing spondylitis; celiac disease; inflammatory bowel disease; side effects from weight loss surgery, gastrectomy, and gastrointestinal bypass procedures; cancer; leukemia; lymphoma; multiple myeloma; sickle cell disease; stroke; Parkinson’s disease; multiple sclerosis; vertebral column injuries; thalasemia; diabetes; hyperparathyroidism; hyperthyroidism; Cushing’s syndrome; thyrotoxicosis;irregular periods; premature menopause; low levels of testosterone and estrogen in men; and combinations thereof.2.23. Method I or Use I, wherein the ENPP1 related disease or disorder is cancer, cardiovascular disorder, neurological disorder, immunological disorder, musculoskeletal disorder, hormonal disorder, hematological disorder, gingivitis, periodontal disease, bone disorder, cartilage disorder or a combination thereof.2.24. Method 1 or Use 1, or any of 2.1-Error! Reference source not found., wherein the method comprises administering an effective amount of a compound of Formula I or any of 1.1-1.24.2.25. Method I or Use I, or any of 2.1-Error! Reference source not found., wherein the method comprises administering an effective amount of Composition I or any of 1.29-1.51.2.26. I or Use I, or any of 2.1-Error! Reference source not found., wherein the method comprises administering an effective amount of Composition I or formula 1.50.2.27. Method I or Use I, or any of 2.16-2.26, wherein the ENPP1 inhibitor is administered via parenteral administration.2.28. Method I or Use I, or any of 2.16-2.26, wherein the ENPP1 inhibitor is administered via subcutaneous administration.Definitions

[0019] “Pharmaceutically acceptable salt” refers to the modification of the original compound by making the acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, hydrochloride salt. Lists of suitable pharmaceutically acceptable salts can be found in Remington’s Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704; and Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

[0020] The terms “treatment” and “treating” refer to the medical management of a subject with the intent to cure, ameliorate, or stabilize one or more symptoms of a disease or disorder. This term includes active treatment toward the improvement of a disease or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease or disorder. It is understood that treatment, whileintended to cure, ameliorate, or stabilize a disease or disorder, need not actually result in the cure, amelioration, or stabilization. The term “prophylaxis” means preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease or disorder. The effects of treatment or prophylaxis can be measured or assessed as described herein and as known in the art as is suitable for the disease or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease or disorder and / or symptoms of a disease or disorder can be reduced to any effect or to any amount.

[0021] The term “5-10 membered heterocycloalkyl” means a 5-10 membered cycloalkyl wherein one or more of the carbon atoms are replaced with one or more heteroatoms such as nitrogen or oxygen, preferably, nitrogen.

[0022] The term “5-10 membered bridged heterocycloalkyl” means a 5-10 membered bridged cycloalkyl wherein one or more of the carbon atoms are replaced with one or more heteroatoms such as nitrogen or oxygen, preferably, nitrogen.

[0023] The term “5-10 membered bridged heterocycloalkenyl” means a 5-10 membered bridged cycloalkyl containing a double bond (bridged cycloalkenyl), wherein one or more of the carbon atoms are replaced with one or more heteroatoms such as nitrogen or oxygen, preferably, nitrogen.

[0024] The weight percent of the ENPP1 inhibitor disclosed herein may be based on the weight of the free base or the salt form of the ENPP1 inhibitor. In a particular embodiment, the weight percent of the ENPP1 inhibitor is based on the salt form of the ENPP1 inhibitor.

[0025] Polylactide is a biodegradable thermoplastic poly(lactic acid) polyester polymer having a backbone formula of (-C(CH3)(H)C(O)O-)n. or [C3H4O2]n. Preferably, the polylactide is a poly(D, L-lactic acid) or poly(D, L-lactide) polymer. Still preferably, the polylactide polymer has an average molecular weight of 75,000-120,000 or about 23,000 to about 45,000 or about 15,000 to about 24,000, preferably 75,000-120,000.

[0026] The compounds in the methods and compositions described herein can be synthesized using methods disclosed in PCT / US2024 / 019100, the contents of which are incorporated herein by reference in their entirety. In particular, the compound PTGN-19cb may be prepared as follows:10.0 eq. TMSBr0-23 degrees C, 2h,in a polar aprotic organic solvent,HCl quench

[0027] As used herein, the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state or disorder being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (such as age, immune system health, etc.), the disease, disorder, and the treatment being affected.ExamplesExample 1: Preparation of biodegradable polymer based controlled delivery system for 15% loading of PTGN-19cb.

[0028] The following sample is prepared the release profile is evaluated as follows: fiber optics micro dissolution apparatus with 100 mg API standard curve generation, prototype sample size of 2 g (0.3g API (PTGN19cb in HC1 salt form) + 1.7 g of 25:75 PLA / NMP mix), sample size 0.2 g / vessel, API concentration of 1.5 mg / mL, 20 mL simulated saliva medium at 37°C, 100 rpm at pH 6.8. n = 3. Dissolution time is 72 hours

[0029] The results are shown in Figures la-c. Figure Id shows release profile of a control (25:75 PLA / NMP without active).Example 2 - Preparation of biodegradable polymer based controlled delivery system with 10% loading of PTGN-19cb.

[0030] 15% drug loading sample prepared above is diluted with 25:75 PLA: NMP polymer solution to provide 10% drug load sample and the release profile is evaluated. The results show that 60-70% of the active is released immediately when injected into dissolution media and then continuously releases until 10-12 hours to reach near 100%. The results are shown in Figures 2a and 2b.Example 3 - Preparation of 10% loading of PTGN-19cb in 35%:65% PLA: NMP.

[0031] 1.4 g of PLA is added into 8 mL scintillation vial. 2.1 g of NMP is added to the vial and the vial is placed on a 40°C shaker @ 1000 RMP for 24 hours to fully dissolve PLA. To a 0.444 g of PTGN-19cb in a 4 mL clear vial is added 0.5 g NMP to dissolve API and vertex mixed until homogeneous. PLA / NMP solution is added to the PTGN-19cb solution and mixed with a spatula. The formulation is held at ambient conditions for 24 hours and manually mixed with a spatula before dosing in dissolution test. The release profile is evaluated using fiber optics micro dissolution apparatus with a sample size of 0.2 g / vessel, pH 6.8 20mL simulated saliva medium @ 37°C, 100 rpm, n = 2; 50 rpm, n = 2; 0 rpm, n = 1, dissolution for 72 hours.

[0032] The results show that 35:65 PLA: NMP with 10% drug loading yields controlled sustained release (Figure 3).Example 4: Non-GLP: Single-Dose and Multi-Dose Efficacy, Toxicity, Maximum Tolerated Dose, and Plasma Toxicokinetics of a Proprietary Treatment (Periodontal Pocket Administration) in a Model of Induced Periodontitis in Beagle Dogs.

[0033] The purposes of this Non-GLP Exploratory study will be to investigate the single-dose and multi-dose efficacy, toxicity, Maximum Tolerated Dose (MTD), and plasma toxicokinetics (TK) of a proprietary treatment for periodontal disease following a single or multiple periodontal pocket administration in a model of induced periodontitis in male and female Beagle dogs.

[0034] The dogs will be observed for 30 days following treatment. The MTD will be based on the number of periodontal pockets created and treated in each animal.

[0035] Twenty-one (21) dogs, 11 (8-12 kg) males and 10 (6-9 kg) females will be divided into a Pilot Phase with a single male dog, and the Main Study Phase with 5 groups of2 male and 2 female dogs each (total 10 M, 10 F; approximately 2 to 3 years old). There will be no vehicle-only Control group in this Non-GLP Pilot study.

[0036] Approximately 11 male and 10 female colony animals (depending on availability) will be screened by a Dentist shortly after the execution of this protocol, for periodontal pocket depth, measuring at least 4 pockets (evenly distributed on each side). Periodontal pocket size of at least 84 measurements will be documented (the “Baseline Pocket Measurement”). One male with the smallest pocket size will be assigned to the pilot phase while the remaining 10 male and 10 female animals will be assigned to the Main Study Phase. Two colony animals will be used prior to baseline for practice collection of images.Pilot Phase;

[0037] This non-GLP exploratory study evaluated the safety, local tolerability, preliminary efficacy, maximum tolerated dose (MTD), and plasma toxicokinetics (TK) of a proprietary periodontal treatment (PTGN-19cb) administered into experimentally induced gingival pockets in a single male beagle dog.

[0038] Periodontal pockets were induced using silk ligatures over a period of approximately 60-75 days to achieve pocket depths of 4-6 mm. The treatment was administered once to four defined pockets, and the animal was monitored for 21 days postdosing. At study end, tissues from the maxilla and mandible were collected for histopathologic assessment, and plasma was sampled for TK analysis.

[0039] No clinical signs of local or systemic toxicity were observed following treatment, and no adverse findings were detected in plasma toxicokinetic analysis.Histologically, the treated sites exhibited gingival hyperplasia and increased subepithelial cellularity, including basophilic cells with prominent nucleoli and features of both epithelial and mesenchymal origin. Surprisingly, evidence of alveolar bone growth was observed adjacent to treated pockets, characterized by basophilic tidemarks and new bone facing the periodontal ligament. Widening of the periodontal ligament and cellular cementum was also noted in treated areas. In contrast, untreated control teeth showed minimal to mild epithelial hyperplasia and overall narrower periodontal ligament widths.

[0040] The proprietary treatment demonstrated excellent local and systemic tolerability in this pilot model, with no observed toxicity and histological evidence of alveolar bone regeneration. These findings support further evaluation in larger, controlled studies and highlight the potential of PTGN-19cb for promoting periodontal repair.OBJECTIVE

[0041] The purposes of this non-GLP exploratory study was to investigate the singledose and multi-dose efficacy, toxicity, MTD, and plasma TK of a proprietary treatment for periodontal disease following a single or multiple periodontal pocket administration in a model of induced periodontitis in male and female beagle dogs. The current pilot study represents a single male dog in which experimental periodontitis creation was formed to include a total of four pockets.MATERIALS AND METHODS

[0042] Pocket induction on the left side was conducted using the silk-wire twisted ligature, while the pockets on the right side were created using silk ligatures alone. The goal was to determine how many days were needed to induce periodontal pockets of 4-6 mm in depth. Periodontal pocket measurement and ligature changes or removal, once desired pocket size was achieved, was estimated by the dentist to be approximately Day 60-75.

[0043] Once the periodontal pocket was of a desired size, the pilot animal was dosed once in each of the four pockets and monitored for 21 days. After 21 days, the animal was humanely euthanized and the maxilla and mandible tissues were collected into 10% neutral buffered formalin to optimize histology methods. The number of days needed to induce the desired pocket depth (around 5 mm) was assigned as the “Induction Day”.

[0044] For clarity, if it takes 42 days, then Day -42 will be known as the Induction Day.On Pilot Day 99, the Pilot animal received test article as follows:Upper Right Quadrant: Tooth #108 Disto-buccal (DB), Disto-lingual (DL) Upper Left Quadrant: Tooth #208 DBLower Left Quadrant: Tooth #308 DL and DBLower Right Quadrant: None identified for dosing.

[0045] On Pilot Day 107, all ligatures were removed and each identified site was dosed with test article. Blood was collected for plasma TK pre-dose and up to 168 hours postdose (post-test article administration).

[0046] Dental cleaning was not performed until the study was complete for this Pilot animal.

[0047] The maxilla and mandible were received at Experimental Pathology Laboratories, Inc. (EPL®) in Sterling, VA. The bones were put into decalcification solutionand once the bone was soft enough to cut, the teeth enumerated above were collected, to include the distal portion of the tooth, with surrounding gingiva, and alveolar bone.

[0048] The tissues were processed routinely and microtomed and stained with hemotoxylin and eosin (H& E) for histopathologic evaluation.

[0049] Histopathologic findings are presented in the Individual Data Listing of Histopathology tables. Findings were graded from one to five, depending upon severity and summarized by treatment group in the Incidence Summary of Microscopic Findings by Sacrifice. An explanation of descriptive severity grades is provided at the end of the tables; equivalent numbered grades are 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. Pristima® version 7.5.0 Build 8 was used to generate the tables listed above.RESULTS AND DISCUSSION

[0050] Gingival pockets were seen on the three teeth, but DL and DB pockets were not clearly differentiated from each other on Tooth #108 and Tooth #308. The pockets were characterized by succal gingival hyperplasia and increased cellularity in the subepithelial gingival lamina. A distinct subepithelial basophilic cell population was observed in association with gingival pocket formation. These cells exhibited prominent nucleoli and somewhat resembled the epithelial rests of Malassez, but were too numerous and located subepithelially, rather than sparsely, along the cementum of the periodontal ligament as described in the literature. Specifically, the nuclei were large and basophilic with prominent nucleoli - suggestive of epithelial origin - while the cytoplasm showed streaming morphology reminiscent of fibroblasts, indicating mesenchymal behavior.

[0051] Given these characteristics and their location within the subepithelial gingival lamina, the findings are consistent with an active regenerative or reparative process in response to periodontal tissue injury.

[0052] The periodontal ligament (PDL) was widened as well as the cellular cementum, usually on the side in which the pocket appeared (Text Table 1). New alveolar bone was also seen, again, usually on the side nearest the pocket and was characterized by one or multiple basophilic tidemarks of the alveolar bone facing the PDL (data not shown). Text Table 1: Measurements (microns) of Tooth Structures - Pilot DogCellularTooth Number New alveolar bone Cementum PDL width to tip PDL width (facing tooth) Width of alveolar bone (range)(widest point)108 DL* 110-300 160 290 25-160108 DB* 40 (tip and buccal 100 100 5-70side of tooth)208 DL 70-160 160 250 25-280 208 DB* 100 60 120 20308 DL* 80b200a700 700308 DB* 40b40 500-700 200-700*Location of pocketa=focal on slide 3 and 3dupl-4 and above the alveolar bone; otherwise varies depending on section, cementum thickened up to 250 u facing PDL in a section likely representing Tooth #309.b=21 slides evaluated and widths vary, likely two separate teeth are represented (Tooth # 309 and treated Tooth #308.) DB=distal buccal, DL=distal lingual

[0053] Text Table 2 reports results from two normal teeth for comparison purposes. No pockets were created or treated with test article. However, each of these teeth had minimal (Tooth No. 209) to mild (Tooth No. 408) gingival sulchar epithelial hyperplasia, and in close association, an increased cellularity of the gingival lamina on the buccal side. Since pockets were not specifically created on these teeth, this buccal side change (specifically related to Tooth No. 408) may have been related to treatment accidentally getting onto these teeth, the dental cleaning, or it might be a normal feature.

[0054] The primary difference in the measurements from the treated teeth is that the normal untreated teeth appeared to have an overall narrower PDL width at the tooth or cementum to the tip of the alveolar bone than the treated teeth (Text Table 1). Otherwise, measurements were variable for both treated and untreated teeth with considerable overlap. Text Table 2: Measurements (microns) of Untreated Tooth Structures - Pilot DogNew alveolar bone CellularTooth Number (facing tooth) Cementum PDL width to tip PDL width Width of alveolar bone (range)(widest point)408 DL* 250 220 60 20-700a408 DB* 100 140 55 20-250209 DL 20 30 250 200-310 209 DB* 30 10 200 30-220*Location of pocket“Widest measurement in area of notch in root before expansion into crown

[0055] Possible reasons for these results include varying sectioning, varying shape of the teeth as presented in the slide section, and possible loss of orientation of buccal versus lingual sides. To control for the latter, careful casetting and labeling needs to be strictly instituted through all steps of histology. Tooth notching is a possibility if it is done a slight distance from the gingiva and is consistently placed. Histology ink inside the created gingival pocket may also help differentiate exactly where the pocket was located, particularly if the pocket slightly wraps around the tooth in the plane of eventual sectioning. One or more untreated teeth from each dog should also be processed for comparison. Also, helpful would be test article processed in gel and paraffin block to illustrate its physical characteristics if there is reason to believe that residual material may be present.Main Study Phase:1 STUDY OBJECTIVE AND DESIGN

[0056] The purposes of this Non-GLP Exploratory study will be to investigate the single-dose and multi-dose toxicity, Maximum Tolerated Dose (MTD), plasma toxicokinetics (TK) and efficacy of a proprietary treatment for periodontal disease following a single or multiple periodontal pocket administration in a model of induced periodontitis in male and female Beagle dogs. The dogs will be observed for 30 days following treatment. The MTD will be based on the number of periodontal pockets created and treated in each animal.

[0057] Twenty (20) dogs, 11 (8-14 kg) males and 9 (6-12 kg) females will be divided into 5 groups of 4 dogs each. Animals will be approximately 3 years old at the time of periodontitis induction.

[0058] There will be no vehicle-only Control group in this Non-GLP Pilot study.

[0059] A previous study, PE-2301 (Pilot Study), employed one dog to determine how many days are needed to induce periodontal pockets of 4-6 mm in depth. In that study, it took -107 days from the time of periodontal induction to achieve a pocket depth of 4-6 mm. This study will aim for pocket depth of 5-7 mm.Pocket Measurements and Group Assignment:

[0060] All animals will be anesthetized and screened by a Dentist prior to induction of periodontitis, for periodontal pocket depth, measuring up to 5 pockets in each quadrant. Baseline periodontal pocket size will be documented and shared with the Study Director and Sponsor. Based on Baseline Pocket Measurement, the Sponsor and Dentist will assign animals to groups based on pocket depth and clinical attachment levels with the goal of completing the study in a timely manner. Animals will be grouped as detailed below.Animals will be reorganized into new groups and assigned new animal numbers based on the table below:Old Animal NewNumbers Animal 4+ mm Treat Treat TreatGroup Numbers pockets ment 1 ment ment Necropsy M F M F (Day 2 - 14 3-28Tl) days dayspost postTl Tl2001 6001 6 Yes3001 6002 3 Yes6 42 days 3002 6003 4 Yes post Tl 5501 6501 4 YesOld Animal NewNumbers Animal 4+ mm Treat Treatm TreatGroup Numbers pockets ment ent 2 - ment Necropsy M F M F 1 14 days 3-28(Day post Tl daysTl) postTl5001 7001 6 Yes Yes5003 7002 7 Yes Yes7 42 days 2502 7501 4 Yes Yes post Tl 3501 7502 4 Yes YesOld Animal NewNumbers Animal 4+ mm Treat Treatm Treat Group Numbers pockets ment ent 2 - ment Necropsy M F M F 1 14 days 3-28(Day post Tl daysTl) postTl1002 8001 7 Yes Yes Yes2002 8002 7 Yes Yes Yes8 42 days 2501 8501 7 Yes Yes Yes post Tl 3502 8502 6 Yes Yes YesOld Animal NewNumbers Animal 4+ mm Treat Treatm Treat Group Numbers pockets ment ent 2 - ment Necropsy M F M F 1 14 days 3-28(Day post Tl daysTl) postTl1001 9001 29 42 days 4002 9002 1 post TlAdditional details:1. The total number of animals for the upcoming dosing phase has changed from 20 to 14, with pocket depths of 4 mm or above.2. The actual number of animals being dosed has changed from 16 to 12 (excluding the 2 negative controls).3. The dosing schedules have changed: one group now has 3 dosing time points, another has 2, and the third has 1.4. As mentioned in the protocol, scaling and polishing / cleaning of the test teeth and the teeth initially measured in that quadrant using an ultrasonic scaler will be conducted under anesthesia on animals in Groups 6-8, as well as of the teeth decided by the dentist for Group 9.5. Pocket depths will be measured for all animals under anesthesia in Groups 6-8 prior to dose on treatment day 1 (Tl) and also for animals in Group 9 prior to dental cleaning.6. Test article will be weighed (g / mg) prior to and after dosing each animal to calculate the amount of test article used per animal.7. Necropsy is now scheduled for 42 days post- first dose, instead of the originally planned 29 days.8. Weekly ligature replacements will be stopped per Sponsor and dentist’ s suggestion.9. CT scans will be performed before the 1stdose and prior to necropsy on Day 42 for Group 8 only (details below).10. Six (6) animals not included in the dosing phase will be removed from the study.Induction of Periodontitis:

[0061] Periodontitis Induction will be conducted in 4 staggers.

[0062] On Stagger Day 1, the dogs will be anesthetized and subjected to induction of periodontitis in the upper and lower premolars and / or molars. The experimental periodontitis will be induced by a Dentist as detailed in the Periodontitis Induction Section below using dental ligatures (based on the results of the Pilot Study PE-2301) placed apical to the interproximal region around selected teeth to promote plaque formation.

[0063] Ligature retention checks will be conducted twice each week. Missing ligatures will be replaced under anesthesia once each week by staff.

[0064] Pocket depths will be measured by the Dentist on approximately Day 45 (+ / - 3 days), Day 90 (+ / - 3 days), and Day 135 (+ / - 3 days) [and if needed up to Day 160]. A Change Order / Budget will be approved by the Sponsor once the actual duration required to achieve the desired pocket depth of 5-7 mm is determined based on data.

[0065] All other study activities will continue per schedule - body weight measurements, clinical observations, ligature retention checks, ligature replacements, etc. until the desired pocket depths are achieved.Test Article Treatment:

[0066] When the periodontal pockets are of desired size (5-7 mm), animals will be considered ready for Test Article Treatment. It is expected that dogs will be ready for test article treatment at different times as periodontitis may progress faster in some animals and slower in others. Test Article Treatment will be conducted in staggers with 4 animals per stagger, maintaining their group assignments.

[0067] On Treatment Day, dogs will be anesthetized, ligatures will be removed for the remainder of the study and each dog will be subjected to scaling and polishing / cleaning of the test teeth and the teeth initially measured in that quadrant using an ultrasonic scaler.Following dental scaling, the test article will be administered directly into the inducedperiodontal pockets until each pocket is filled. Animals in Group 4 will have a second test article treatment 15 days following the first treatment.Dental Imaging:

[0068] Only dogs in Group 8 (3-dose group) will be subjected to dental imaging under anesthesia using a CT prior to test article administration on Treatment Day 1 (first dose), and again prior to sacrifice on the day of necropsy. Dental images will be evaluated by the Dentist and a dental subreport will be provided which will be added as an appendix to the final report, to include assessment of any changes in cementum, alveolar bone, dentin or other tooth structures.

[0069] Images will not be obtained from animals in Groups 6, 7, and 9.Blood Collection for Pharmacokinetics (PK):

[0070] Animals in Groups 6, 7, and 8 (12 dogs only), will have blood collected (~3 ml, K2EDTA, cephalic or jugular vein) for plasma TK.

[0071] Blood will be collected from Group 6 animals on dosing day (T0 Day; only one single dose), from Group 7 animals on 2nddosing day (T14 Day; 2nddose), and from Group 8 animals on last dosing day (T28 Day; 3rddose) at following timepoints: pre-dose and 0.5, 1, 2, 3, 6, 24, and 48 hours post-dose, 4 days post dose (92-100 hours post-dose), and 7 days post-dose (164-172 hours post-dose).

[0072] In addition, blood samples will be collected once on the day of scheduled necropsy from Group 6, 7, and 8 animals. There will be total 11 timepoints and 132 samples. Blood will be processed to obtain plasma and shipped for Bioanalysis and PK evaluation. A bioanalysis subreport along with PK evaluation subreport will be provided and included in the final report as appendices.Clinical Observations:

[0073] Detailed clinical observations will be conducted once weekly for the duration of the study. In addition, detailed clinical observations will be conducted once daily for 7 consecutive days post periodontitis induction as well as test article treatment including the day of induction and dosing in all groups. During clinical observations, special attention will be provided to gingiva, teeth, and for any signs of inflammation or local toxicity / irritation as well as damage or changes in tooth structure.Body Weights:

[0074] Body weights will be recorded once each week including on the day of baseline pocket measurement, the day of periodontitis induction, the day of test article administration, and the day of sacrifice.Feed:

[0075] Animals will be fed LabDiet, Laboratory Canine Diet 5006 soaked in water for 48 hours post- induction procedure as well as post-test article administration. Animals will be fed LabDiet, Laboratory Canine Diet 5006 for the remainder of the study duration. Chew toys will be withheld for 48 hours post-test article administration.Fasting:

[0076] Animals will be fasted the evening before for procedures involving anesthesia / sedation and for 3 hours post placement of the test article. Water will be provided ad libitum.Euthanasia, Necropsy, and Tissue Collection:

[0077] Twenty-nine (29) days following Treatment Day, the dogs will be humanely euthanized subjected to a necropsy including standard organ weights. Organs weighed will include brain, liver (with gall bladder), kidneys (paired), adrenals (paired), spleen, heart, thymus, testes (paired), epididymis (paired), and ovaries (paired). These tissues will not be saved for pathology.

[0078] The maxilla and mandible will be collected into 10% NBF and sent for histopathology including measurements of cementum and alveolar bone. A histopathology subreport along will be provided and included in the final report as an appendix.Anesthesia for probing, induction, CT scans, treatment, and ligature replacements:

[0079] Anesthesia for pocket depth measurements / probing: Animals will be sedated with dexmedetomidine hydrochloride (0.03 mg / kg) for pocket depth measurement. Isoflurane anesthesia will be used as needed. After the procedure, sedation will be reversed with atipamezole hydrochloride (0.3 mg / kg) and animals will be monitored and returned to their kennel upon recovery.

[0080] Anesthesia for periodontitis induction: On Stagger Day 1, the dogs will be sedated with acepromazine (0.05 mg / kg SQ), then anesthetized with propofol (1-6 mg / kg IV to effect). Animals will be intubated and maintained on Isoflurane anesthesia for the duration of the procedure. The animals will be recovered from anesthesia and returned to their home pens upon recovery.

[0081] Anesthesia for ligature replacements: Animals will be sedated with dexmedetomidine hydrochloride (0.03 mg / kg) for ligature replacements. Isoflurane anesthesia will be used as needed. After the procedure, sedation will be reversed with atipamezole hydrochloride (0.3 mg / kg) and animals will be monitored and returned to their kennel upon recovery.

[0082] Anesthesia for dosing: On Treatment Day, the dogs will be sedated with acepromazine (0.05 mg / kg SQ), then anesthetized with propofol (1-6 mg / kg IV to effect). Animals will be intubated and maintained on Isoflurane anesthesia for the duration of the procedure. The animals will be recovered from anesthesia and returned to their home pens upon recovery.

[0083] Anesthesia for dental imaging: On Treatment Day, dental imaging will be conducted under anesthesia mentioned above for dosing (acepromazine and propofol) prior to treatment administration. On the day of necropsy, animals will be sedated with dexmedetomidine hydrochloride (0.03 mg / kg) for dental imaging. Isoflurane may be used for maintenance.

[0084] Animals will be euthanized while under sedation / anesthesia prior to necropsy.2 MATERIALS AND METHODS2.1 Test Article2.1.1 Test Article Identification and Storage

[0085] The test article, a proprietary treatment for periodontal disease, will be supplied by the sponsor. PTGN-19-cb is a small molecule of MW 415.43 and molecular formula C21H26N3O4.

[0086] The test article will be stored at 2-8°C (refrigerated) upon receipt. Test article receipt information (if received) including manufacturer, Lot Number, Expiration Date and storage will be maintained, and included in the raw data, and detailed in the final report.2.1.2 Vehicle Identification, Manufacturer, and Lot Number

[0087] Test article will be mixed with PLA / NMP Biopolymer system solution provided by the Sponsor. Vehicle will be stored at ambient at room temperature upon receipt Manufacturer, Lot Number, Expiration Date and storage will be maintained, and included in the raw data, and detailed in the final report. The pre- weighed test article will be provided in a capped syringe designated syringe B. The PLA / NMP will be provided in a separate syringe also capped and pre-weighed designated syringe A. There will be 4-5 sets of syringes A plus B provided depending on the dosage requirements, syringe size, and overage. Adapters and blunt tipped needles (25G and larger) will also be supplied.2.1.3 Dose Formulation PreparationInstructions:FormulationsFormulation Syringe A Syringe B Formulation Strength PTGN-19CB 10% w / w in PLA / NMP PTGN-19CB 10% PLA / NMP biopolymer system BiopolymersystemFormulation Storage□ Store all the formulation syringes B containing the test article (drug powder) at 2-8°C (refrigerated) protected from light in and ensuring they remained capped until ready for use to exclude moisture until ready to use. Store the syringes A containing PLA / NMP at room temperature also keeping them capped until ready for use.Formulation Dosing Preparation1. Bring the syringe B containing the test article to room temperature.2. Uncap syringe B (drug powder) and syringe A (liquid delivery system (PLA / NMP) and couple both syringes together using a supplied adapter.3. Inject the liquid contents syringe A into syringe B (drug powder) and push the contents back into syringe A. This operation is considered one mixing cycle.4. Complete additional mixing cycles until all of the powder is dissolved and the mixture appears homogeneous. Lees then 10 mixing cycles areestimated to be needed.5. The coupled syringes can be stored briefly maximum 2 days) if necessary.After storage remix before use.6. The contents will be contained in syringe A. Hold the coupled syringes vertically with syringe A at the bottom. Pull back on the syringe A plunger and allow the contents to flow down the barrel for several seconds.7. Uncouple the two syringes and attach one of the provided cannulae 25G or larger to syringe A.8. The product is now ready for application.2.2 Test System2.2.1 Species and StrainDogs, Beagle, Non-Naive (MWR Colony)2.2.2 Number, Sex, Body Weight, and AgeTwenty (20) dogs, 11 (8-14 kg) males and 9 (6-12 kg) females will be divided into 5 groups of 4 dogs each. Animals will be approximately 3 years old at the time of periodontitis induction.2.2.3 SourceMWR ColonyOriginally from:Ridglan Farms MountHoreb, WI 608-437-86702.2.4 Identification

[0088] Each dog will have a unique ear tattoo and an assigned animal number on the cage card. The cages will be identified with at least the study number, IACUC number, animal number, and dose information.2.2.5 Dropouts and Replacements

[0089] Animals will not be replaced once enrolled in the study.2.2.6 Justification for Test System, Number of Animals, and Dose Level

[0090] Dogs have been selected for this study based on their traditional use as a second species in toxicology studies and are the typically second species of choice by the FDA.

[0091] Twenty dogs, 8-14 kg males and 6-12 kg females will be divided into 5 groups of 4 animals. Animals will be approximately 3 years old at the time of periodontitis induction.

[0092] Four animals per group will be enrolled in each group with the number of pockets created ranging from 2 to 6 per animal. The number of pockets created followed by subsequent administration of test article (or control) will help determine the toxicity, efficacy, PK, and provide biologically meaningful results and also allow comparison that relates to different number of pockets created per animals.

[0093] The activities specified in this study do not unnecessarily duplicate any previous experiment.2.3 Environment and Husbandry2.3.1 Housing

[0094] All animal housing and research procedures involving live animals will be conducted at MWR. The standards for animal husbandry and care to be followed are those found in the Guide for the Care and Use of Laboratory Animals, 8th Edition, Revised 2011. Animal welfare for this study will be in compliance with the U. S. Department of Agriculture’s (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3), the Guide for the Care and Use of Laboratory Animals, and MWR SOPs.

[0095] During the duration of the study, the dogs will be single or group housed in modern elevated kennels. Each kennel will use automatic waters. Food will be fed in stainless steel feeders attached to the kennels fronts. The kennels will conform to standards set forth in the Guide for the Care and Use of Laboratory Animals.l2JToys will be provided for enrichment except for 48 hours after test article administration.2.3.2 Veterinary CareThe Attending Veterinarian or a staff Veterinarian will be on-site or on-call during the live animal phase of this study.2.3.3 Environmental Monitoring2.3.3.1 Temperature

[0096] Environmental controls will be set to maintain temperatures from 65° to 81°F. A calibrated 24- hour electronic temperature recorder will be observed once daily by study personnel who will record the high and low temperature since the last reading (approximately every 24 hours).

[0097] Transient excursions are expected during pen and floor cleaning operations, will not require a protocol deviation. Animals will be housed in with heating, exhaust air exchange, and swamp- cooler type cooling.2.3.3.2 Humidity

[0098] Humidity will not be controlled in the farm-type building due to the high volume of air exchanges; however, a calibrated 24-hour electronic humidity recorder will be observed once daily by study personnel who will record the high and low humidity since the last reading (approximately every 24 hours). Transient excursions are expected during pen and floor cleaning operations, will not require a protocol deviation. Animals will be housed in with heating, exhaust air exchange, and swamp-cooler type cooling.2.3.3.3 Light

[0099] The light source will be fluorescent lighting on a 12 hr / 12 hr ON / OFF cycle except as required for specimen collection.2.3.4 Concurrent Medication

[0100] No concurrent medication will be given while animals are on study unless deemed necessary by a Veterinarian and approved by the Study Director. The Sponsor will be notified.

[0101] All dogs will be administered meloxicam, 0.2 mg / kg IM, on the day of periodontitis induction and once a day for the next 3 days after periodontitis induction.2.3.5 Feed

[0102] LabDiet, Laboratory Canine Diet 5006 will be available ad libitum, except when being fasted for anesthesia, for the duration of the study. The expiration date(s) and lot number(s) will be maintained in the study raw data.

[0103] Feed will be soaked in water for 48 hours post-induction procedure as well as post-test article administration.

[0104] Animals will be fasted the evening before anesthesia / sedation. Food will be returned upon recovery from anesthesia. On the day of Test Article Treatment food will be returned and hours post placement of the test article.2.3.6 Water

[0105] Tap water from the Fort Collins municipal water supply will be pressure-reduced and passed through a particulate filter, then a carbon filter prior to supply to an automatic watering system. Water is supplied ad libitum. Reports for municipal water testing of total dissolved solids, hardness, specified microbiological content, and environmental contaminants are received monthly and kept on file at MWR.2.3.7 Contamination Statement

[0106] There are no known contaminants in the feed or water that would be expected to interfere with this study.2.3.8 Sanitation

[0107] Animal rooms and equipment sanitation procedures are conducted in accordance with USDA Animal Welfare Regulations, CFR Title 9, Chapter 1, Subchapter A and in accordance with the Guide for the Care and Use of Laboratory Animals.2.4 Experimental Design2.4.1 Duration of Study

[0108] -137 days (-107 days for pocket creation, +30 days for treatment)2.4.2 RandomizationNo randomization plan will be used. Animals will be assigned to groups by the Sponsor and Dentist based on Baseline Pocket Measurement.2.4.3 Periodontitis Induction

[0109] Periodontitis Induction will be conducted in 4 staggers. On Stagger Day 1, the dogs will be sedated with Acepromazine (0.05 mg / kg SQ), then anesthetized with Propofol (1-6 mg / kg IV to effect). Animals will be intubated and maintained on Isoflurane anesthesia for the duration of the procedure. Animals will be subjected to induction of periodontitis in the upper and lower premolars and / or molars as detailed below.Group 1: 2 males and 2 females, 2 periodontal pockets created per animal (1 on each side).Group 2: 2 males and 2 females, 4 periodontal pockets created per animal (2 on each side).Group 3: 2 males and 2 females, 6 periodontal pockets created per animal (3 on each side).Group 4: 2 males and 2 females, 2 periodontal pockets created per animal (1 on each side).Group 5: 3 males and 1 female, 2 periodontal pockets created per animal (1 on each side).

[0110] The experimental periodontitis will be induced by a Dentist using 2-0 silk suture ligatures (based on the results of the Pilot Study PE-2301) placed apical to the interproximal region around selected teeth to promote plaque formation. This procedure is cited many times in the literature and standard practice for experimental periodontal pocket induction in laboratory animals. The procedure will include preparation of the pocket sites to a periodontal pocket depth of approximately 3mm Following undermining of the periodontal ligaments with an Orban knife (periodontal instrument), shallow notches will be made in the mesial and distal cervical region of each applicable tooth with a straight fissure dental bur; the notches will hold the ligature in place.

[0111] In order to increase ligature retention (after Day 45), ligatures on teeth #108 and #208 will be placed through the mesio-buccal gingiva, in the sulus, secured on the distal enamel notch and tied on the buccal surface of the tooth.

[0112] This procedure will be conducted under anesthesia - animals will be sedated with dexmedetomidine hydrochloride (0.03 mg / kg). Isoflurane anesthesia will be used as needed. After the procedure, sedation will be reversed with atipamezole hydrochloride (0.3 mg / kg) and animals will be monitored and returned to their kennel upon recovery. The procedure may be repeated under anesthesia during the live phase to ensure ligature retention. All dogs will be administered meloxicam, 0.2 mg / kg IM, on the day of procedure.

[0113] Pocket depths will be measured under anesthesia prior to sacrifice on the day of necropsy.

[0114] In addition, to help with orientation for histology, a small notch (divet) with a bur on the enamel will be placed (under anesthesia) at the gingival margin for histologic orientation prior to sacrifice on the day of necropsy; if that surface does not already have a ligature retention notch.

[0115] The animals will be recovered from anesthesia and returned to their home pens upon recovery..2.4.4 Ligature Replacement

[0116] Missing ligatures will be replaced once weekly by MWR staff. Animals will be sedated with dexmedetomidine hydrochloride (0.03 mg / kg) for ligature replacement. Isoflurane anesthesia will be used as needed. After the procedure, sedation will be reversed with atipamezole hydrochloride (0.3 mg / kg) and animals will be monitored and returned to their kennel upon recovery.2.4.5 Test Article Administration

[0117] When the periodontal pockets are of desired size (5-7 mm), animals will be considered ready for Test Article Treatment. It is expected that dogs will be ready for test article treatment at different times as periodontitis may progress faster in some animals and slower in others. Test Article Treatment will be conducted in staggers with 4 animals per stagger.

[0118] On Treatment Day, the dogs will be sedated with Acepromazine (0.05 mg / kg SQ), then anesthetized with Propofol (1-6 mg / kg IV to effect). Animals will be intubated and maintained on Isoflurane anesthesia for the duration of the procedure. Ligatures will be removed for the remainder of the study and each dog will be subjected to scaling and polishing / cleaning of the test teeth and the teeth initially measured in that quadrant using an ultrasonic scaler. Following dental scaling, the test article will be administered directly into the induced periodontal pockets until each pocket is filled. Animals in Group 4 will have a second test article treatment 15 days following the first treatment. The animals will be recovered from anesthesia and returned to their home pens upon recovery.

[0119] Animals in different groups will receive test article treatments in the periodontal pockets as detailed below.Group 1: 2 males and 2 females. Two periodontal pockets per animal (one on each side). All pockets will be filled once with the test article.Group 2: 2 males and 2 females. Four periodontal pockets per animal (two on each side). All pockets will be filled once with the test article.Group 3: 2 males and 2 females. Six periodontal pockets per animal (three on each side). All pockets will be filled once with the test article.Group 4: 2 males and 2 females. Two periodontal pockets per animal (one on each side). All pockets will be filled with the test article on Day 1 (1stdose). On Day 16, all pockets will be filled again with test article (2nddose).Group 5: 3 males and 1 female. Two periodontal pockets per animal (one on each side). The dogs will be subjected to all study procedures except no treatment will be instilled into the pockets.2.5 Observations, Measurements, and Specimens2.5.1 Physical Examinations

[0120] Prior to assignment to the study, a qualified study investigator or staff Veterinarian will conduct general physical examinations. The general examination will include assessment of the coat, mobility, external orifices, respiration, and reaction to external stimuli. Animals in obvious poor health will not be used in this study. The criteria of health will include the absence of abnormal clinical signs and a normal physical exam.2.5.2 Mortality Check

[0121] Animals will be checked at least once daily by Animal Care staff for mortality, moribundity, and general signs of toxicity.2.5.3 Clinical Observations

[0122] Detailed clinical observations will be conducted once weekly for the duration of the study. In addition, detailed clinical observations will be conducted once daily for 7 consecutive days post periodontitis induction as well as test article treatment including the day of induction and dosing in all groups. During clinical observations, special attention will be provided to gingiva, teeth, and for any signs of inflammation or local toxicity / irritation as well as damage or changes in tooth structure.2.5.4 Body Weights

[0123] Body weights will be recorded once each week including on the day of baseline pocket measurement, the day of periodontitis induction, the day of test article administration, and the day of sacrifice.2.5.5 Food Consumption

[0124] During the course of the study, food consumption will be subjectively monitored during clinical observations as objective weight data are un-reliable due to dogs digging in, or otherwise contaminating their food.2.5.6 Ligature Retention Checks and Replacement

[0125] Ligature retention checks will be conducted twice each week. Missing ligatures will be replaced under anesthesia once each week by MWR staff as detailed in Section 9.5.4.2.5.7 Periodontal Pocket Measurement

[0126] All animals will be anesthetized and screened by a Dentist prior to induction of periodontitis, for periodontal pocket depth, measuring up to 5 pockets in each quadrant. In addition, pocket depths will be measured on approximately Day 45 (+ / - 3 days) and Day 90 (+ / - 3 days) [and if needed up to Day 120]. If pocket depths are not 5-7 mm by Day 90, a Change Order / Budget will be approved by the Sponsor.

[0127] Pocket depth measurements and clinical attachment levels will be measured at 4 sites per tooth: Mesio-buccal, disto-buccal, mesio-lingual and disto-lingual. Some calculus may need to be removed to gain accurate probing depth measurements. Missing teeth will be recorded on the dental chart and no measurements will be taken.Quadrant Teeth / Pockets to bemeasuredRight Maxilla 104-108Right Mandible 405-409Left Maxilla 204-208Left Mandible 305-309

[0128] Baseline periodontal pocket size will be documented and shared with the Study Director and Sponsor. Based on Baseline Pocket Measurement, the Sponsor and Dentist will assign animals to groups based on pocket depth and clinical attachment levels with the goal of completing the study in a timely manner.

[0129] Animals will be fasted overnight prior to pocket measurements. Animals will be sedated with dexmedetomidine hydrochloride (0.03 mg / kg) for pocket depth measurement. Isoflurane anesthesia will be used as needed. After the procedure, sedation will be reversed with atipamezole hydrochloride (0.3 mg / kg) and animals will be monitored and returned to their kennel upon recovery.

[0130] The Dentist will assess the condition and healing of the periodontal pockets including measurements of the periodontal pockets as well as clinical attachment. Thecondition of the periodontal pockets along with dental efficacy and toxicity will be described in detail (including focus on cementum, alveolar bone and dentin changes) in the dental subreport.2.5.8 Dental Imaging

[0131] Dogs will be subjected to dental imaging under anesthesia using a CT prior to test article administration on Treatment Day, and prior to sacrifice on the day of necropsy. Dental images will be evaluated by the Dentist and a dental subreport will be provided which will be added as an appendix to the final report, to include assessment of any changes in cementum, alveolar bone, dentin or other tooth structures.2.5.9 Plasma Pharmacokinetics

[0132] On Treatment Day, animals in Groups 1, 2, and 3 (12 dogs only), will have blood collected (~3 ml, K2EDTA, cephalic or jugular vein) for plasma TK pre-dose and 0.5, 1, 2, 3, 6, 24, and 48 hours post-dose, 4 days post dose (92-100 hours post-dose), and 7 days post-dose (164-172 hours post-dose). In addition, blood samples will be collected once on the day of scheduled necropsy from animals in Groups 1, 2, and 3. There will be total 11 timepoints and 132 samples.

[0133] Blood will be processed to obtain plasma. Plasma samples will be split into 2 aliquots and stored at -60°C or lower until shipped to Aliri Bioanalysis at the end of the live phase on dry ice by overnight shipment. Bioanalysis and PK evaluation will be conducted by Aliri Bioanalysis. A bioanalysis subreport along with PK evaluation subreport will be provided to MWR and included in the final report as appendices. Back-up samples may be stored at MWR for up to 3 months after the end of the live phase and will then be discarded unless arranged by the Sponsor to send them to a location of the Sponsor’ s choice for longterm storage.2.5.10 Necropsy and Tissue Collection

[0134] Twenty-nine (29) days following Treatment Day, the dogs will be humanely euthanized subjected to a necropsy including standard organ weights. Organs weighed will include brain, liver (with gall bladder), kidneys (paired), adrenals (paired), spleen, heart, thymus, testes (paired), epididymis (paired), and ovaries (paired). These tissues will not be saved for pathology.

[0135] The maxilla and mandible will be collected into 10% NBF and sent for histopathology including measurements of cementum and alveolar bone. A histopathology subreport will be provided and included in the final report as an appendix.2.5.10.1 Euthanasia and Animal Disposition

[0136] Animals will be humanely euthanized using an AVMA accepted method and properly disposed according to MWR SOP.2.5.11 Animals Found Dead or Moribund

[0137] Any moribund animal as determined by the Study Director or Staff Veterinarian and agreed upon with the Sponsor (if the sponsor can be contacted immediately) or an animal found dead will be subjected to a gross necropsy in an effort to determine the cause of morbidity or death.6. Results:

[0138] Using the protocol described or similarly described above, the study is conducted and the results are shown in Figure 4, showing a mean pocket depth change of -2mm over 42 days with a starting mean pocket depth of 5mm.

Claims

1. We claim:

1. A pharmaceutical composition comprising an ENPP1 inhibitor having a structure defined by Formula I:

4. 6.Formula I7.in free or pharmaceutically acceptable salt form, wherein:8.T is a heteroaryl (e.g., quinolinyl, quinazolinyl, isoquinolinyl) optionally substituted with one or more hydroxy, C1-6alkoxy (e.g., methoxy) and / or cyano;9.Li is absent (i.e., a bond connecting T and B), -OC1-6alkylene (e.g., -OCH2-) or -N(R5)-C1-6alkylene wherein R5 is H or C1-6alkyl (for example, L1 is -N(H)-CH2-); B is aryl (e.g., phenyl), Cs-Cioheterocycloalkyl, a bridged Ce-C 10 heterocycloalkyl or bridged Ce-C 10 heterocycloalkenyl;10.L2 is absent (i.e., a bond connecting T and B) or a C1-6alkylene; or11.L2 has the structure:

13. 15.d and di are points of attachment to B and HG, respectively, each Ra, Rb, Rc, and Rd are independently hydrogen, C1-6alkyl (e.g., ethyl, n-propyl, n-butyl), haloC1-6alkyl (trifluoropropyl) or Ra, Rb, and the carbon atom to which they are attached together form a Ca-Cecycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclopropyl or cyclobutyl);16.m and n are independently 0 or 1, preferably both m and n are 1;17.HG is phosphonate or boronic acid,18.in admixture with a biopolymer system comprising poly(lactic acid) (PLA) polymer and N-methyl-2-pyrrolidone (NMP) present in a ratio of 20:80 to 50:50.

2. The composition of claim 1, wherein the PLA has a molecular weight of 75,000- 120,000.

3. The composition of claim 1 or 2, wherein the PLA to NMP ratio is 35:65.

4. The composition of any of claims 1-3, wherein the percentage of ENPP1 inhibitor loading is 1-25 wt% of the total weight of the composition, preferably 10 wt%, of the total weight of the composition.

5. The composition of any of claims 1-4, wherein: (i) the ENPP1 inhibitor or a pharmaceutically acceptable salt thereof is present in an amount equal to 10 wt% to 15 wt%, preferably 10 wt%, of the total weight of the composition, and (ii) PLA present in the PLA / NMP admixture in an amount between 25 wt% to 35 wt%, preferably 35 wt%, of the total weight of the PLA / NMP admixture, and / or (iii) NMP present in the PLA / NMP admixture in an amount between 65 wt% to 75 wt%, preferably 65 wt%, of the total weight of the PLA / NMP admixture.

6. The composition of any of claims 1-5, wherein the composition provides controlled release of the active over one to seven days, e.g., one to two days, for example over 40 hours.

7. The composition of any of claims 1-6, wherein the ENPP1 inhibitor and the PLA: NMP solution are mixed shortly before administration.

8. The composition of any composition of any of claims 1-7, wherein the composition is in a flowable liquid or gel form, e.g., for application (injection) via a syringe.

9. The composition of any of claims 1-8, wherein the composition may be administered via a syringe optionally with a needle e.g., a 14-25 Gauge needle, for example 25 Gauge, 23 Gauge, 16 Gauge or 14 Gauge needle.

10. The composition of any of claims 1-9, wherein upon contact with an aqueous medium, such as physiological fluids or water, the composition exhibits an increase in viscosity to form a semi-solid or solid material (e.g., pellet).

11. The composition of any of claims 1-10, wherein:28.T is a heteroaryl (e.g., quinazolinyl) optionally substituted with one or more Ci- ealkoxy (e.g., methoxy); Li is absent (i.e., a bond connecting T and B);29.B is a 6-10 membered bridged heterocycloalkenyl (e.g., 8-azabicyclo[3.2.1]oct-2-en-8yl);30.LT has the structure:

32. 34.d and di are points of attachment to B and HG, respectively, each Rc, and Rd are hydrogen, and Ra, Rb, and the carbon atom to which they are attached together form a C3-C6cycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclopropyl or cyclobutyl); m and n are 1; and35.HG is phosphonate.

12. The composition of any of claims 1-11, wherein the ENPP1 inhibitor is a compound selected from:37.,. O„.o.v38.A.-N\39." f pH OH40.<1 J* 'OH5O ^ OH41.BO-P-O HO P:: O Hfr'M42.OH on OH44.

45. PET-104-PCT47.

48. in free or pharmaceutically acceptable salt form.

13. The composition of any of claims 1-10, wherein the ENPP1 inhibitor is the compound:

51. 53.in free or pharmaceutically acceptable salt form.

14. A method for the treatment or prophylaxis of an ENPP1 related disease or disorder, e.g., selected from cancer, cardiovascular disorder, neurological disorder, immunological disorder, musculoskeletal disorder, hormonal disorder, hematological disorder, gingivitis, periodontal disease, bone disorder, cartilage disorder, autoimmune disorders; inflammatory disorders; metabolic disorders; digestive and gastrointestinal disorders; side effects from medical procedures; hematologic / blood disorders; neurological / nervous system disorders; bone marrow disorders; endocrine disorders; bone disorders associated with ageing; and combinations thereof, for example, periodontal disease (periodontitis), rheumatoid arthritis; lupus; multiple sclerosis; ankylosing spondylitis; celiac disease; inflammatory bowel disease; side effects from weight loss surgery, gastrectomy, and gastrointestinal bypass procedures; cancer; leukemia; lymphoma; multiple myeloma; sickle cell disease; stroke; Parkinson’s disease; multiple sclerosis; vertebral column injuries; thalasemia; diabetes; hyperparathyroidism; hyperthyroidism; Cushing’s syndrome; thyrotoxicosis; irregular periods; premature menopause; low levels of testosterone and estrogen in men; and combinations thereof, in a subject, comprising administering an effective amount of the composition of any of claims 1-13, to a subject in need thereof.

15. The method of claim 14, wherein the ENPP1 related disease or disorder is periodontitis (e.g., chronic periodontitis, for example chronic adult periodontitis).

16. The method of any of claims 14-15, wherein the ENPP1 inhibitor is administered via subgingival application (e.g., periodontal pocket of the affected tooth).

17. The method of any of claims 14-16, further comprising administering water (e.g., mist of water) following the application of the composition.

18. Any of the foregoing methods, wherein the ENPP1 inhibitor is administered once a month, twice a month or once every two, three, four, six, nine or twelve months.

19. Any of the foregoing methods, wherein the method promotes production of the cememtum, alveolar bone or dentin relative to baseline, e.g., as measured by pocket depth reduction and / or clinical attachment levels gain (mesio-buccal, disto-buccal, mesio-lingual and disto-lingual), e.g., via dental imaging such as CT scan or C-arm scan, as well as bleeding on probing.

20. Any of the foregoing methods, further comprising administering one or more other active agent, e.g., antimicrobial agent or antibiotic such as doxycycline or doxycycline hyclate (e.g., sequentially or simultaneously, separately on in the same composition as the ENPP1 inhibitor) or one or more other therapies such as scaling and root planning (sequentially or simultaneously).

21. A kit comprising the composition according to any of claims 1-13, optionally further comprises one or more syringes, preferably a two syringe mixing system, preferably one syringe comprises ENPP1 inhibitor and the other syringe comprises the polymer system.

22. A method for the treatment or prophylaxis of periodontitis in a subject, comprising administering an effective amount of a compound of formula I:

63. 65.Formula I66.in free or pharmaceutically acceptable salt form, wherein:67.T is a heteroaryl (e.g., quinolinyl, quinazolinyl, isoquinolinyl) optionally substituted with one or more hydroxy, C1-6alkoxy (e.g., methoxy) and / or cyano;68.Li is absent (i.e., a bond connecting T and B), -OC1-6alkylene (e.g., -OCH2-) or -N(Rs)-C1-6alkylene wherein R5 is H or C1-6alkyl (for example, Li is -N(H)-CH2-);69.B is aryl (e.g., phenyl), Cs-Cioheterocycloalkyl, a bridged Ce-Cio heterocycloalkyl or bridged Ce-Cioheterocycloalkenyl;70.L2 is absent (i.e., a bond connecting T and B) or a C1-6alkylene; or L2has the structure: d and di are points of attachment to B and HG, respectively, each Ra, Rb, Rc, and Rd are independently hydrogen, C1-6alkyl (e.g., ethyl, n-propyl, n- butyl), haloC1-6alkyl (trifluoropropyl) or Ra, Rb, and the carbon atom to which they are attached together form a C1-C6cycloalkyl (e.g., Ra, Rb, and the carbon atom to which they are attached together form a cyclopropyl or cyclobutyl);71.m and n are independently 0 or 1, preferably both m and n are 1;72.HG is phosphonate or boronic acid,

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