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as) United States a2) Patent Application Publication (10) Pub. No.: US 2018/0236130 Al MORADIAN-OLDAK et al. US 20180236130A1 (43) Pub. Date: Aug. 23, 2018 64) (I) (72) Ql) (22) (62) (60) AMELOGENIN-CHITOSAN HYDROGEL FOR DENTIN HYPERSENSITIVITY Applicant! UNIVERSITY OF SOUTHERN CALIFORNIA, Los Angeles, CA (US) Inventors: JANET MORADIAN-OLDAK, LOS ANGELES, CA (US), QICHAO RUAN, ALHAMBRA, CA (US); KAUSHIK MUKHERJEE, LOS ANGELES, CA (US) Appl. No.: 15/952,947 Filed: Apr. 13, 2018 Related U.S. Application Data Division of application No. 15/202,882, filed on Jul. 6, 2016, now Pat. No. 9,962,465. Provisional application No. 62/189.356, filed on Jul. 7, 2015. Publication Classification (51) Int. Cl. AGIL 27/22 (2006.01) COBL 5/08 (2006.01) CO7K 14/47 (2006.01) AGLL 27/44 (2006.01) AGIL 27/52 (2006.01) AOLL 27/46 (2006.01) (52) US. Cl. CPC we AOLL 27/227 (2013.01); COBL 5/08 (2013.01); CO7K 14/47 (2013.01); A6IL 2430/12 (2013.01); A6IL 27/52 (2013.01); AGIL 27/46 (2013.01), AGIL 27/446 (2013.01) (57) ABSTRACT A method to regrow a protective layer over exposed/demin- eralized dentin that includes a step of identifying a subject having exposed/demineralized dentin. The demineralized dentin is contacted with a remineralization composition that includes an amelogenin and derived peptides, a chitosan, water, and a sufficient amount of a pH adjusting component such that the composition has a pH greater than about 6.0 such that dentinal tubules are occluded with apatite crystals and enamel is regrown on the dentinal tubules. Specification includes a Sequence Listing.
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US 2018/0236130 Al Aug. 23, 2018 Sheet 1 of 20 Patent Application Publication
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US 2018/0236130 Al Aug. 23, 2018 Sheet 2 of 20 Patent Application Publication uoyeBuoje jEISAID A SON@ULY UOHeZIRIOUIAY A Builpurg omedy ‘SUISIGIN SARS Ui SUIBMIOP SATSY Z ‘bly Zed B 9d VOIGE~ <quIUEbojeuy jo Buayds yn eaneUoyy dvae1 vans TaINY Sue7-HIN4 BOse.- (20x) tenar-h p= 5 alee | PLL-SPL JOULE} HOOD ayrydoupAy paAsasuod AjYBIY a t L Aa " " 4889 1@ OS _-Buroyds vNM 1 ‘ woneMiNeso ud N fd) (vou) (uo) 2 ne fEURLIO] as09 a1qoydoipAy aIPPIW — %3ny- aIgoydoipAy pesuesuce AjyyBis JOSIND@Id NU TAWY
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Patent Application Publication Aug. 23,2018 Sheet 3 of 20 US 2018/0236130 Al intensity (Counts) Intensity (Counts) After 5 days 60H. — 002 (300) eee Wen 50 hy names ad 40} On sctunipliiche oss 30%, 30}, P26 oa SU a , x Sound dentin ae A acne een Oe ce OT cag Ma A Demin dentin eel a acattenndN pek QT 10 20 30 40 50 60 Two-Theta (deg) Fig. 3A After 10 days °° A (on 50 ral ‘ oe \ sen LEAP, 401 Be Ns | : \ ee P32 30 Same, ee Sf i 30 Nat, rind mal gsc P26 Pee S\ Sounddentin | 20 A are ada Seal net ncanienneel a“ Nn orn death POET OE yee et a Sa a a es ek ee ee ee Oe 10 20 30 40 50 60 Two-Theta (deg) Fig. 3B
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Patent Application Publication Intensity (Counts) Intensity (Counts) 60 50 30- 30 20 Aug. 23,2018 Sheet 4 of 20 = US 2018/0236130 Al After 5 days Ma 40", linia innaiant a vation (002) (300) Besant Mi. RC os Tulcea apienieid Macca sendin fh Chitosan a ee ad inal boar AES earn Staaten cB ON IE a gl LRAP-CS j P32-C8 P26-CS “aarti neta eanasi titel OOo taper tetan nies _ AS mS pe Vemnine a Sound dentin a Demin dentin Sy, Pinstripes 60 50 40 30 305 20 ret a 30 40 50 60 Two-Theta (deg) Fig. 3C After 1Odays SS ‘. Readies Pap an manannar nana Sa inp & Se Sf ca ‘. Jnr se A Sound dentin Panini ced \ age ON, (00 (300) LRAP-CS I ened P32 CS capi winnatrtatecned pasos amarante Las a ae K Chitesan MW Sect ee nl WN A haga ae Demin dentin a_i i ee ae rinitorunierVinennst a CETTE Eee L TPP Pp ey Phe Pt 40 20 30 40 50 60 Two-Theta (deg) Fig. 3D
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Patent Application Publication Aug. 23,2018 Sheet Sof 20 US 2018/0236130 Al Fig. 4A
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Patent Application Publication Aug. 23,2018 Sheet 6 of 20 US 2018/0236130 Al
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Patent Application Publication Aug. 23,2018 Sheet 7 of 20 US 2018/0236130 Al Fig. 6B
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Patent Application Publication Aug. 23,2018 Sheet 8 of 20 US 2018/0236130 Al
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Patent Application Publication Aug. 23,2018 Sheet 9 of 20 US 2018/0236130 Al
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Patent Application Publication Aug. 23,2018 Sheet 10 of 20 US 2018/0236130 Al Fig. 9A Fig. 9B
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Patent Application Publication Aug. 23,2018 Sheet 11 of 20 US 2018/0236130 Al
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Patent Application Publication Aug. 23,2018 Sheet 12 of 20 US 2018/0236130 Al ee Fig. 11
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Patent Application Publication Aug. 23,2018 Sheet 13 of 20 US 2018/0236130 Al Fig. 12
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Patent Application Publication Aug. 23,2018 Sheet 14 of 20 US 2018/0236130 Al Fig. 13
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Patent Application Publication Aug. 23,2018 Sheet 15 of 20 US 2018/0236130 Al
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Patent Application Publication Aug. 23,2018 Sheet 16 of 20 US 2018/0236130 Al Fig. 15A Fig. 15B
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Patent Application Publication Aug. 23,2018 Sheet 17 of 20 US 2018/0236130 Al Fig. 16A
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Patent Application Publication Aug. 23,2018 Sheet 18 of 20 US 2018/0236130 Al Prior Art § ee Fig. 17B
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Patent Application Publication Modulus (GPa) Modulus (GPa) 60 80 40 30 20 10 4 60 50 40 30 20 10 Aug. 23, 2018 Sheet 19 of 20 US 2018/0236130 Al After § days aa rm Healthy Demin LRAP LRAP-CS P32 P32CS P26 P26-CS Dentin Dentin Fig. 18A After 10 days i HR coy ‘ia i 7 Healthy Demin Dentin Dentin T ¥ ¥ ¥ T "y . “po r ChitosanLRAPLRAP-CS P32 P32-CS P26 P26-CS only Fig. 18B
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Patent Application Publication Aug. 23,2018 Sheet 20 of 20 US 2018/0236130 Al After 5 days 2.0 y 18 1.6 + s 14 oO 1.2 I 2 1.0 i ® 08 p+= ay g 0.6 i : zr 0.44 z = ' 02+, bi) * ie 0 T T : T T T T : t Healthy Demin LRAP LRAP-CS P32 P32-CS P26 P26-CS Dentin Dentin Fig. 18C After 10 days 2.8 1.8 1.6 t vt i s 1.4 = an & 0.87 . | Healthy Demin Chitosan LRAPLRAP-CS P32 P92-CS P26 P28.C8 Dentin Dentin only Fig. 18D
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US 2018/0236130 Al AMELOGENIN-CHITOSAN HYDROGEL FOR DENTIN HYPERSENSITIVITY CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. provi- sional application Ser. No. 62/189,356 filed Jul. 7, 2015, the disclosure of which is hereby incorporated in its entirety by reference herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] The invention was made with Government support under Contract No. (301)594-7703 awarded by the National Institute of Dental and Craniofacial Research. The Govern- ment has certain rights to the invention. TECHNICAL FIELD [0003] In at least one aspect, the present invention is related to the methods of reconstructing enamel. BACKGROUND [0004] Enamel is the exterior layer of the mammalian tooth. Dentin is the underlying calcified tissue, similar in composition to bone, that constitutes the bulk of the tooth structure. Dentin is secreted by the odontoblasts to form a large number of closely packed S-shaped dentinal tubules in a mineralized collagen matrix. Together with the overlying enamel, it provides resilience and rigidity to the tooth structure against masticatory and shearing forces. Exposure of dentinal tubules to the oral environment may occur due to several factors like dental caries, erosion, abrasion, attrition, failed restorations, cracked cusps and gingival recession. This can prompt a sharp acute pain response termed dentinal hypersensitivity explained by the increased dentinal tubular fluid flow, which activates the nerve fibers for pain. Cur- rently, no clinically viable strategies are available to regrow an enamel-like tissue on a dentin substrate. SUMMARY [0005] In at least one embodiment, a method to regrow a protective layer over demineralized dentin is provided. The method includes a step of identifying a subject having exposed and/or demineralized dentin. The demineralized/ exposed dentin is contacted with a remineralization compo- sition that includes an amelogenin, a chitosan, water, and a sufficient amount of a pH adjusting component such that the composition has a pH greater than about 6.0 (e.g., about 6.5) such that enamel is regrown on the dentinal tubules. The present embodiment is a peptide-based biomimetic approach that uses a chitosan/amelogenin-derived peptide (i.e. LRAP, P26, and P32) hydrogel to regrow a protective layer over exposed dentin. Advantageously, the method restores the integrity of the tissue against future acid attacks thereby providing a treatment for dentinal hypersensitivity, root caries and cases of severe enamel loss associated with deep caries and dental erosion. [0006] Advantageously, the present method enables the occlusion of dentinal tubules that were exposed due to physical and physiological forces acting within the oral cavity and/or the remineralization of demineralized collagen fibers with growth of a barrier layer of tightly adherent Aug. 23, 2018 enamel-inspired crystalline material. This remineralization prevents any external stimulus from direct contact and activation of the nerve fibers for pain. The involvement of amelogenin-derived peptides (LRAP) modulates the apatite crystals to growth similar to the enamel crystal formation in vivo thus greatly increasing the strength and toughness of such material. The cell free method of the invention allows control of the product in terms of: the type of mineral, crystal orientation, and crystal morphology by controlling local pH, and ion concentrations (Mg, Ca, PO,, F) and most impor- tantly amelogenin and its derived concentration. Finally, since the composition used in the present embodiment is a cell free system, problems associated with contamination by cell component, which may cause severe immunological reactions, are avoided. BRIEF DESCRIPTION OF THE DRAWINGS [0007] FIG. 1 provides a model for peptide-mediated dentin mineralization of a tooth having carious or non- carious cervical lesions. [0008] FIG. 2 provides a rationale for the design of amelogenin-inspired peptides from full-length amelogenin. [0009] FIGS. 3A, 3B, 3C and 3D provide 4 XRD patterns of dentin slices treated with peptide only (A, B) and peptide- chitosan hydrogel (C, D) in artificial saliva for 5 and 10 days. [0010] FIGS. 4A, 4B, and 4C provide SEM images show- ing the open ended dentinal tubules in healthy dentin (A), widened tubules in 3 day demineralized dentin (B) and chitosan gel treated dentin for 10 days (positive control) (C). FIG. 4C (inset) depicts heterogenous distribution of small needle-like crystallites closing the openings to the dentinal tubules in parts of the dentin surface. Peritubular (arrow) and intertubular (asterisk) dentin have also been shown in the images (A, B). [0011] FIGS. 5A and 5B provide SEM images of the remineralized dentin slices after 5 days of treatment with LRAP (200 ig/ml). [0012] FIGS. 6A and 6B provide SEM images of the remineralized dentin slices after 5 days of treatment with polypeptide P32 (200 ug/ml). [0013] FIGS. 7A and 7B provide SEM images of the remineralized dentin slices after 5 days of treatment with polypeptide P26 (200 pg/ml). [0014] FIGS. 8A and 8B provide SEM images of the remineralized dentin slices after 10 days of treatment with LRAP (200 1g/ml). [0015] FIGS. 9A and 9B provide SEM images of the remineralized dentin slices after 10 days of treatment with polypeptide P32 (200 pg/ml). [0016] FIGS. 10A and 10B provide SEM images of the remineralized dentin slices after 10 days of treatment with polypeptide P26 (200 pg/ml). [0017] FIG. 11 provides SEM images of the remineralized dentin slices after 5 days of treatment with peptide-chitosan hydrogel LRAP-CS (200 p1g/ml). [0018] FIG. 12 provides SEM images of the remineralized dentin slices after 5 days of treatment with peptide-chitosan hydrogel P32-CS (200 ug/ml). [0019] FIG. 13 provides SEM images of the remineralized dentin slices after 5 days of treatment with peptide-chitosan hydrogel P26-CS (200 pg/ml).
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US 2018/0236130 Al [0020] FIGS. 14A and 14B provide SEM images of the remineralized dentin slices after 10 days of treatment with peptide-chitosan hydrogel LRAP-CS (200 pg/ml). [0021] FIGS. 15A and 15B provide SEM images of the remineralized dentin slices after 10 days of treatment with peptide-chitosan hydrogel P32-CS (200 g/ml). Areas of transition have been shown via dotted arrows as the miner- alization advances from the occlusion of the tubules to the regeneration of enamel-like apatitic crystallites on the dentin surface. [0022] FIGS. 16A and 16B provide SEM images of the remineralized dentin slices after 10 days of treatment with peptide-chitosan hydrogel P26-CS (200 t1:g/ml). [0023] FIG. 17A is a TEM image depicting the uniformly- dispersed spherical assemblies of amelogenin-inspired pep- tide (P32) formed at pH 7.4, in HEPES buffer, at 25° C. [0024] FIG. 17B is a TEM image of a prior art assembly pattern of full-length recombinant porcine amelogenin (172). [0025] FIGS. 18A, 18B, 18C, and 18D provide nanoin- dentation test results showing changes in the modulus (A, B) and hardness (C, D) of the newly regenerated layer after being treated with peptide only and peptide-chitosan hydro- gel for 5 and 10 days. DETAILED DESCRIPTION, [0026] Reference will now be made in detail to presently preferred compositions, embodiments and methods of the present invention, which constitute the best modes of prac- ticing the invention presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the invention and/or as a representative basis for teaching one skilled in the art to variously employ the present invention. [0027] Except in the examples, or where otherwise expressly indicated, all numerical quantities in this descrip- tion indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word “about” in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: percent, “parts of” and ratio values are by weight; the term “polymer” includes “oligomer,” “copolymer,” “terpolymer,” and the like; molecular weights provided for any polymers refers to weight average molecular weight unless otherwise indicated; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not nec- essarily preclude chemical interactions among the constitu- ents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutan- dis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, Aug. 23, 2018 measurement of a property is determined by the same technique as previously or later referenced for the same property. [0028] It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodi- ments of the present invention and is not intended to be limiting in any way. [0029] It must also be noted that, as used in the specifi- cation and the appended claims, the singular form “a,” “an,” and “the” comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components. [0030] The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps. [0031] The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole [0032] The phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. [0033] The terms “comprising”, “consisting of”, and “con- sisting essentially of’ can be alternatively used. Where one of these three terms is used, the presently disclosed and claimed subject matter can include the use of either of the other two terms. [0034] Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Abbreviations [0035] [0036] [0037] [0038] tide. [0039] [0040] “CS” means chitosan. “demin” means demineralized. “HAP” means hydroxyapatite. “LRAP” means leucine rich amelogenin polypep- “P32” means peptide 32. “P26” means peptide 26. [0041] “XRD” means X-ray diffraction. [0042] The term “hydrogel” refers to a gel in which the dispersion medium is water. [0043] The term “subject” refers to a human or animal, including all mammals such as primates (particularly higher primates) having dental caries, early dental carious and erosive lesions as well as enamel defects resulting from genetic diseases. [0044] The term “polypeptide” refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a poly- peptide. This term also does not exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the defini-
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US 2018/0236130 Al Aug. 23, 2018 tion are, for example, polypeptides containing one or more sequence may be compared to a reference sequence of the analogs of an amino acid (including, for example, unnatural same number of contiguous positions after the two amino acids, etc.), polypeptides with substituted linkages as gequences are aligned optimally. In a refinement, the com- well as other medifications known in the art, both naturally parison window is from 15 to 30 contiguous positions in and non-naturally occurring. ee tte s which a sequence may be compared to a reference sequence [0045] The terms “sequence identity” or “identity” refers . an : : : ca of the same number of contiguous positions after the two to a specified percentage of residues in two nucleic acid or . : . wa sequences are aligned optimally. In another refinement, the amino acid sequences that are identical when aligned for : : . ° vp : comparison window is usually from about 50 to about 200 maximum correspondence over a specified comparison win- : reeeen : . ‘ contiguous positions in which a sequence may be compared dow, as measured by sequence comparison algorithms or by . ‘ A . wpe A to a reference sequence of the same number of contiguous visual inspection. When sequences differ in conservative ae . . an ea . positions after the two sequences are aligned optimally. substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the sub- [0047] The term “amelogenin” refers to the closely related stitution. Sequences that differ by such conservative substi- polypeptides involved in the formation of enamel and iso- tutions are said to have “sequence similarity” or “similarity.” _ forms thereof. Amelogenin is exemplified by SEQ ID NOs Means for making this adjustment are well known to those __ set forth in Table 1. In a refinement, this definition includes of skill in the art. Typically this involves scoring a conser- _ alternatively spliced isoforms such as LRAP (leucine rich vative substitution as a partial rather than a full mismatch, amelogenin polypeptide). It should be appreciated that this thereby increasing the percentage sequence identity. definition includes polypeptides having | to 10 conservative [0046] The term “comparison window” refers to a seg- substitutions of SEQ ID NOs: 1-9 as set forth below in more ment of at least about 20 contiguous positions in which a _—_ detail. TABLE 1 Amelogenin Polypeptide Sequences Species Sequence SEQ ID NO: 1 Sus scrofa MGTWILFACLLGAAFSMPLPPHPGHPGYINFSYEVLTPLK WYQNMIRHPYTS YGYEPMGGWLHHOT I PVVSQQTPOSH ALQPHHHIPMVPAQQPGI POQPMMPLPGQHSMTPTQHH QPNLPLPAQOPF QPOPVOPOPHOPLOPQSPMHPIQPLLPQ PPLPPMFSMQSLLPDLPLEAWPATDKTKREEVD SEQ ID NO: 2 Bos taurus FACLLGAAYSMPLPPHPGHPGYINFSYEVLTPLEWYON MLRYPYPSYGYEPVGGWLHHOI I PVVSOQQSPONHALOPH HHNPMVPAQOPVVPQOPMMPVPGQHSMTP IQHHOPNLP LPAQOSFOPQP1 QPOPHOPLOPOPPVHPIORLPPOPPLPPIF PMQPLPPVLPDLPLEAWPATDKTKREE SEQ ID NO: 3 Homo MGTWILFACLLGAAFAMPLPPHPGHPGYINFSYEVLTPL sapiens KWYQSIRPPYPSYGYEPMGGWLHHQTIPVLSQQHPPTHT LOPHHHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQHHOP NLPPPAQQPYQPQPVQPQPHOPMQPQPPVHPMQPLPPQP PLPPMFPMQPLPPMLPDLTLEAWPSTDKTKREEVD SEQ ID NO: 4 Pongo ACLLGAAFAMPLPPHPGHPGYINFSYENSHSQAINVDRT pygmaeus ALVLTPLKWYQSIRPPYPSYGYEPMGGWLHHOTIPVLSQ QHPPTHTLOPHHHI PVVPAQQPV IPOQQPMMPVPGOQHSMT PTQHHQPNLLPPAQQPYQPOPVOPQPHOPMOPQPPVHP MOQPLPPQPPLPPMFPMOPLPPMLPDLTLEAWPSTDKTKR EEVD SEQ ID NO: 5 Pan MGTWILFACLLGAAFAMPLPPHPGHPGYINFSYENSHSQ troglodytes AINVDRTALVLTPLKWYQSIRPPYPSYGY EPMGGWLHH QTIPVLSQQHPPTHTLOPHHHIPVVPAQOPVIPQOPMMPV PGQHSMTP1QHHOPNLPPPAQOP YOPOPVOPOPHOPMOP QPPVHPMQPLPPQPPLPPMFPMQPLPPMLPDLTLEAWPST DKTKREEVD SEQ ID NO: 6 Rattus MGTWILFACLLGAAFAMPLPPHPGSPGYINLSYEVLTPL norvegicus KWYQSMIRQPHPPSHTLOPHHHLPVVPAQQPVAPQOPM MPVPGHHSMTPTQHHOPNIPPSAQOPFOOPFOPOAI PPOS HOPMQPQSPLHPMQPLAPQPPLPPLFSMQPLSPILPELPLE ANPATDKTKREEVAFS PMKWYQGTARHPLNMETTTEK SEQ ID NO: 7 Mus MGTW1LFACLLGAAFAMPLPPHPGSPGYINLSYEKSHSQ musculus AINTDRTALVLTPLKWYQSMIRQPYPSYGYEPMGGWLH HOT IPVLSQOHPPSHTLOPHEIRLPVVPAQOPVAPQOPMM PVPGHHSMTPTQHHOPNI PPSAQQPFQQPFQPQAIPPQSH QPMQPOSPLHPMOPLAPQPPLPPLFSMOPLSPILPELPLEA WPATDKTKREEVD
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US 2018/0236130 Al TABLE 1-continued Aug. 23, 2018 Amelogenin Polypeptide Sequences Species Sequence SEQ ID NO: 8 Sug scrofa MPLPPHPGHPGYINFSYEVLTPLKWYQNMIRHPSLLPDLL EAWPATDKTKREEVD SEQ ID NO: 9 Homo MPLPPHPGHPGYINFSYEVLTPLKWYOS IRPPPLPPMLPD sapiens LTLEAWPSTDKTKREEVD (LRAP (+P) [0048] In another variation, the term “amelogenin” also includes “amelogenin-derived polypeptides” having the ability to form enamel. Examples of amelogenin-derived polypeptides include the previously unknown polypeptides having SEQ ID NO: 10 and SEQ ID NO: 11 as set forth in Table 2 and variations of these polypeptides having conser- vative substitutions as set forth below in more detail. TABLE 2 Artificial Amelogenin-derived polypeptides Description Sequence SEQ ID Artificial MPLPSYEVLTPLKWPVHPMOPSTDKT NO: 10 (Peptide 32) KREEVD SEQ ID Artificial | MPLPSYEVLTPLKWPSTDKTKREEVD NO: 11 (Peptide 26) [0049] In an embodiment of the present invention, a method to regrow a protective layer over exposed or demin- eralized dentin is provided. The method includes a step of identifying a subject having demineralized dentin (e.g., exposed dentinal tubules). The exposed or demineralized dentin is contacted with a remineralization composition that includes an amelogenin, a chitosan, water, and a sufficient amount of a pH adjusting component such that the compo- sition has a pH greater than about 6.0 (e.g., 6.5) such that enamel is regrown on the exposed or demineralized dentin. Typically, the composition is allowed to air dry. The present method is advantageously used to remineralize dental lesions such as non-carious cervical lesions and class V caries. [0050] In a variation, the method further includes a step contacting the demineralized dentin with a component selected from the group consisting of a calcium-containing compound, a phosphate containing compound, and combi- nations thereof. In some variations, the exposed/demineral- ized dentin is contacted with saliva or a solution including water and a component selected from the group consisting of electrolytes, mucus, glycoproteins, enzymes, antibacterial compounds, and combinations thereof. [0051] In one refinement, the method also includes a step of contacting the substrate with a base (e.g., NaOH) for a first period of time. In a further refinement, the exposed or demineralized dentin is contacted with a component selected from the group consisting of calcium-containing compound, a phosphate containing compound, and combinations thereof for a second period of time. In still a further refinement, the exposed or demineralized dentin is contacted with saliva or a solution including water and a component selected from the group consisting of electrolytes, mucus, glycoproteins, enzymes, antibacterial compounds, and com- binations thereof. [0052] With reference to FIG. 1, a schematic illustration showing the remineralization of exposed dentin is provided. Tooth 10 is observed to have a cervical lesion 12 or class V caries 14 in which dentinal tubules 16 are exposed. Odon- toblasts 18 are also depicted in FIG. 1. The exposed dentinal tubules are treated with the remineralization composition as either an amelogenin only peptide solution 20 or an amelo- genin-chitosan gel 22. In either case, the composition also includes calcium ions 24 and phosphate ions 26 as set forth below in more detail. Peptide mediated agglomeration and nucleation of the calcium and phosphate ions occurs in region 30. After about 5 days occlusion of the dentinal tubules is observed as indicated by item number 30. After about 10 days, thin layer 32 of well-organized apatite crystals is regrown over the dentin. [0053] Details of the remineralization compositions used in the methods set forth herein are provided in U.S. patent application Ser. No. 14/142,086 filed Dec. 27, 2013; the entire disclosure of which is hereby incorporated by refer- ence. In particular, the chitosan has a formula described by a partially acetylated polysaccharide having formula I: HO. HO. ta) OH 0 0 OH Nib HO Nib wherein n is from about 500 to 2500. In a refinement, the partially acetylated polysaccharide having formula I has a degree of acetylation less than about 35 percent. In a further refinement, the partially acetylated polysaccharide having formula I has a degree of acetylation from about 5 to 35 percent. The chitosan in general has a viscosity average molecular weight (Mv) from about 150,000 to 400,000 Daltons. In a refinement, the chitosan has an Mv from about 190,000 to 310,000 Daltons. [0054] Examples of suitable amelogenins include, but are not limited to, porcine amelogenin rp172, mouse amelogen- ins, human amelogenins, etc., recombinant variations thereof, and isoforms thereof, and combinations thereof. Such isoforms include truncated amelogenin rP147 or an alternatively spliced isoform such as LRAP (leucine rich
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US 2018/0236130 Al amelogenin polypeptide) having 59 amino acids. SEQ ID NOs: 1-9 provide specific examples of amelogenins. In a variation, the amelogenins used in the methods herein are polypeptides having an amino acid sequence that is at least 80 percent identical to the polypeptides forth as SEQ ID NOs: 1-9. In other refinements, the amelogenin used in the methods herein are polypeptides having an amino acid sequence that is at least, in order of increasing preference, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5% or 99% percent identical to the amino acid sequence set forth as SEQ ID NOs: 1-9. Thus, the present invention also encompasses the use of sequences having a degree of sequence identity with the SEQ ID NOs: 1-9. Herein, the term “sequence identity” means a polypeptide having a certain similarity with the subject amino acid sequence. The similar amino acid sequence should provide a polypeptide which retains the functional activity of the sequence in re-mineralizing exposed dentin and forming enamel. [0055] In another variation, the amelogenins used in the methods herein are amelogenin-derived polypeptides having an amino acid sequence that is at least 80 percent identical to the polypeptides forth as SEQ ID NOs: 10-11. In other refinements, the amelogenins used in the methods herein are amelogenin-derived polypeptides having an amino acid sequence that is at least, in order of increasing preference, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5% or 99% percent identical to the amino acid sequence set forth as SEQ ID NOs: 1-9. These similar amino acid sequences should provide a polypeptide which retains the functional activity of the sequence in re-mineralizing exposed dentin and forming enamel. In still another refine- ment, a polynucleotide that encodes the amino acid sequences forth as SEQ ID NOs: 10-11 is also provided. [0056] Ina refinement, sequence identity comparisons are conducted as is well known in the art using sequence comparison computer programs that use algorithms to align two or more sequences using a scoring system that rewards alignment of identical or similar amino acids and penalizes substitutions of non-similar amino acids and gaps. Computer programs for carrying out alignments include, but are not limited to, BLASTP which is publicly available from the National Center for Biotechnology Information (www.ncbi. nim.nih.gov/). These programs calculate percent sequence identity and report the determined value. It is preferred to use the default values when using such software for amino acid sequence alignments. BLASTP is preferred to deter- mine amino acid sequence identity between the subject polypeptide sequences SEQ ID NOs: 1-11 and a candidate polypeptide sequence according to the present invention. Details of the BLASTP algorithm are set forth in D. W. Mount “Bioinformatics: Sequence and Genome Analysis,” Cold Spring Harbor Press (2004). A particularly preferred set of parameters for the BLASTP alignment includes a Blossum 62 scoring matrix with a gap penalty of 11, a gap extend penalty of 1, and conditional adjustments set to conditional compositional score matrix adjustment. Other preferred parameters for the BLASTP alignment are an expected threshold of 10 and a word size of 3. [0057] | In other variations, the amelogenins (including the amelogenin-derived polypeptides), in particular the poly- peptides, described by SEQ ID NOs: 1-11, include deletions, insertions or substitutions of amino acid residues which result in a functionally equivalent protein. Preferably, the substitutions are conservative being similar with respect to Aug. 23, 2018 polarity, charge, solubility, hydrophobicity, hydrophiliticity, and/or the amphipathic nature of the residues, while pre- serving the functionality of forming enamel. Conservative substitutions that may be made are, for example, substitu- tions between aliphatic amino acids (alanine, valine, leucine, isoleucine), polar amino acids (glutamine, asparagine, ser- ine, threonine), acidic amino acids (glutamic acid and aspar- tic acid), basic amino acids (arginine, lysine and histidine), aromatic amino acids (phenylalanine, tryptophan and tyro- sine), large amino acids (phenylalanine and tryptophan), small amino acids (glycine, alanine) and hydroxyl amino acids (serine, threonine). In a refinement, the amelogenins include polypeptides having 1 to 10 (i.e., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10) conservative substitutions of SEQ ID NOs: 1-11. In another refinement, the amelogenins include polypeptides having 1 to 5 conservative substitutions of SEQ ID NOs: 1-11. [0058] Typically, the amelogenin is present in an amount from about 0.02 percent to about 0.4 percent of the total weight of the first composition and the chitosan is present in an amount from about 0.2 to about 3 percent of the total weight of the first composition with the balance being water [0059] In a variation, the remineralization composition also includes a calcium-containing compound (e.g., Ca, (PO,),, CaCl,) that provides calcium ions when dissolved in water. In a further refinement, the remineralization compo- sition can further include a phosphate-containing compound that provides phosphate and/or hydrogen phosphate ions when dissolved in water. Examples of such phosphate- containing compound include, but are not limited to, Na,HPO, which provides hydrogen phosphate ions (HPO,”). Typically, the amount of the phosphate-contain- ing compound is present in an amount from about 0.01 percent to about 0.2 percent of the total weight of the remineralization composition. The pH of the remineraliza- tion composition is set to a pH equal to or greater than 6.0. A base such as NaOH may be used to adjust the pH. In one variation, the remineralization composition is advanta- geously a hydrogel that is useful for re-construction of enamel while inhibiting bacterial growth. [0060] In a variation, the remineralization composition includes an amelogenin, chitosan, a calcium-containing compound that provides calcium ion when dissolved in water (e.g., CaCl), a phosphate-containing compound (e.g., Na,HPO,) that provides phosphate ions and/or hydrogen phosphate ions when dissolved in water, and water. Examples of suitable amelogenins are set forth above. Typically, the amelogenin is present in an amount from about 0.02 percent to about 0.2 percent of the total weight of the second composition and the chitosan is present in an amount from about 0.2 to about 3 percent of the total weight of the second composition, and the calcium containing compound is present in an amount from about 0.01 percent to about 0.2 percent of the total weight of the second composition with the balance being water. The details regarding the chitosan are also set forth above. In a refine- ment, the remineralization composition further includes a compound such as Na, HPO, providing hydrogen phosphate ions (HPO,?-). Typically, the amount of the compound providing phosphate ions is present in an amount from about 0.01 percent to about 0.2 percent of the total weight of the remineralization composition. The pH of the second com- position is set to a pH equal to or greater than 6.0 (e.g., about 6.5). A base such as NaOH may be used to adjust the pH to
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US 2018/0236130 Al a value of 6.0 or greater. The remineralization composition is advantageously a hydrogel that is useful for re-construc- tion of enamel while inhibiting bacterial growth. [0061] The remineralization composition is advanta- geously used to re-construct enamel by contacting an exposed dentin substrate (e.g., a tooth) with the composition and then allowing the composition to air dry. The dentin coated substrate is then optionally rinsed and then contacted with saliva for an extended period of time (>24 hours). [0062] In one or more variations of the remineralization composition, additional protein components are included. Examples of such additional components include, but are not limited to, enamelin, ameloblastin, enamel proteases, chitosanolytic enzymes, and combinations thereof. Suitable enamel proteases include, but are not limited to, Kallikrein- related peptidase 4 (KLK-4) and matrix metalloproteinase- 20(MMP-20). In a refinement, these additional components are individually or collectively present in an amount from about 0.005 percent to about 0.1 percent of the total weight of the first composition. [0063] In another embodiment, a method for remineraliz- ing a variety of dental lesions with remineralization com- positions that include a polypeptide having SEQ ID NO: 1 or 2 is provided. Advantageously, this method can reminer- alize dental caries, early dental carious and erosive lesions, and enamel defects in addition to lesions having exposed dentin. The method includes a step of identifying a subject having a dental lesion and then contacting the dental lesion with a remineralization composition. The method also includes a step of allowing the remineralization composition to air dry. Characteristically, the remineralization composi- tion includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, with up to 10 conservative amino acid substitutions (or identity with) as set forth above. The remineralization composition also includes an optional chitosan, water (balance), a suflicient amount of a pH adjusting component such that the compo- sition has a pH greater than about 6.0. The details of the chitosan are set forth above. Advantageously, enamel is regrown on the dental lesions. [0064] Additional details of the remineralization compo- sition for this embodiment are provided in U.S. patent application Ser. No. 14/142,086 filed Dec. 27, 2013 with the peptides replacing the amelogenin in that reference. In particular, the remineralization composition can include a calcium-containing compound that provides calcium ion when dissolved in water and a phosphate containing com- pound that provides phosphate ions when dissolved in water. In one refinement, the method also includes a step of contacting the dental lesion with a base (e.g., NaOH) for a first period of time. In a further refinement, the dental lesion is contacted with a component selected from the group consisting of calcium-containing compound, a phosphate containing compound, and combinations thereof for a sec- ond period of time. In still a further refinement, the dental lesion is contacted with saliva or an aqueous solution that includes water and a component selected from the group consisting of electrolytes, mucus, glycoproteins, enzymes, antibacterial compounds, and combinations thereof. In a refinement, these additional components are individually or collectively present in an amount from about 0.005 percent to about 0.1 percent of the total weight of the aqueous solution. Aug. 23, 2018 [0065] The composition and methods of the invention are further illustrated by the following examples. These are provided by way of illustration and are not intended in any way to limit the scope of the invention. Polypeptide Design [0066] As illustrated in FIG. 2, the polypeptides having the amino acid sequence with SEQ ID NO: 10 and SEQ ID NO: 11 were inspired peptides as potential biomimetic regrowth strategies based on a critical understanding of the apatite binding and mineralization promoting domains of the native protein. The amino acid sequences SEQ ID NO: 10 and SEQ ID NO: 11 were synthesized by Chempeptide Limited, Shanghai, China. The amino acid SEQ ID NO: 11 retains the last 12 mers of the amelogenin C-terminus and includes about 50% of the charged residues of the full-length amelogenin. The close proximity of the hydrophilic C-ter- minus of amelogenin to the HAP surface has been directly implicated in mediating crystal nucleation and growth pro- cesses through a highly specific protein-crystal interaction. The 14 amino acid residues from the N-terminus (residues 1-4 and 16-25) with a phosphorylated Serine at the N-ter- minal were preserved. It has been shown through in vitro mineralization studies that the role of the N-terminal (+P) is to regulate crystal shape and stabilize ACP formation, thus playing a vital role in crystal morphology and apatite phase transition. For the amino acid sequence having SEQ ID NO: 10, two polyproline repeat regions (PXX/PXQ) were retained from the middle hydrophobic core of native amelo- genin to observe whether addition of proline repeat length to the C-terminus would promote crystal elongation and growth as suggested in previous literature. Treatment of a Tooth with the Remineralization Composition. [0067] Human third molars were sectioned transversally into 2 mm thick slices at the mid-coronal region of the tooth. A window of sound dentin measuring 2x2 mm each was delineated by applying two coats of acid-resistant nail varnish on the tooth surfaces. To mimic carious lesions, the samples were immersed in a demineralizing solution (pH 4.6, 30 ml ) at 37° C. for 3 days. Twenty \l of peptide only (LRAP, P32, P26) and peptide-chitosan hydrogel (200 ug/ml) were applied separately on the demineralized win- dows (2 applications: Days 1 & 5) and the tooth slices were immersed in artificial saliva (pH 7, 5 ml ) at 37° C. for 5 and 10 days. The artificial saliva includes 1.2 mM Ca?*, 0.2 mM Mg?*0.7 mM H.PO,-, 16 mM KCl, 4.5 mM NH,Cl and 50 mM Hepes in water. The artificial saliva solution (5 ml) was changed daily from days 1-10. Characterization of the Remineralization [0068] FIG. 3 provides XRD patterns of the demineralized dentin, sound dentin, control samples (treated in artificial saliva and chitosan only), peptide only (A, B) and peptide chitosan hydrogel (C, D) treated dentin slices after being immersed in artificial saliva for 5 and 10 days. [0069] FIGS. 4A, 4B, and 4C provide SEM images show- ing the open ended dentinal tubules in healthy dentin (A), widened tubules in 3 day demineralized dentin (B) and chitosan gel treated dentin for 10 days (positive control) (C). FIG. 4C (inset) depicts heterogenous distribution of small needle-like crystallites closing the openings to the dentinal
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US 2018/0236130 Al tubules in parts of the dentin surface. Peritubular (arrow) and intertubular (asterisk) dentin have also been shown in the images (A, B). [0070] FIGS. 5A and 5B provide SEM images of the remineralized dentin slices after 5 days of treatment with LRAP. FIGS. 6A and 6B provides SEM images of the remineralized dentin slices after 5 days of treatment with polypeptide P32. FIGS. 7A and 7B provides SEM images of the remineralized dentin slices after 5 days of treatment with polypeptide P26. Needle-like HAP crystals densely are observed to cover the entire surface of demineralized dentin with complete occlusion of the dentinal tubules. The smaller images are enlarged details and cross-sections of the dentin slices. [0071] FIGS. 8A and 8B provide SEM images of the remineralized dentin slices after 10 days of treatment with LRAP. FIGS. 9A and 9B provides SEM images of the remineralized dentin slices after 10 days of treatment with polypeptide P32. FIGS. 10A and 10B provide SEM images of the remineralized dentin slices after 10 days of treatment with polypeptide P26. A well-organized newly grown layer was observed forming a robust interface with underlying native dentin. Apatite crystals grew from the walls of the dentinal tubules up to the dentin surface. [0072] FIG. 11 provides SEM images of the remineralized dentin slices after 5 days of treatment with peptide-chitosan hydrogel LRAP-CS. FIG. 12 provides SEM images of the remineralized dentin slices after 5 days of treatment with peptide-chitosan hydrogel P32-CS. FIG. 13 provides SEM images of the remineralized dentin slices after 5 days of treatment with peptide-chitosan hydrogel P26-CS. FIGS. 14A and 14B provide SEM images of the remineralized dentin slices after 10 days of treatment with peptide-chitosan hydrogel LRAP-CS. FIGS. 15A and 15B provide SEM images of the remineralized dentin slices after 10 days of treatment with peptide-chitosan hydrogel P32-CS. Areas of transition have been shown via dotted arrows as the miner- Aug. 23, 2018 alization advances from the occlusion of the tubules to the regeneration of enamel-like apatitic crystallites on the dentin surface. FIGS. 16A and 16B provides SEM images of the remineralized dentin slices after 10 days of treatment with peptide-chitosan hydrogel P26-CS. Areas of transition have been shown via dotted arrows as the mineralization advances from the occlusion of the tubules to the regenera- tion of small crystallites on the dentin surface. Narrowing of the peritubular dentin was observed followed by occlusion of the tubules. There was no apatitic layer formed at the end of 5 days of treatment with peptide-gel. [0073] FIG. 17A provides a TEM image depicting the uniformly-dispersed spherical assemblies of amelogenin- inspired peptide (P32) formed at pH 7.4, in HEPES buffer, at 25° C. This characteristic morphology is similar to the assembly pattern of full-length recombinant porcine amelo- genin (rP172) as seen in previous studies (FIG. 17B). (Ref: doi:10.1016/j jsb.2011.07.016). [0074] FIGS. 18A, 18B, 18C, and 18D provide nanoin- dentation test results showing changes in the modulus (A, B) and hardness (C, D) of the newly regenerated layer after being treated with peptide only and peptide-chitosan hydro- gel for 5 and 10 days. Treatment with LRAP and P32 restored surface hardness of dentin lesions in 5 days. A highly mineralized dense layer was formed with superior mechanical properties than healthy dentin. Treatment with peptide-chitosan gel took ~10 days to significantly improve the mechanical properties of dentin lesions. [0075] While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limi- tation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implement- ing embodiments may be combined to form further embodi- ments of the invention. SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 11 <210> SEQ ID NO 1 <211> LENGTH: 189 <212> TYPE: PRT <213> ORGANISM: Sus scrofa <400> SEQUENCE: 1 Met Gly Thr Trp Tle 1 5 Leu Phe Ala Leu 10 cys Gly Ala Pro Lew Pro His Pro Gly His Gly Tyr Ile Glu Val 35 Thr Pro Leu Lys ln 40 Asn Met 45 Trp Tyr Pro Tyr Thr elu 50 Ser Tyr Tyr Pro Met 55 Gly ¢ly 60 Gln Ile Ile Pro val Gln Gln Thr Pro Ser Gln His 85 Pro His Pro Met Val Pro 90 Ala Gln Gin Gln Gln Pro Met Met Pro Leu Pro Gly Gln His Ser Ala Phe 15 Asn Phe 30 Ile Arg His Leu His His His Ala Pro Gly 95 Met Thr
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US 2018/0236130 Al Aug. 23, 2018 -continued 100 105 110 Thr Gln His His Gln Pro Asn Leu Pro Leu Pro Ala Gln Gln Pro Phe 115 120 125 Gln Pro Gln Pro Val Gln Pro Gln Pro His Gln Pro Leu Gln Pro Gln 130 135 140 Ser Pro Met His Pro Ile Gln Pro Leu Leu Pro Gln Pro Pro Leu Pro 145 150 155 160 Pro Met Phe Ser Met Gln Ser Leu Leu Pro Asp Leu Pro Leu Glu Ala 165 170 175 Trp Pro Ala Thr Asp Lys Thr Lys Arg Glu Glu Val Asp 180 185 <210> SEQ ID NO 2 <211> LENGTH: 184 <212> TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 2 Phe Ala Cys Leu Leu Gly Ala Ala Tyr Ser Met Pro Leu Pro Pro His 1 5 10 15 Pro Gly His Pro Gly Tyr Ile Asn Phe Ser Tyr Glu Val Leu Thr Pro 20 25 30 Leu Lys Trp Tyr Gln Asn Met Leu Arg Tyr Pro Tyr Pro Ser Tyr Gly 35 40 45 Tyr Glu Pro Val Gly Gly Trp Leu His His Gln Ile Ile Pro Val Val 50 55 60 Ser Gln Gln Ser Pro Gln Asn His Ala Leu Gln Pro His His His Asn 65 70 75 80 Pro Met Val Pro Ala Gln Gln Pro Val Val Pro Gln Gln Pro Met Met Pro Val Pro Gly Gln His Ser Met Thr Pro Ile Gln His His Gln Pro 100 105 110 Asn Leu Pro Leu Pro Ala Gln Gln Ser Phe Gln Pro Gln Pro Ile Gln 115 120 125 Pro Gln Pro His Gln Pro Leu Gln Pro Gln Pro Pro Val His Pro Ile 130 135 140 Gln Arg Leu Pro Pro Gln Pro Pro Leu Pro Pro Ile Phe Pro Met Gln 145 150 155 160 Pro Leu Pro Pro Val Leu Pro Asp Leu Pro Leu Glu Ala Trp Pro Ala 165 170 175 Thr Asp Lys Thr Lys Arg Glu Glu 180 <210> SEQ ID NO 3 <211> LENGTH: 191 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Met Gly Thr Trp Ile Leu Phe Ala Cys Leu Leu Gly Ala Ala Phe Ala 1 5 10 15 Met Pro Leu Pro Pro His Pro Gly His Pro Gly Tyr Ile Asn Phe Ser 20 25 30 Tyr Glu Val Leu Thr Pro Leu Lys Trp Tyr Gln Ser Ile Arg Pro Pro
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US 2018/0236130 Al Aug. 23, 2018 -continued 35 40 45 Tyr Pro Ser Tyr Gly Tyr Glu Pro Met Gly Gly Trp Leu His His Gln Ile Ile Pro Val Leu Ser Gln Gln His Pro Pro Thr His Thr Leu Gln 65 70 75 80 Pro His His His Ile Pro Val Val Pro Ala Gln Gln Pro Val Ile Pro 85 90 95 Gln Gln Pro Met Met Pro Val Pro Gly Gln His Ser Met Thr Pro Ile 100 105 110 Gln His His Gln Pro Asn Leu Pro Pro Pro Ala Gln Gln Pro Tyr Gln 115 120 125 Pro Gln Pro Val Gln Pro Gln Pro His Gln Pro Met Gln Pro Gln Pro 130 135 140 Pro Val His Pro Met Gln Pro Leu Pro Pro Gln Pro Pro Leu Pro Pro 145 150 155 160 Met Phe Pro Met Gln Pro Leu Pro Pro Met Leu Pro Asp Leu Thr Leu 165 170 175 Glu Ala Trp Pro Ser Thr Asp Lys Thr Lys Arg Glu Glu Val Asp 180 185 190 <210> SEQ ID NO 4 <211> LENGTH: 198 <212> TYPE: PRT <213> ORGANISM: Pongo pygmaeus <400> SEQUENCE: 4 Ala Cys Leu Leu Gly Ala Ala Phe Ala Met Pro Leu Pro Pro His Pro 1 5 10 15 Gly His Pro Gly Tyr Ile Asn Phe Ser Tyr Glu Asn Ser His Ser Gln 20 25 30 Ala Tle Asn Val Asp Arg Thr Ala Leu Val Leu Thr Pro Leu Lys Trp 35 40 45 Tyr Gln Ser Ile Arg Pro Pro Tyr Pro Ser Tyr Gly Tyr Glu Pro Met 50 55 60 Gly Gly Trp Leu His His Gln Ile Tle Pro Val Leu Ser Gln Gln His 65 70 75 80 Pro Pro Thr His Thr Leu Gln Pro His His His Ile Pro Val Val Pro Ala Gln Gln Pro Val Tle Pro Gln Gln Pro Met Met Pro Val Pro Gly 100 105 110 Gln His Ser Met Thr Pro Thr Gln His His Gln Pro Asn Leu Leu Pro 115 120 125 Pro Ala Gln Gln Pro Tyr Gln Pro Gln Pro Val Gln Pro Gln Pro His 130 135 140 Gln Pro Met Gln Pro Gln Pro Pro Val His Pro Met Gln Pro Leu Pro 145 150 155 160 Pro Gln Pro Pro Leu Pro Pro Met Phe Pro Met Gln Pro Leu Pro Pro 165 170 175 Met Leu Pro Asp Leu Thr Leu Glu Ala Trp Pro Ser Thr Asp Lys Thr 180 185 190 Lys Arg Glu Glu Val Asp 195
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US 2018/0236130 Al Aug. 23, 2018 10 -continued <210> SEQ ID NO 5 <211> LENGTH: 205 <212> TYPE: PRT <213> ORGANISM: Pan troglodytes <400> SEQUENCE: 5 Met Gly Thr Trp Ile Leu Phe Ala Cys Leu Leu Gly Ala Ala Phe Ala 1 5 10 15 Met Pro Leu Pro Pro His Pro Gly His Pro Gly Tyr Ile Asn Phe Ser 20 25 30 Tyr Glu Asn Ser His Ser Gln Ala Ile Asn Val Asp Arg Thr Ala Leu val Leu Thr Pro Leu Lys Trp Tyr Gln Ser Ile Arg Pro Pro Tyr Pro 50 55 60 Ser Tyr Gly Tyr Glu Pro Met Gly Gly Trp Leu His His Gln Ile Ile 65 70 75 80 Pro Val Leu Ser Gln Gln His Pro Pro Thr His Thr Leu Gln Pro His His His Ile Pro Val Val Pro Ala Gln Gln Pro Val Ile Pro Gln Gln 100 105 110 Pro Met Met Pro Val Pro Gly Gln His Ser Met Thr Pro Ile Gln His 115 120 125 His Gln Pro Asn Leu Pro Pro Pro Ala Gln Gln Pro Tyr Gln Pro Gin 130 135 140 Pro Val Gln Pro Gln Pro His Gln Pro Met Gln Pro Gln Pro Pro Val 145 150 155 160 His Pro Met Gln Pro Leu Pro Pro Gln Pro Pro Leu Pro Pro Met Phe 165 170 175 Pro Met Gln Pro Leu Pro Pro Met Leu Pro Asp Leu Thr Leu Glu Ala 180 185 190 Trp Pro Ser Thr Asp Lys Thr Lys Arg Glu Glu Val Asp 195 200 208 <210> SEQ ID NO 6 <211> LENGTH: 195 <212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 6 Met Gly Thr Trp Ile Leu Phe Ala Cys Leu Leu Gly Ala Ala Phe Ala 1 5 10 15 Met Pro Leu Pro Pro His Pro Gly Ser Pro Gly Tyr Ile Asn Leu Ser 20 25 30 Tyr Glu Val Leu Thr Pro Leu Lys Trp Tyr Gln Ser Met Ile Arg Gln 35 40 45 Pro His Pro Pro Ser His Thr Leu Gln Pro His His His Leu Pro Val 50 55 60 Val Pro Ala Gln Gln Pro Val Ala Pro Gln Gln Pro Met Met Pro Val 65 70 15 80 Pro Gly His His Ser Met Thr Pro Thr Gln His His Gln Pro Asn Ile 85 90 95 Pro Pro Ser Ala Gln Gln Pro Phe Gln Gln Pro Phe Gln Pro Gln Ala 100 105 110
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US 2018/0236130 Al Aug. 23, 2018 11 -continued Ile Pro Pro Gln Ser His Gln Pro Met Gln Pro Gln Ser Pro Leu His 115 120 125 Pro Met Gln Pro Leu Ala Pro Gln Pro Pro Leu Pro Pro Leu Phe Ser 130 135 140 Met Gln Pro Leu Ser Pro Ile Leu Pro Glu Leu Pro Leu Glu Ala Trp 145 150 155 160 Pro Ala Thr Asp Lys Thr Lys Arg Glu Glu Val Ala Phe Ser Pro Met 165 170 175 Lys Trp Tyr Gln Gly Thr Ala Arg His Pro Leu Asn Met Glu Thr Thr 180 185 190 Thr Glu Lys 195 <210> SEQ ID NO 7 <211> LENGTH: 210 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 7 Met Gly Thr Trp Ile Leu Phe Ala Cys Leu Leu Gly Ala Ala Phe Ala 1 5 Met Pro Leu Pro Pro His Pro Gly Ser Pro Gly Tyr Ile Asn Leu Ser 20 25 30 Tyr Glu Lys Ser His Ser Gln Ala Ile Asn Thr Asp Arg Thr Ala Leu 35 40 45 Val Leu Thr Pro Leu Lys Trp Tyr Gln Ser Met Ile Arg Gln Pro Tyr 50 55 60 Pro Ser Tyr Gly Tyr Glu Pro Met Gly Gly Trp Leu His His Gln Ile 65 70 75 80 Ile Pro Val Leu Ser Gln Gln His Pro Pro Ser His Thr Leu Gln Pro His His His Leu Pro Val Val Pro Ala Gln Gln Pro Val Ala Pro Gln 100 105 110 Gln Pro Met Met Pro Val Pro Gly His His Ser Met Thr Pro Thr Gln 115 120 125 His His Gln Pro Asn Ile Pro Pro Ser Ala Gln Gln Pro Phe Gln Gln 130 135 140 Pro Phe Gln Pro Gln Ala Ile Pro Pro Gln Ser His Gln Pro Met Gln 145 150 155 160 Pro Gln Ser Pro Leu His Pro Met Gln Pro Leu Ala Pro Gln Pro Pro 165 170 175 Leu Pro Pro Leu Phe Ser Met Gln Pro Leu Ser Pro Ile Leu Pro Glu 180 185 190 Leu Pro Leu Glu Ala Trp Pro Ala Thr Asp Lys Thr Lys Arg Glu Glu 195 200 205 val Asp 210 <210> SEQ ID NO 8 <211> LENGTH: 55 <212> TYPE: PRT <213> ORGANISM: Sus scrofa <400> SEQUENCE: 8
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US 2018/0236130 Al Aug. 23, 2018 -continued Met Pro Leu Pro Pro His Pro Gly His Pro Gly Tyr Ile Asn Phe Ser 1 5 10 15 Tyr Glu Val Leu Thr Pro Leu Lys Trp Tyr Gln Asn Met Ile Arg His 20 25 30 Pro Ser Leu Leu Pro Asp Leu Leu Glu Ala Trp Pro Ala Thr Asp Lys 35 40 45 Thr Lys Arg Glu Glu Val Asp 50 55 <210> SEQ ID NO 9 <211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 Met Pro Leu Pro Pro His Pro Gly His Pro Gly Tyr Ile Asn Phe Ser 1 5 10 15 Tyr Glu Val Leu Thr Pro Leu Lys Trp Tyr Gln Ser Ile Arg Pro Pro 20 25 30 Pro Leu Pro Pro Met Leu Pro Asp Leu Thr Leu Glu Ala Trp Pro Ser 35 40 45 Thr Asp Lys Thr Lys Arg Glu Glu Val Asp 50 55 <210> <21l> <212> <213> <220> <223> SEQ ID NO 10 LENGTH: 32 TYPE: PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: Amelogenin-derived polypeptide <400> SEQUENCE: 10 Met Pro Leu Pro Ser Tyr Glu Val Leu Thr Pro Leu Lys Trp Pro Val 1 5 10 15 His Pro Met Gln Pro Ser Thr Asp Lys Thr Lys Arg Glu Glu Val Asp <210> <211> <212> <213> <220> <223> 20 25 30 SEQ ID NO 11 LENGTH: 26 TYPE: PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: Amelogenin-derived polypeptide <400> SEQUENCE: 11 Met Pro Leu Pro Ser Tyr Glu Val Leu Thr Pro Leu Lys Trp Pro Ser 1 Thr Asp Lys Thr Lys Arg Glu Glu 20 5 10 Val Asp 25 15 1. A method comprising: a) identifying a subject having exposed or demineralized dentin; and b) contacting the exposed or demineralized dentin with a remineralization composition including: an amelogenin; a chitosan; water; and a sufficient amount of a pH adjusting component such that the remineralization composition has a pH greater than about 6.0 such that enamel is regrown on the exposed or demineralized dentin. 2. The method of claim 1 wherein the subject exhibits exposed dentinal tubules. 3. The method of claim 1 further comprising allowing the remineralization composition to dry.
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US 2018/0236130 Al 4. The method of claim 1 wherein the remineralization composition further includes: a calcium-containing compound that provides calcium ions when dissolved in water; and a phosphate containing compound that provides phos- phate ions when dissolved in water. 5. The method of claim 1 further comprising contacting, the exposed or demineralized dentin with a base for a first period of time. 6. The method of claim 5 further comprising contacting the demineralized dentin with a component selected from the group consisting of calcium-containing compound, a phosphate containing compound, and combinations thereof for a second period of time. 7. The method of claim 5 further comprising contacting the demineralized dentin with saliva or a solution including water and a component selected from the group consisting of electrolytes, mucus, glycoproteins, enzymes, antibacterial compounds, and combinations thereof. 8. The method of claim 1 wherein the amelogenin includes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. 9. The method of claim 1 wherein the amelogenin includes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 with up to 10 conservative amino acid substitutions. 10. The method of claim 1 wherein the amelogenin includes a polypeptide having the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, with up to 10 conservative amino acid substitutions. 11. (canceled) 12. A polynucleotide encoding the polypeptide of claim 11. Aug. 23, 2018 13-21. (canceled) 22. A method comprising: a) identifying a subject having a dental lesion; and b) contacting the dental lesion with a remineralization composition including: a polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, with up to 10 conservative amino acid substitutions; an optional chitosan; water; a suflicient amount of a pH adjusting component such that the remineralization composition has a pH greater than about 6.0; and b) allowing the remineralization composition to air dry. 23. The method of claim 22 further comprising: acalcium-containing compound that provides calcium ion when dissolved in water; and a phosphate containing compound that provides phos- phate ions when dissolved in water. 24. The method of claim 22 further comprising contacting the dental lesion with a base for a first period of time. 25. The method of claim 24 further comprising contacting the dental lesion with a component selected from the group consisting of calcium-containing compound, a phosphate containing compound, and combinations thereof for a sec- ond period of time. 26. The method of claim 25 further comprising contacting the dental lesion with saliva or a solution including water and a component selected from the group consisting of electrolytes, mucus, glycoproteins, enzymes, antibacterial compounds, and combinations thereof. 27. The method of claim 22 wherein the dental lesion is selected from the group consisting of dental caries, early dental carious and erosive lesions, and enamel defects. ce
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