Regeneration of connective tissue function and phenotype through NPAS2 suppression

Abstract

The present invention provides a pharmaceutical composition for improving or promoting wound healing. [Solution] A pharmaceutical composition is provided comprising an agent selected from reserpine, tetrabenazine, dutetrabenazine, dihydrotetrabenazine, resinnamine, benzoylreserpine, 3-methoxybenzoylreserpine, 4-methoxybenzoylreserpine, 3,4-dimethoxybenzoylreserpine, 3,5-dimethoxybenzoylreserpine, methylenedioxyreserpine, cinnamoylreserpine, deserupidine, methylreserpic acid, or silosingopine, for use in a subject to improve or promote the healing of skin or periodontal wounds, or for topical use to reduce the wound area size of an open skin or periodontal wound site.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Application No. 62 / 895,821, filed on September 4, 2019, the content of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for improving or promoting wound healing in a subject, comprising administering an agent that suppresses the expression of a clock gene to a wound of a subject that needs it, wherein the clock gene is Neuronal PAS domain protein 2 (Npas2). The present invention also relates to a method for regenerating alveolar bone, comprising administering an agent that suppresses the expression of Npas2 to a bone loss site of a subject that needs it. The present invention further relates to a method for regenerating connective tissue at a wound site of a subject that requires regeneration of connective tissue at the wound site, comprising administering a therapeutically effective amount of an Npas2 expression inhibitor to the wound. The present invention also relates to a method for reducing the size of a wound area, comprising topically administering an agent that suppresses the expression of Npas2 to an open wound site of a subject.

Background Art

[0003] Open wounds in the skin and oral cavity pose a major threat to current medical and dental treatments. The treatment goals for soft tissue wound management are infection control and wound closure. When Eliason and McLaughlin published their classic review on postoperative wound complications in 1934, their focus was mainly on surgical site infections. Through the effective use of antibiotics and aseptic techniques, the risk of surgical site infection has been significantly reduced today. However, the problem of suppressing scarring still remains. The current approach to achieving wound closure has essentially remained unchanged for over a century and involves the use of sutures and adhesives (4).

[0004] The face and head are the areas where injuries most frequently occur due to accidents, assaults, or battlefield injuries. Facial injuries account for 4%–7% of all emergency department visits, and nearly 90% of facial soft tissue injuries are treated in the emergency department, thanks to the availability of various wound closure techniques for clinicians. While major facial injuries, such as facial cancer, burns, or fractures, obviously have many social impacts on patients, even minor facial injuries can have significant psychosocial effects, potentially leading to decreased life satisfaction, altered perception of body image, and an increased incidence of post-traumatic stress disorder, alcoholism, imprisonment, unemployment, or marital problems.

[0005] The main lesion of periodontitis in the oral cavity is the formation of open spaces called periodontal pockets between the gums and the tooth surface, which provide an abnormal environment for the oral microbiome, leading to the proliferation of pathogenic bacteria. Closing periodontal pockets is currently only done through surgery to reduce the size of the pockets. The U.S. National Health and Nutrition Examination Survey of non-inpatient civilian populations reported that 46% of adults with teeth, representing 64.7 million people, suffer from periodontitis. The prevalence of periodontitis is positively correlated with age, with 8.9% of people developing severe or aggressive periodontitis. Similarly, periodontitis is the most common oral disease in companion dogs. According to the American Veterinary Dental College, periodontitis is the most common clinical manifestation, with prevalence rates exceeding 90% in some canine bloodlines, and a large number of veterinary patients are not receiving adequate care.

[0006] Therefore, when administered to the target wound, particularly open skin wounds in the skin and oral cavity, it promotes wound healing, regenerates alveolar bone, and / or regenerates connective tissue in the wound. Identifying effective medications is extremely important. [Overview of the project]

[0007] In one embodiment, the present invention provides a method for improving or promoting wound healing of a target, comprising administering a drug that suppresses the expression of a clock gene to a wound of a target requiring such suppression, wherein the clock gene is neuron PAS domain protein 2 (Npas2).

[0008] In another embodiment, the present invention provides a method for regenerating alveolar bone, comprising administering a drug that suppresses Npas2 expression to a bone loss site in a subject requiring regeneration.

[0009] In a further embodiment, the present invention provides a method for regenerating connective tissue in a wound site requiring the regeneration of connective tissue at the wound site, the method comprising administering a therapeutically effective amount of an Npas2 expression inhibitor to the wound.

[0010] In another embodiment, the present invention provides a method for reducing wound area size, comprising topically administering a drug that suppresses Npas2 expression to a target open wound site.

[0011] Other features and advantages of the present invention will become apparent from the following detailed description, examples, and drawings. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, it should be understood that while specific embodiments of the present invention are shown, the detailed description and specific embodiments are shown for illustrative purposes only. Where appropriate, any embodiment of the present invention may be combined with one or more other embodiments of the present invention, even if that embodiment is described under different aspects of the present invention. [Brief explanation of the drawing]

[0012] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure, and the invention can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. This patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication containing color drawings are available from the Office upon request and payment of the required fees.

[0013] [Figure 1A-1C] This study demonstrates accelerated healing of full-thickness skin punch wounds in Npas2- / - mice. Figure 1A shows standardized photographs of skin wounds obtained 0–12 days post-surgery, showing progress in wound closure and contraction. Figure 1B shows relative wound area values ​​calculated on days 2, 4, 6, and 12. Npas2 KO mice showed a significantly smaller wound area than WT mice on day 12 (**P<0.01). Figure 1C shows histological observation of the wound on day 7, indicating granulation tissue (GT) formation and recovery of epithelial integrity (EP), with a clearly visible wound margin (dotted line). On day 14, the wound margin, highlighted by hair follicles (HF), became indistinct and approached granulation tissue (GT). [Figure 2A-2C] The characteristics of WT, Npas2+ / -, and Npas2- / - skin fibroblasts are shown. Figure 2A shows the genotype of each fibroblast batch determined by PCR of genomic DNA. The wild-type Npas2 allele produced a PCR product of 250 bp, while the mutant allele produced a PCR product of 350 bp. Figure 2B shows that the WST-1 assay demonstrated increased cell proliferation in Npas2 KO fibroblasts (**P<0.01, significant difference compared to WT at time by Tukey analysis). Figure 2C shows the expression of core clock genes, with the LacZ reporter gene measured every 6 hours for 48 hours by RT-PCR (P values ​​in the figure: two-way ANOVA of the interaction between time and genotype factors. *P<0.05, **P<0.01, significant difference compared to WT at time by Tukey analysis) (Figure 2C). [Figure 3A-3H(c)] This report presents in vitro wound healing experiments using WT, Npas2+ / -, and Npas2- / - fibroblasts. Figure 3A shows slow-speed micrographs capturing the progress of the abrasion healing assay. Figure 3B shows that the number of migrating cells within the abrasion area was significantly higher in the Npas2 KO group at 12 and 24 hours (**P<0.01). Figure 3C shows an image of a standardized suspended collagen gel showing increased collagen gel contraction in the Npas2 KO fibroblast group. Figure 3D shows that the area of ​​the collagen gel decreased over time. The gel contraction rate was faster in Npas2 KO fibroblasts (**P<0.01, significant difference only when compared to WT). Figure 3E is a schematic diagram of FLECS-based single-cell contraction. Figure 3F shows an increased proportion of contracting cells in Npas2 KO fibroblasts. Figure 3G shows that the Npas2 KO mutation did not affect the gene expression of β-actin (Actb) and α-SMA (Acta2) in dermal fibroblasts. Figures 3H(a) to 3H(c) show that the steady-state gene expression levels of integrin subunits αV (ItgaV), β3 (Itgb3), and β5 (Itgb5) in dermal fibroblasts were not affected by the Npas2 KO mutation. [Figure 4A-4C] Figure 4A shows collagen synthesis in vitro by WT, Npas2+ / -, and Npas2- / - fibroblasts. Figure 4A shows gene expression of type I (Col1a1 and Col1a2), type III (Col3a1), type XII (Col12a1), and type XIV (Col14a1) collagen (**P<0.01, *P<0.05, significant difference compared only to WT). FACIT collagen of type XII and XIV showed significantly increased steady-state mRNA levels in Npas2+ / - and Npas2- / - fibroblasts. Figure 4B shows images of cultured fibroblasts with collagen fiber synthesis highlighted by picrosilius red staining. Figure 4C shows that collagen fiber deposition in vitro was measured by picrosilius red staining (**P<0.01, one-way ANOVA with post-hoc Holm test). [Figures 5A-5C] This shows the evaluation of the collagen fiber structure at the wound healing site. Figure 5A shows a confocal laser scanning microscope image showing the collagen fiber structure stained with picrosilius red 14 days post-surgery. Figure 5B shows the measurement of the wound closure ratio using the wound closure area (WCA), which was calculated as the width of ISA(a) between cutaneous muscle layers (PC) after subtracting the granulation tissue area (GT:b) standardized by ISA. Figure 5C shows that the wound closure ratio was higher at 14 days in Npas2+ / - and Npas2- / - mice, but statistical significance was observed only between the WT group and the Npas2- / - group. [Figures 6A-6E] This shows alveolar bone regeneration induced by tooth extraction in Npas2 KO mice. In Figure 6A, C57Bl6J (B6) wild-type (WT) mice underwent extraction of the maxillary left first molar, allowing wound healing in both the oral mucosa and alveolar bone (arrows). Npas2 KO mice with a B6 background showed rapid wound closure and robust bone regeneration in the extraction socket. Figure 6B shows MicroCT images of extraction wound healing at week 2. Figure 6C shows MicroCT-based three-dimensional data analysis (BV / TV) of three root sockets showing rapid bone filling in Npas2 KO mice at week 1 (W1) and week 2 (W2). Tukey's multiple comparison test, *: p<0.05; **: p<0.01. Figure 6D shows the mineralization effect of bone marrow MSCs in vitro. After 28 days of culture in osteogenic medium, MSCs synthesized alizarin red-positive mineralized nodular regions, which were significantly increased in Npas2 KO BMSCs. Figure 6E shows BMP-2 expression by RT-PCR. Npas2 KO MSCs (bone marrow-derived mesenchymal stromal / stem cells) showed a strong increase in BMP-2 expression after incubation in osteogenic medium. Statistical analysis: Figures 6C and 6D show Tukey's multiple comparison test, *: p<0.05; **: p<0.01, and Figure 6E shows Student's t-test, **: p<0.01. [Figures 7A-7B]This shows the effect of reserpine on the osteogenic differentiation of bone marrow stromal cells. Figure 7A shows that wild-type BMSCs (Dwn-C) cultured in osteogenic medium supplemented with reserpine showed increased in vitro mineralization activity, positive for Alizarin Red, on day 21 of culture. Bar: Tukey analysis, p<0.05. Figure 7B shows the results of real-time RT-PCR on total RNA prepared from BMSCs after 21 days of culture for osteopontin (Opn), osteocalcin (Ocn), and housekeeping genes (Gapdh). Expression levels were normalized using RNA from day 0. [Figure 8] This shows that skin punches on the back of mice were treated with reserpine-encapsulated DNV. [Figures 9A-9C] This shows periodontal tissue regeneration in a mouse model of periodontitis. Figure 9A shows MicroCT images of alveolar bone resorption induced by periodontitis and ligature-induced periodontitis in mice. Figure 9B is a flowchart of a mouse model of ligature-induced periodontitis in which reserpine + DNV was locally applied after ligature removal. Figure 9C shows that ligature placement induced severe inflammation, epithelial hyperplasia, and collagen disturbance in connective tissue, consistent with periodontitis. Ligature removal calmed the inflammatory response, but epithelial and connective tissue abnormalities remained. Alveolar bone height remained unchanged (black arrows). In the reserpine + DNV treatment group, epithelial and connective tissue normalized. Clear signs of alveolar bone regeneration were observed (between white and black arrows). The reserpine + DNV treatment group showed collagen rearrangement in gingival connective tissue, similar to the control group, and alveolar bone regeneration was also observed. [Figure 10] The titration assay for DwnC, the top inhibitory compound, is shown. Dwn1 was serially diluted from 100 μM to 0.2 nM and applied to MSC Npas2-LacZ. The effective concentration (EC) was measured by LacZ expression, and the inhibitory concentration (IC) was measured by cell viability using calcein AM / Hoechst 33342 staining. Both DwnC and Dwn1 are reserpines. [Figure 11A-11D(b)]The results of an in vitro biological assay of the Npas2 inhibitory compound Dwn1 (reserpine) are shown. Figure 11A shows that the in vitro mineralization effect of MSCs increased in a dose-dependent manner with Dwn1 supplementation. Dwn1 (1 μM) showed a similar effect to BMP-2 (100 ng / ml) supplementation. Figure 11B shows the expression of osteocalcin (OCN) from D21, whose expression level was elevated by Dwn1. Figure 11C shows that Dwn1 did not affect Bmal1 expression. Figures 11D(a) to 11D(b) show that Npas2+ / - MSCs responded to Dwn1, while Npas2- / - did not, suggesting that the effect of Dwn1 was mediated through Npas2 inhibition. **: p<0.01 compared to untreated control by Tukey analysis. [Figure 12A-12H] This study demonstrates the effect of Dwn1 on modified ligature-induced periodontitis in mice. Figure 12A shows the placement of a 5.0 silk suture in the maxillary left second molar (M2) for 14 days, followed by removal. Figure 12B shows gingival swelling, indicating ligature-induced periodontitis. Figure 12C shows RT-PCR results of gingival tissue confirming the expression of inflammatory cytokines. Figure 12D shows a MicroCT scan illustrating the progression of alveolar bone loss. Figure 12E shows the application of a deformable nanoscale vesicle (DNV) to the palatine gingiva using an oral instrument. Transoral drug administration was demonstrated by fluorescent bisphosphonates on the alveolar bone. Figure 12F shows the application of Dwn1 / DNV to the palatine gingiva after suture removal. The vehicle control showed abnormal epithelial thickening (white arrow). Figure 12G shows a MicroCT scan demonstrating increased bone height on the palatal side treated with Dwn1, but no increase on the untreated buccal side. Figure 12H shows tissue sections stained with H&E (top) and Sirius Red (bottom), illustrating alveolar bone regeneration (top) and gingival / PDL connective tissue regeneration by Sharpey's fibers (SF) (bottom). *: p<0.05, *: p<0.01. [Figure 13A-13C]This section presents unbiased chemogenetic analysis used to determine the molecular mechanisms underlying implant osseointegration. Figure 13A is a flowchart of the chemogenetic analysis using BMSCs carrying the Npas2-LacZ reporter system. Figure 13B shows high-throughput screening of LOPAC1280 compounds for Npas2-LacZ expression in mouse BMSCs. Hit compounds were identified as z-scores >2.5 or <-2.5. Figure 13C shows validation of Npas2-LacZ expression by hit compounds in three sets of experiments. Compounds (black bars) significantly modulated Npas2-LacZ expression compared to untreated controls (white bars) (p<0.05). [Figure 14A-14D] This shows that Npas2 KO mice responded to bone injury through bone regeneration. Figure 14A shows the results of monitoring in vivo microCT for 4 weeks after treating the skulls of C57Bl6J wild-type (WT) and B6 background Npas2- / - mice, which were damaged to the limit size and treated with a collagen sponge carrying 325 ng of BMP2. Figure 14 shows that the amount of regenerated bone in Npas2- / - mice was significantly greater than that in WT mice. Figure 14C shows extraction-induced alveolar bone regeneration in Npas2 KO mice. WT mice were treated by extraction of the maxillary first molar to induce wound healing in both the oral mucosa and alveolar bone (arrows). Npas2 KO mice showed rapid wound closure and robust bone regeneration in the extraction socket. Figure 14D shows MicroCT data analysis (BV / TV) in three root sockets showing rapid bone filling in Npas2 KO mice at 2 weeks. Figure 14B shows Student's t-test, **: p<0.01. Figure 14D shows Tukey's multiple comparison test, *: p<0.05, *: p<0.01. [Figures 15A-15G]This study demonstrates ligature-induced periodontitis in mice and alveolar bone regeneration after ligature removal in Npas2- / - mice. Figure 15A shows that ligature placement around the maxillary second molar (M2) on day 14 (D) progressed gingival inflammation (dotted line). Figure 15B shows the tissue expression of inflammatory cytokines, such as increased IL-17a, on the ligature-placed side of the palatal gingiva. Figure 15C shows a gradual increase in alveolar bone loss as monitored by microCT. Figure 15D shows a gradual increase in Npas2 expression in the gingiva. Figure 15E shows ligature removal on day 14, mimicking scaling and root planing (SRP). Gingival inflammation subsided on day 28. Gingival defect (white arrow) around M2 was observed in WT mice, but this was not prominent in Npas2- / - mice. Figure 15F shows that before suture removal on day 14, both WT and Npas2- / - mice exhibited similar alveolar bone loss induced by periodontitis. Figure 15G shows that while WT mice remained at a low alveolar bone height, Npas2- / - mice showed an increase in bone height, suggesting bone regeneration. *: p<0.05, ***: p<0.001. [Figures 16A-16D] This study demonstrates the identification of alveolar bone regeneration by the Npas2 inhibitory compound (Dwn1) in HTS. Figure 16A shows that the ligature was removed at D14 (Figure 15E) and Dwn1 was applied topically to the palatal gingiva. At D28, gingival defects observed in control mice (cont.) were not prominent in Dwn1-treated mice. Figure 16B shows attenuation of alveolar bone loss on the palatal side where Dwn1 was applied. Figure 16C shows normalized gingiva (white arrow) at the cemento-enamel junction and new bone (red arrow) on resorbed alveolar bone (black arrow) on the palatal side where Dwn1 was applied. Figure 16D shows that Dwn1 treatment resulted in the alignment of Sirius Red-stained gingival collagen, normalized to have Sharpey's fibers (SF) beneath the epithelial (Ep) attachment to the alveolar bone (B). *:p<0.05 [Figure 17]It shows that HTS data was applied to chemical genomics analysis. Most of the drug targets overlapped within the chemical space of monoamine-related receptors, transporters, and signal transduction pathways. [Figure 18] It shows that MSCs expressed neuronal monoamine transporters, vesicular monoamine transporters (VMAT), plasma membrane monoamine transporters (PMAT), extracellular monoamine transporters (EMT), dopamine transporters (DAT), serotonin transporters (SERT), and norepinephrine transporters (NET). [Figure 19] It shows that Dwn1 (a pan-monoamine transporter inhibitor) increased the mineralization effect in vitro in a dose-dependent manner at a level equivalent to BMP2 supplementation (100 ng / ml). **: p < 0.01 compared to the control (white bar). [Figures 20A-20C] It shows the behavior of MSCs from Npas2 KO mice. Figure 20A shows that MSCs were exposed to differentiation media for osteogenesis, chondrogenesis, and adipogenesis. Npas2 - / - MSCs showed higher pluripotent differentiation ability than WT MSCs. Figure 20B shows that the self-renewal activity was increased in Npas2 - / - MSCs. Figure 20C shows that the expression of the stem cell markers Nanog and KLF4 was maintained at a high level in Npas2 - / - MSCs. [Figure 21] It shows hypertrophic scars, which are characterized by excessive deposition of collagen (center) and raised scars (left), and the high-density collagen fibers are strongly stained blue in Masson's trichrome staining. [Figure 22]This paper demonstrates the relationship between circadian rhythms and wound healing, applying the findings of Hoyle et al., Sci Transl Med., 2017, which were incorporated herein by reference, and showed that burns occurring at night in humans require a longer healing period than those occurring during the day. Wound healing requires the migration of fibroblasts (FBs). To test fibroblast migration, Hyde et al. used an in vitro abrasion model, a common method for in vitro wound healing. Skin FBs were cultured on plates, and abrasions were applied to the plates at night or during the day. As shown, FBs abraded during the day migrated faster than FBs abraded at night. This suggests that faster migration implies better healing. [Figure 23] This study demonstrates that the clock molecule Npas2 plays a crucial role in implant-mediated wound healing. Small titanium implants were surgically placed in the femurs of rats. Four weeks later, whole-genome microarrays were performed on the peri-implant tissue. Npas2 was identified as the most important clock molecule in the role of implant-mediated wound healing. Npas2 knockout mutant mice were created, and implant surgery was performed using the same methods as in previous literature (Mengatto et al, PlosOne, 2011 and Morinaga et al, Biomaterials, 2019, respectively, which are incorporated herein by reference). In the wild-type group, high-density collagenous tissue around the implant is beneficial for bone integration. Surprisingly, Npas2 knockout mice did not form high-density collagenous fibrous tissue. High-density collagenous fibrous tissue is a common structure of hypertrophic scars. We hypothesized that suppression of Npas2 reduces "fibrosis" formation. [Figure 24] Knockout (KO) of Npas in mice improves wound healing and minimizes scarring in a mouse model of skin punch wound healing (see Sasaki H, et al., Anat Rec (Hoboken). 2019, which is incorporated herein by reference in its entirety). [Figure 25]This study demonstrates the inhibitory effect of Npas2 on dermal fibroblasts in vitro. Abrasion healing and collagen gel contraction assays, known as in vitro wound healing models, were performed. Npas2 knockout fibroblasts exhibited high cell migration and contractile capacity. These results indicate that Npas2 knockout dermal fibroblasts improve wound healing in an in vitro model. [Figure 26] This document presents a platform for searching for Npas2 inhibitory compounds. First, mouse dermal fibroblasts containing a reporter gene were generated, and a high-throughput screening (HTS) was initiated using over 1,000 FDA-approved compounds. After screening, 10 hit compounds that downregulated Npas2 were identified. Some hit compounds downregulated Npas2 due to their toxicity. Cell viability assays were combined with HTS to successfully eliminate false positives, and the top five hit compounds were obtained. [Figure 27] This study demonstrates that Dwn1 promotes fibroblast migration and gel contraction in vitro. The in vitro wound healing capacity of Dwn1 was tested using the method described above. [Figure 28] This study demonstrates that Dwn1 improved the healing of dehiscence wounds with minimal scarring. To test in vivo wound healing using Dwn1, a 1.5 × 10 mm skin dehiscence model with a central suture was created and designed in three groups. This model closely resembles the clinical state of a skin wound. When the skin was sutured alone, there was no visible wound healing effect. In clinical observation, the control group with sutures and a 10% DMSO vehicle showed significantly better wound healing than the group with sutures alone. It can be hypothesized that the moisture content of the vehicle may improve wound healing. In the third group, Dwn1 in 10% DMSO showed the best wound healing compared to the other two groups. [Figure 29]This study demonstrates that Dwn1 improved the healing of dehiscence wounds with minimal scarring. Masson's trichrome staining was used to stain collagen deposition blue. Dark blue collagen deposition was observed not only in granulation tissue but also in peripheral wounds. Histological staining of the vehicle control using 10% DMSO was similar to the control. Dark collagen deposition was observed. In contrast, Dwn1 in 10% DMSO resulted in a very small area of ​​collagen deposition. These results indicate that Dwn1 improved the healing of dehiscence wounds with minimal scarring. [Figure 30] We demonstrate that intracellular circadian rhythms are regulated by transcription-translational feedback loops of various clock genes, including the basic helix-loop helix genes BMAL1, CLOCK, and Npas2, as well as repressor genes such as Per or Cry (see Sci Rep 8:11996, 2018 and Morinaga et al, Biomaterials, 2019, respectively, whose entirety is incorporated herein by reference). To date, these core circadian genes have been studied as therapeutic targets. However, BMAL1 or CLOCK-deficient mice exhibit abnormal phenotypes or several significant phenomena. On the other hand, no significant phenotypes have been reported in Npas2-deficient mice. Therefore, Npas2 is a safer molecular target. [Figure 31]The mechanism of action of reserpine (Res) is described below. Res blocks vesicle monoamine transporters (VMATs), which are primarily expressed in neurons (see Endocrinology: Adult and Pediatric 2016, Science Direct, the entire article of which is incorporated herein by reference). Blocking neuronal VMATs inhibits the uptake of monoamine neurotransmitters such as norepinephrine, dopamine, serotonin, and histamine in synaptic vesicles. The relationship between monoamine neurotransmitters and the circadian clock, and the transcription of monoamine oxidase A (which maintains monoamine balance), is regulated by the clock genes BMAL1, Npas2, and Per2. Per2 mutant mice show reduced activity of monoamine oxidase A, as described in Hampp G. et al, Current Biology 2008, the entire article of which is incorporated herein by reference. This indicates that the circadian clock regulates neurotransmitters. [Figures 32A-32B] This paper presents the hypothesized mechanism of reserpine (Res) and the upregulation of serotonin for fibroblast function (see Wang C, et al., PloS ONE, 2013, which is incorporated herein by reference in its entirety). Although the mechanism of action of reserpine in the skin is not yet fully understood, it is hypothesized that reserpine inhibits extraneuronal monoamine transporters (EMTs), similar to VMATs. When Res blocks EMTs, monoamines such as serotonin accumulate in the extracellular environment. A previous study by Sadiq et al., Int J Mol Sci 2018, which is incorporated herein by reference in its entirety, showed that increased serotonin can upregulate fibroblast function. Therefore, it is theorized that EMT blocking by reserpine promotes fibroblast (FB) function and wound healing. [Figures 33A-33F]Figure 33A shows a linear wound / scar model of the dorsal skin of a mouse. Figure 33A is a schematic diagram of the animal model used in the study. Longitudinal wounds (10 × 1.5 mm) were made on both sides using a double-edged scalpel. The center of the wound was ligated once with 5-0 nylon suture. Figure 33B shows the Visual Analog Scale (VAS) scored daily up to postoperative day 7 using gross images of the wound / scar. Figure 33C shows the postoperative gross image of the wound / scar using a ruler. The ruler unit is mm. Figure 33D shows histological images of the center (left) and lateral (right) of the wound / scar on postoperative day 7. The top two were stained with hematoxylin-eosin (HE). The bottom two were stained with Masson's trichrome (MT). The yellow dotted line indicates granulation tissue. The scale bar is 1000 μm. Figure 33E shows the scar index evaluated using HE-stained slices. Figure 33F shows the area % of fibrous tissue evaluated using MT-stained slices. * indicates p<0.05. [Figure 34A-34C] This report describes the selection and evaluation of candidate compound Dwn1 for Npas2 inhibition in dermal fibroblasts in vitro. Figure 34A shows a scatter plot of an in vitro high-throughput drug screening assay using an FDA-approved compound library at UCLA's MSSR. High absolute values ​​of the negative Npas2 Z score indicate highly downregulated Npas2 expression (X axis). High cell viability Z scores indicate high fibroblast viability (Y axis). Candidate compounds (Dwn1) were selected in descending order of absolute value of the negative Npas2 Z score and viability. Figure 34B shows the evaluation of circadian Npas2 expression in mouse dermal fibroblasts treated with Dwn1 (1 μM or 10 μM) compared to the control. Figure 34C shows the evaluation of cell migration in mouse dermal fibroblasts treated with Dwn1. * indicates p<0.05. [Figures 35A-35B]This shows the in vitro effect of Dwn1 on collagen synthesis in mouse dermal fibroblasts. Figure 35A shows picrosilius red staining of mouse dermal fibroblasts 7 days after Dwn1 treatment. AA: l-ascorbic acid. OD: optical density. CTRL: cells treated in control medium without AA. Figure 35B shows gene expression of collagen (Col) types 1a1, 1a2, 3a1, and 14a1 3 and 7 days after Dwn1 treatment. * indicates p<0.05. [Figures 36A-36E] The effect of Dwn1 on a linear wound / scar model on the dorsal side of a mouse is shown. Figure 36A shows gross images on day 0 (D0), day 2 (D2), day 5 (D5), and day 7 (D7) postoperatively and after the start of topical application of Dwn1 to the wound. Veh: Vehicle. The vehicle or Dwn1 + vehicle was applied every 24 hours postoperatively. Figure 36B shows a visual analog score scale for wounds treated with vehicle or vehicle + Dwn1. Figure 36C shows histological images of lateral wounds / scars treated with vehicle or vehicle + Dwn1 on day 7 postoperatively. The yellow dotted line indicates granulation tissue. The two on the left are stained with HE, and the two on the right are stained with MT. The scale bar is 1000 μm. Figure 36D shows the evaluation of the scar index using HE-stained slices. Figure 36E shows the area percentage of fibrous tissue evaluated using MT-stained slices. * indicates p<0.05. [Figures 37A-37C] This study demonstrates the molecular biological effects of Dwn1 on a linear dorsal wound / scar model in mice. Figure 37A shows typical laser-captured microdissection (LCM) images. Slides were briefly stained with hematoxylin and eosin before LCM. G: granulation tissue, W: wound tissue. Figure 37B shows gene expression of Col1a1, Col1a2, Col3a1, Col14a1, Tgfβ1, and Acta2 in granulation tissue (G) and wound tissue (W). Gapdh was used as an internal control. * indicates p<0.05. Figure 37C shows in vivo immunohistochemical staining of αSMA at 7 days postoperatively in wounds treated with vehicle or vehicle + Dwn1. The yellow dotted line indicates granulation tissue. Scale bar is 100 μm. [Modes for carrying out the invention]

[0014] In the following detailed description, numerous specific details are provided to give a complete understanding of the invention. However, it will be understood by those skilled in the art that the invention may be carried out without these specific details. In other cases, well-known methods, procedures, and components are not described in detail so as not to obscure the invention.

[0015] Alveolar bone loss is a characteristic feature of periodontitis progression in humans and companion animals. The height of the alveolar bone crest is measured in the cementoenamel junction (CEJ) of healthy subjects. It is located approximately 2 mm below the root. The top of the alveolar bone undergoes bone resorption during the pathological development of periodontitis. Symptoms of moderate and severe periodontitis are defined by radiographic loss of 25% to 50% and more than 50% of the alveolar bone, i.e., from the root tip to the cephalic junction (CEJ). In conventional treatment, tooth extraction was often the clinical option because the lost alveolar bone does not regenerate.

[0016] Treatment options that can predictably regenerate lost alveolar bone remain a major clinical need. Therapeutic stimulation of osteoblast proliferation and differentiation has been studied for bone regeneration and the clinical application of recombinant growth factors. The biological basis for growth factor therapy lies in the process of embryonic development. For example, mouse knockout mutations in bone morphogenetic protein (BMP) signaling pathway molecules resulted in significant skeletal defects such as spontaneous fractures and fracture repair disorders. Since adult tissue regeneration at least partially replicates the embryonic development process, the application of growth factors is thought to induce signaling pathways necessary for bone regeneration in periodontal defects and induce osteogenesis in extracted sockets, known as socket preservation.

[0017] Current bone regeneration therapies utilize peptide biological agents and growth factors, namely Emdogain® (a porcine enamel matrix derivative product containing amelogenin), Infuse® (recombinant human BMP-2), GEM21S® (recombinant human platelet-derived growth factor-bb), fibroblast growth factor-basic 154 (recombinant human fibroblast growth factor-2), Forteo® (teriparatide, recombinant human N-terminal parathyroid hormone), and rhGDF-5 (recombinant human growth differentiation factor-5, BMP-14, Phase I / II completed).

[0018] Over the past several decades, recombinant peptide therapies have played a significant role in medical and dental practice, with over 60 peptide drugs approved in the U.S. and other major markets. The FDA has published specific guidelines for safety monitoring of recombinant peptide products, which must include rigorous monitoring of adverse events related to anaphylactic reactions. When antibodies are produced against recombinant human peptides that share the sequence of endogenous proteins, they can potentially trigger serious autoimmune reactions. Unexpected safety issues are a major challenge for recombinant peptide therapies. In 2008, the FDA issued a black-box warning for BMP-2 due to the presence of a risk of excessive inflammation. Additional safety monitoring can impact the time and cost of drug development. Recombinant peptide products for dental regenerative therapy are currently a major obstacle to providing dental care to patients due to their high cost and the presence of potential side effects.

[0019] Circadian rhythms (also known as the circadian clock) are known as the endogenous, autonomous, and cell-autonomous 24-hour rhythm in mammalian cells and are involved in a wide range of physiological homeostatic functions. Disruption of this rhythm leads to chronic diseases such as cardiovascular disease, diabetes, metabolic and sleep disorders, infertility, and impaired wound healing. Previous studies, based on human burn databases, have reported that wounds sustained at night (rest period) heal more slowly than wounds acquired during the day (activity period). These results suggest a regulatory role of circadian rhythms in wound healing, although the mechanisms by which circadian rhythms contribute to skin wound healing are still unclear.

[0020] The circadian clock has been reported to regulate physiological tissue regeneration in adult animals. The circadian clock (rhythm) is strictly maintained in the midbrain by the suprachiasmatic nucleus (SCN) of the hypothalamus, which acts as a circadian pacemaker. Clock molecules, the transcription factors Clock, Npas2, and Bmal1, induce the expression of Per and Cry genes, and their protein products inhibit the transcriptional activity of Clock, Npas2, and Bmal1. In addition to the core circadian clock (the feedforward / back system of the SCN), peripheral tissues such as bone, liver, skin, and heart are also affected. It maintains its own circadian clock (including the expression of clock molecules). Cultures of mouse cranial vault bone organs showed circadian-cycle bone mineral deposition. Microarray analysis of mouse cranial vault revealed the presence of peripheral circadian rhythms in the bone and that the daily expression of approximately 30% of all genes follows a 24-hour cycle (also known as clock-regulating genes (CCGs)). Peripheral circadian clocks have been shown to play a regulatory role in the healing of skin wounds and fractures.

[0021] Neuron PAS domain protein 2 (Npas2), one of the circadian rhythm core regulators, is a member of the basic helix-loop-helix (bHLH)-PAS family of transcription factors and a paralog of the circadian locomotorcycle kaput (CLOCK). Npas2, or CLOCK, dimerizes with Arnt-like protein-1 (BMAL1) in the brain and muscle, and regulates the transcription of period (PER) and cryptochrome (CRY), genes of two other circadian gene clusters. PER and CRY, in turn, repress the expression of Npas2, CLOCK, and BMAL1 through a transcription / translation feedback loop system. Previous studies have shown that Npas2 expression occurs in the mammalian forebrain and midbrain but not in the SCN. However, clear expression of Npas2 has been reported in peripheral tissues such as the heart, liver, vascular system, and skin.

[0022] Mouse dermal fibroblasts have been reported to express Npas2, which may compensate for the lack of Clock expression. Npas2 was identified by microarray analysis among genes that were significantly upregulated in aged human skin. In summary, we hypothesized that Npas2 in dermal fibroblasts plays a crucial role in maintaining homeostasis and is therefore a key factor in skin wound healing. As described in the examples, the object of the present invention is to address this hypothesis using Npas2 knockout mice.

[0023] Recently, ectopic upregulation of Npas2 in the liver has been found to be associated with fibrosis formation. Npas2 is an orthologue molecule of Clock, and in the absence of Clock, Npas2 substitutes for peripheral clock function in fibroblasts. Therefore, the pathological mechanism of substituted Npas2 may contribute to fibrosis formation in the peripheral tissues of Clock knockout mice. Recently, it was reported that Npas2 knockout (KO) mice showed much faster skin wound healing with minimal fibrosis.

[0024] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as generally understood by those skilled in the art according to the present invention. Furthermore, unless specifically required by the context, singular terms shall include the plural form and plural terms shall include the singular form.

[0025] This invention relates to the application of small molecule compounds targeting circadian clock molecules for the regeneration of alveolar bone. Synchronizing the circadian rhythm regulates many molecular, physiological, and biological processes. Dysregulation of circadian rhythms has been reported not only in neuropsychiatric disorders but also in metabolic diseases and cancer. For example, there are increasing reports suggesting that circadian clock molecules, such as Bmal1, may be therapeutic targets in cases of malignant pleural mesothelioma and Alzheimer's disease. The therapeutic potential of small molecules that regulate the circadian system has been proposed as a new approach to "chronotherapy." This invention also provides small molecule-based chronotherapy for effective, safe, and affordable dental tissue regeneration, including but not limited to alveolar bone regeneration, to patients who need it.

[0026] One of the main challenges in chronotherapy is selecting the target clock molecules. Because most, though not all, cells possess a circadian clock mechanism, therapeutic modification can lead to a wide range of side effects. For example, Bmal1 or Clock knockout mutations result in various pathological phenotypes in peripheral bone tissue and also cause premature aging symptoms (sarcopenia, cataracts, organ shrinkage). In contrast, Npas2 knockout mutations did not result in embryogenetic pathology of the jawbone, vertebrae, or limb bones. Npas2 expression levels in SCNs are low and contribute little to the central circadian rhythm. Instead, increased Npas2 expression manifests in pathological peripheral tissues. As described in Mengatto CM, et al., PLoS One. 2011;6(1):e15848 and Hassan N, et al., PLoS One. 2017;12(8):e0183359, respectively, the expression of Npas2 in bone tissue and MSCs was significantly increased upon exposure to titanium (Ti) biomaterials in vivo and in vitro, respectively. Npas2 expression in peripheral tissues may be induced by “ad hoc” bases stimulated by environmental factors, including wounds. Quantitative gene co-expression analysis has shown that Npas2 is not regulated along with other circadian clock genes, as described by Hassan N, et al., PLoS One. 2017;12(8):e0183359, respectively, which is incorporated herein by reference in its entirety. Npas2 KO MSCs maintained normal expression of other core clock genes, as described by Morinaga K, et al., Biomaterials. 2018;192:62-74, which is incorporated in its entirety herein by reference.

[0027] The present invention provides a method for healing wounds using agents that suppress the expression of the clock gene neuron PAS domain protein 2 (Npas2) (also known as Npas2 expression inhibitors or Npas2 inhibitors), specifically a method for improving and / or accelerating wound repair and healing, for regenerating alveolar bone at bone loss sites, for regenerating connective tissue at wound sites, and for reducing the size of wound areas, comprising administering an Npas2 expression inhibitor to an open wound site and / or bone loss site of a target. The administered Npas2 expression inhibitor(s) may be compounds, synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene, or a combination thereof.

[0028] The inventors of this application have found that an Npas2 expression inhibitor regenerates wounded or chronically inflamed connective tissue, regenerates epithelial (skin) wounds and periodontal tissue wounds, and promotes alveolar bone regeneration in areas of bone loss.

[0029] In one embodiment, the present invention provides a method for improving or promoting wound healing of a target, comprising administering a drug that suppresses the expression of a clock gene to a wound of a target requiring such suppression, wherein the clock gene is neuron PAS domain protein 2 (Npas2).

[0030] In one embodiment, administration is by a route selected from topical, transdermal, and / or subcutaneous administration. In another embodiment, the wound is a cutaneous wound. In some embodiments, the cutaneous wound is a periodontal wound. In certain embodiments, the periodontal wound involves degeneration of gingival connective tissue or resorption of alveolar bone. In certain embodiments, a drug that suppresses Npas2 expression accelerates the migration of human cutaneous fibroblasts in a cell migration assay.

[0031] In some embodiments, the Npas2 expression inhibitor is selected from norepinephrine, dopamine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants.

[0032] In certain embodiments, the agent that suppresses Npas2 expression is an adrenergic reuptake inhibitor that inhibits the reuptake of the monoamine neurotransmitters norepinephrine (noradrenaline), dopamine, and serotonin into presynaptic storage vesicles. In one embodiment, the norepinephrine, dopamine, and serotonin reuptake inhibitor is reserpine, a catecholamine-depleting sympathetic nerve blocker, which has the following chemical structure: [ka]

[0033] Reserpine is derived from Rauwolfia serpentine and other Rauwolfia species and is synthesized by the methods described in Woodward RB et al., J.Am.Chem.Soc. 1956 78,2023 and Tetrahedron 1958,2,1, which are incorporated herein by reference in their entirety, or by methods such as those described more recently in Storck, G. et al., J.Am.Chem.Soc. 2005, 127,16255-16262. Reserpine irreversibly blocks the H+-conjugated vesicle monoamine transporters VMAT1 and VMAT2. Blocking of VMAT2 expressed in neurons by reserpine inhibits the uptake of the monoamine neurotransmitters norepinephrine, dopamine, serotonin, and histamine into the neuronal presynaptic vesicles and reduces their storage. Reserpine has traditionally been used as an antihypertensive, antipsychotic, and tranquilizer.

[0034] In additional embodiments, the agent that suppresses Npas2 expression is one of the reserpine derivatives and analogues of resinnamine, benzoylreserpine, 3-methoxybenzoylreserpine, 4-methoxybenzoylreserpine, 3,4-dimethoxybenzoylreserpine, 3,5-dimethoxybenzoylreserpine, methylenedioxyreserpine, cinnamoylreserpine, deserupidine, methylreserpic acid, silosingopine, and evodiamine.

[0035] Resinnamine is also obtained from Rauwolfia serpentine and other Rauwolfia species and is used as an antihypertensive agent. The pharmaceutically active properties of resinnamine are similar to those of reserpine, including its sedative and antihypertensive effects. In one embodiment, the agent that inhibits Npas2 expression is resinnamine, which has the following chemical structure: [ka]

[0036] In another embodiment, the agent that suppresses Npas2 expression is benzoylreserpine, which has the following chemical structure: [ka]

[0037] In a further embodiment, the agent that suppresses Npas2 expression is 3-methoxybenzoylreserpine, which has the following chemical structure: [ka]

[0038] In another embodiment, the agent that suppresses Npas2 expression is 4-methoxybenzoylreserpine, which has the following chemical structure: [ka]

[0039] In one embodiment, the agent that suppresses Npas2 expression is 3,4-dimethoxybenzoylreserpine, which has the following chemical structure: [ka]

[0040] In another embodiment, the agent that suppresses Npas2 expression is 3,5-dimethoxybenzoylreserpine, which has the following chemical structure: [ka]

[0041] In yet another embodiment, the agent that suppresses Npas2 expression is methylenedioxyreserpine, which has the following chemical structure: [ka] .

[0042] In one embodiment, the agent that suppresses Npas2 expression is cinnamoyl reserpine, which has the following chemical structure: [ka]

[0043] Decerupidine is also a sympathetic nerve blocker, meaning it inhibits the sympathetic nervous system, possessing antihypertensive, sedative, and antipsychotic properties. While deserupidine is derived from Rauwolfia canescens L. and Apocyanaceae, it can also be synthesized from reserpine. In certain embodiments, the agent that suppresses Npas2 expression is deserupidine, which has the following chemical structure: [ka]

[0044] In some embodiments, the agent that suppresses Npas2 expression is methyl reserpate, which has the following chemical structure: [ka]

[0045] In one embodiment, the agent that suppresses Npas2 expression is silosingopine, which has the following chemical structure: [ka]

[0046] In one embodiment, the agent that suppresses Npas2 expression is an evodiamine, which has the following chemical structure: [ka]

[0047] In another embodiment, the agent that inhibits Npas2 expression is tetrabenazine, which is a drug similar to reserpine. Tetrabenazine reversibly inhibits VMAT2, which transports dopamine, serotonin, norepinephrine, and histamine to synaptic vesicles, thereby reducing monoamine uptake and depleting monoamine stores. Tetrabenazine also reversibly depletes monoamines, particularly dopamine, by reversibly inhibiting the uptake of monoamines into vesicles of presynaptic neurons. Tetrabenazine has been used as an antipsychotic and is currently used to treat signs of motor disorders such as various hyperkinetic disorders, chorea associated with Huntington's disease, and tardive dyskinesia, a side effect of antipsychotics. The chemical structure of tetrabenazine is as follows: [ka] In some embodiments, the agent that suppresses Npas2 expression is a tetrabenazine enantiomer or one of eight stereoisomers of dihydrotetrabenazine, which are also VMAT2 inhibitors, and their preparations are described in Yao, Z., et al., Eur J Med Chem. 2011 May;46(5):1841-8, which are incorporated herein by reference in their entirety.

[0048] In one embodiment, the drug that suppresses Npas2 expression is deutetrabenazine, an isotopic isomer of tetrabenazine in which six hydrogen atoms are replaced by deuterium atoms. The chemical structure of dutetrabenazine is as follows: [ka] Dutetrabenazine also inhibits vesicle monoamine transporter 2 (VMAT2) and is used to treat Huntington's disease and chorea associated with tardive dyskinesia.

[0049] In an additional embodiment, the agent that suppresses Npas2 expression is chlorpromazine, an oxidative phosphorylation inhibitor. Chlorpromazine inhibits oxidative phosphorylation but does not reduce norepinephrine and serotonin levels. Chlorpromazine is structurally unrelated to reserpine and has the following chemical structure: [ka]

[0050] In another embodiment, the agent that suppresses Npas2 expression is bromopromazine (bromopromazine hydrochloride), a chlorpromazine analog, which has the following chemical structure: [ka]

[0051] Drugs containing 1,4-thiazine similar to chlorpromazine include promethazine, trimeprazine, prochlorperazine, trifluoperazine, metotrimeprazine, and thioproperazine, whose respective chemical structures are as follows (1) to (6). [ka] [ka]

[0052] In some embodiments, the agents that suppress Npas2 expression are antimycin A, niflumic acid, morindone hydrochloride, and mefexamide hydrochloride.

[0053] In a specific embodiment, the Npas2 expression inhibitor is antimycin A, which has the following chemical structure: [ka] Antimycin A is produced by fungi of the genus Streptomyces. Antimycin A is an inhibitor of oxidative phosphorylation, disrupting the electron transport chain by inhibiting cytochrome c, thereby stopping ATP production. Antimycin A is used in fisheries and aquaculture as an insecticide and fish poison, promoting catfish production by killing small, sensitive fish species. Also known as antimycin A1, antimycin A is also used as an antifungal, insecticide, and acaricide.

[0054] In one embodiment, the Npas2 expression inhibitor is antimycin A2, and the following chemical composition It has a structure. [ka]

[0055] In some embodiments, the agent that suppresses Npas2 expression is a derivative or analog of antimycin A, e.g., US2005 / 0239873 (especially 2-methoxyantimycin A derivatives), Batra, PP, et al., J. Biological Chemistry, Vol.246, No.23, Issue of December 10, pp.7125-7130, 1971 (especially di- and tri-acetates of antimycin A), Chevalier A., ​​et al. References include al., Org. Lett. 2016, 18, 2395-2398 (especially acylated antimycin A derivatives), Abidi, SL, J. Chromatogr. 464 (1989) 453-458 (especially homologs of antimycin A or methyl or dansyl derivatives of antimycin A), and Abidi, SL, J. Chromatogr. 447 (1988) 65-79 (especially subcomponents of antimycin A, namely A1a, A1b, A2a, A2b, A3a, A3b, A4a, and A4b, as well as dansyl or methylated derivatives of antimycin A1a, A1b, A2a, A2b, A3a, A3b, A4a, and / or A4b), each of which is incorporated herein by reference in its entirety.

[0056] In one embodiment, the agent that suppresses Npas2 expression is antimycin A3 (also known as blastomycin and blastomycin), which has the following chemical structure: [ka]

[0057] In another embodiment, the agent that suppresses Npas2 expression is antimycin A4, which has the following chemical structure: [ka]

[0058] In one embodiment, the Npas2 expression inhibitor is diflumic acid, and diflumic acid, which is a cyclooxygenase-2 inhibitor, has the following chemical structure: [ka]

[0059] In another embodiment, the agent that suppresses Npas2 expression is talniflumate, a prodrug of diflumic acid, which has the following chemical structure: [ka]

[0060] In one embodiment, the drug that is an Npas2 expression inhibitor is morindone hydrochloride, an antipsychotic agent, which is a dopamine D2 / D5 receptor antagonist and has the following chemical structure: [ka]

[0061] In another embodiment, the Npas2 expression inhibitor is pikindone (pikindone hydrochloride), a rigid analog of the atypical antipsychotic morindone hydrochloride, which is a selective D2 receptor antagonist and has the following chemical structure: [ka]

[0062] In an additional embodiment, the Npas2 expression inhibitor is mefexamide hydrochloride (mefexamide), a psychotherapeutic agent having a central nervous system stimulating effect, and has the following chemical structure: [ka]

[0063] In various embodiments, agents that suppress Npas2 expression are selected from econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolol, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R,S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium salt.

[0064] In one embodiment, the agent that suppresses Npas2 expression is a nonsteroidal anti-inflammatory drug (NSAID), aceclofenac, an analog of diclofenac. Aceclofenac has anti-inflammatory and analgesic effects and is used to treat rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. Aceclofenac inhibits the cyclooxygenase enzyme (COX). The chemical structure of aceclofenac is as follows: [ka]

[0065] In another embodiment, the agent that suppresses Npas2 expression is pravastatin, a hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor used as an anticholesterolemia agent to lower plasma cholesterol and lipoprotein levels. The chemical structure of pravastatin is as follows: [ka]

[0066] In some embodiments, the agent that inhibits Npas2 expression is tyroxapole, an alkylaryl polyether alcohol type nonionic liquid polymer. Tyloxapole is used as a nonionic surfactant in bronchial and pulmonary studies for liquefaction and removal of mucus secretions. Tyloxapole has also been shown to produce dose- and time-dependent cytotoxicity and induce apoptosis. The chemical structure of tyroxapole is as follows: [ka]

[0067] In one embodiment, the agent that suppresses Npas2 expression is isosorbide mononitrate, which is the mononitrate form of isosorbide, an organic nitrate with vasodilatory activity. Isosorbide mononitrate is used as a coronary vasodilator to treat angina pectoris and heart failure, and is also used to treat diffuse esophageal spasms. The chemical structure of isosorbide mononitrate is as follows: [ka]

[0068] In another embodiment, the agent that suppresses Npas2 expression is MS-1500387, also known as mercaptopurine, 6-mercaptopurine, 6-MP, and SPECTRUM1500387, which is a purine antimetabolite, specifically an antitumor agent, i.e., used as an anticancer agent for treating leukemias such as acute lymphoblastic leukemia and chronic lymphoblastic leukemia, as well as an immunosuppressant for treating autoimmune diseases such as ulcerative colitis, and is a thiopurine derivative antimetabolite. The academic structure is as follows: [ka]

[0069] In certain embodiments, the agent that suppresses Npas2 expression is (S)-(-)-atenolol, i.e., the (S)-enantiomer of atenolol, also known as esatenolol and (S)-atenolol. (S)-(-)-atenolol is a β-adrenergic blocker and is used as a β-blocker to treat hypertension, angina pectoris, and improve survival rates after heart attack. The chemical structure of (S)-(-)-atenolol is as follows: [ka]

[0070] In another embodiment, the agent that inhibits Npas2 expression is butenafine hydrochloride, which is the hydrochloride salt form of the synthetic benzylamine butenafine hydrochloride is an antifungal compound. Butenafine hydrochloride interferes with the biosynthesis of ergosterol, an important component of fungal cell membranes, by inhibiting squalene epoxidase, an enzyme necessary for sterol formation required for fungal cell membranes. The chemical structure of butenafine hydrochloride is as follows: [ka]

[0071] In an additional embodiment, the agent that suppresses Npas2 expression is acekidine hydrochloride, also known as glaucostat®, a non-selective muscarinic acetylcholine receptor partial agonist. Acekidine hydrochloride is used to treat narrow-angle glaucoma. The chemical structure of acekidine hydrochloride is as follows: [ka]

[0072] In one embodiment, the agent that suppresses Npas2 expression is atropine sulfate monohydrate, a natural alkaloid isolated from the plants Atropa belladona L., Datura stramonium L., and other plants of the Solanaceae family. Atropine functions as a sympathetic competitive antagonist of muscarinic cholinergic receptors. Atropine sulfate monohydrate is a cholinergic receptor antagonist. Atropine sulfate monohydrate also acts as an antispasmodic, but does not show any detectable effect on the central nervous system (CNS). Atropine sulfate monohydrate has the following chemical structure: [ka]

[0073] In some embodiments, the agent that suppresses Npas2 expression is trimathione, an anticonvulsant used to treat epileptic symptoms in patients who have not responded well to other medications, and the chemical structure of trimathione is as follows: [ka]

[0074] In various embodiments, the agent that suppresses Npas2 expression is chlorphensin carbamate, a centrally acting skeletal muscle relaxant used to treat muscle spasms, and the chemical structure of chlorphensin carbamate is as follows: [ka]

[0075] In one embodiment, the agent that suppresses Npas2 expression is mafenide hydrochloride, which has the following chemical structure: [ka] Mafenide hydrochloride is a sulfonamide drug that inhibits the enzyme carbonic anhydrase, and is used as a topical antibiotic, particularly in the treatment of burns.

[0076] In one embodiment, the agent that suppresses Npas2 expression is artikaine hydrochloride, which has the following chemical structure: [ka] Articaine hydrochloride, the hydrochloride form of articaine, is an amide-type local anesthetic that is typically used in combination with the vasoconstrictor epinephrine to relieve pain in minor surgical procedures.

[0077] In one embodiment, the Npas2 expression inhibitor is nifenazone, which has the following chemical structure: [ka] Nifenazon is a nonsteroidal anti-inflammatory drug that also has therapeutic effects such as analgesia, fever reduction, and platelet inhibition.

[0078] In another embodiment, the Npas2 expression inhibitor is theobromine, which has the following chemical structure: [ka] Theobromine (3,7-dimethylxanthine) is a purine alkaloid derived from the cocoa plant. It is an adenosine receptor antagonist and is used as a bronchodilator and vasodilator. Theobromine is also used as a diuretic and cardiac stimulant.

[0079] In additional embodiments, the Npas2 expression inhibitor is a compound structurally and pharmaceutically similar to theobromine. In one embodiment, the theobromine-related compound is theophylline, which has the following chemical structure: [ka]

[0080] In another embodiment, the theobromine-related compound is caffeine and has the following chemical structure: [ka]

[0081] In one embodiment, the Npas2 expression inhibitor is nifloxazide, an antibiotic used as an intestinal antibacterial agent for treating diarrhea and colitis in humans, and the chemical structure of nifloxazide is as follows: [ka]

[0082] In some embodiments, the Npas2 expression inhibitor is SAM001246626, also known as atomoxetine hydrochloride, and has the following chemical structure: [ka] Atomoxetine hydrochloride is a norepinephrine reuptake inhibitor that inhibits the presynaptic norepinephrine transporter, thereby inhibiting the reuptake of norepinephrine at the presynaptic cleft and delaying norepinephrine activity. For this reason, atomoxetine hydrochloride is used to treat attention deficit hyperactivity disorder (ADHD).

[0083] In one embodiment, the Npas2 expression inhibitor is dropropidine (R,S), also known as dropropidine or dipropidine, which is a cough suppressant, and the chemical structure of dropropidine is as follows: [ka]

[0084] In another embodiment, the Npas2 expression inhibitor is diethylcarbamazine citrate, an anthelmintic used to treat filariasis, and the chemical structure of diethylcarbamazine citrate is as follows: [ka]

[0085] In a further embodiment, the Npas2 expression inhibitor is MS-1501214, also known as enalapril maleate, which is the maleate form of enalapril. Enalapril maleate is an angiotensin-converting enzyme (ACE) inhibitor used to treat hypertension, congestive heart failure, and renal disease of diabetes, and has the following chemical structure: [ka]

[0086] In one embodiment, the Npas2 expression inhibitor is drasetron mesylate, also known as drasetron (mesylate hydrate), and drasetron mesylate. Dracetron mesylate is a selective serotonin 5-HT3 receptor antagonist with antiemetic properties and is used to treat nausea and vomiting after chemotherapy. The chemical structure of drasetron mesylate hydrate is as follows: [ka]

[0087] In another embodiment, the Npas2 expression inhibitor is estrone, also known as estrone, which is a synthetically prepared or naturally occurring steroid estrogen, specifically an agonist of estrogen receptors ER-α and ER-β. The chemical structure of estrone is as follows: [ka]

[0088] In additional embodiments, the Npas2 expression inhibitor is prednisolone, a synthetic glucocorticoid with anti-inflammatory and immunomodulatory properties, and prednisolone acts as a corticosteroid hormone receptor agonist. The chemical structure of prednisolone is as follows: [ka]

[0089] In one embodiment, the Npas2 expression inhibitor is daunorubicin hydrochloride, also known as daunorubicin and daunomicin, which is the hydrochloride salt of an anthracycline antibiotic having antitumor activity used to treat leukemia, lymphoma, and other cancers. The chemical structure of daunorubicin hydrochloride is as follows: [ka]

[0090] In some embodiments, the Npas2 expression inhibitor is cycloheximide, an antibiotic and antifungal agent produced by the bacterium Streptomyces griseus. The chemical structure of cycloheximide is as follows: [ka]

[0091] In another embodiment, the Npas2 expression inhibitor is monensin sodium salt, also known as monensin sodium, an antiparasitic agent produced by Streptomyces cinnamonensis. The chemical structure of monensin sodium is as follows: [ka]

[0092] In one embodiment, the agent that suppresses Npas2 expression is an oxidative phosphorylation inhibitor. In a particular embodiment, the oxidative phosphorylation inhibitor is antimycin A, which has the following chemical structure. [ka]

[0093] In another embodiment, the agent is an Npas2 downregulator selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors. In one embodiment, the cytoskeleton / ECM inhibitor is brefelzin A, colchicine, podophyllotoxin, or 5175348. In another embodiment, the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952.

[0094] In certain embodiments, transdermal administration is the application of deformable nanoscale vesicles that encapsulate a drug to a wound. In one embodiment, transdermal administration is the application of a transdermal delivery system to a wound, selected from the group consisting of drug-coated microneedles, a solid polymer matrix having an incorporated drug inside, a transdermal patch comprising a reservoir and semipermeable membrane for storing the drug, a transdermal gel containing a dissolved drug inside, a transdermal spray containing a dissolved drug inside, and a quantitative transdermal spray containing a dissolved drug inside.

[0095] In certain embodiments, the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene. In some embodiments, the siRNA is administered by a route selected from the group consisting of microneedle arrays, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods. In various embodiments, the siRNA is chemically modified at the 2' position of the ribose saccharide ring, the phosphate backbone, the nucleic acid base and ribose sugar, or at the 5' end modification or conjugation. In one embodiment, the ribose saccharide ring is guanosine or uridine, and the 2' position modification is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE). In another embodiment, the phosphate backbone is modified with a phosphorodithioate, triazole dimer, amide, or boranophosphate. In some embodiments, the nucleic acid base and ribose sugar modifications are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphorodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine.In certain embodiments, 5'-terminus modifications or conjugations include palmitic acid conjugation at the 5' end of siRNA, reverse thymidine (idT) coupling to the 3' end of siRNA, and topalmitic acid conjugation at the 5' end, conjugation of siRNA with cell-permeable peptides (CPPs), conjugation of siRNA with aromatic compounds selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with urea / thiourea-crosslinked aromatic compounds; polyethylene glycol (PEG) conjugation at the 3' end of the sense and antisense strands; and cholesterol conjugation of siRNA.

[0096] In another embodiment, the present invention provides a method for regenerating alveolar bone, comprising administering an agent that suppresses Npas2 expression to a site of bone loss in a subject requiring regeneration. In some embodiments, administration is by a route selected from topical, transdermal, and / or subcutaneous administration. In one embodiment, the wound is a skin wound. In another embodiment, The skin wound is a periodontal wound. In further embodiments, the periodontal wound includes degeneration of gingival connective tissue or resorption of alveolar bone.

[0097] In one embodiment, a drug that suppresses Npas2 expression accelerates the migration of human dermal fibroblasts in a cell migration assay. In some embodiments, the drug is selected from norepinephrine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants. In various embodiments, the drug is reserpine. In some embodiments, the drug is antimycin A, niflumic acid, morindone hydrochloride, and mefexamide hydrochloride. In certain embodiments, the drug is selected from econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolo, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R,S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium salt. In certain embodiments, the agent is an Npas2 downregulatory compound selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors. In one embodiment, the cytoskeleton / ECM inhibitor is brefelzin A, colchicine, podophyllotoxin, or 5175348. In another embodiment, the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952.

[0098] In certain embodiments, transdermal delivery is by deformable nanoscale vesicles that encapsulate the drug. In certain embodiments, transdermal delivery is the application to a wound of a transdermal delivery system selected from the group consisting of drug-coated microneedles, a solid polymer matrix having an incorporated drug inside, a transdermal patch comprising a reservoir and semipermeable membrane for storing the drug, a transdermal gel containing a dissolved drug inside, a transdermal spray containing a dissolved drug inside, and a quantitative transdermal spray containing a dissolved drug inside.

[0099] In various embodiments, the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene. In one embodiment, the siRNA is administered by a route selected from the group consisting of microneedle arrays, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods. In another embodiment, the siRNA is chemically modified at the 2' position of the ribose ring, the phosphate backbone, the nucleic acid base and ribose sugar, or at the 5' end modification or conjugation. In some embodiments, the ribose ring is guanosine or uridine, and the 2' modification is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE). In one embodiment, the phosphate backbone is modified with a phosphorodithioate, triazole dimer, amide, or boranophosphate. In another embodiment, the nucleic acid base and ribose sugar modifications are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphorodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine. In one embodiment, the 5' end modification or conjugate is used. Conjugations include palmitic acid conjugation at the 5' end of siRNA, reverse thymidine (idT) coupling to the 3' end of siRNA, and topalmitic acid conjugation at the 5' end; conjugation of siRNA with cell-permeable peptides (CPPs); conjugation of siRNA with aromatic compounds selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with urea / thiourea-crosslinked aromatic compounds; polyethylene glycol (PEG) conjugation at the 3' end of the sense and antisense strands; and cholesterol conjugation of siRNA.

[0100] In certain embodiments, transdermal delivery is by deformable nanoscale vesicles that encapsulate the drug. In some embodiments, transdermal delivery is the application to a wound of a transdermal delivery system selected from the group consisting of drug-coated microneedles, a solid polymer matrix having an incorporated drug inside, a transdermal patch comprising a reservoir and semipermeable membrane for storing the drug, a transdermal gel containing a dissolved drug inside, a transdermal spray containing a dissolved drug inside, and a quantitative transdermal spray containing a dissolved drug inside.

[0101] In a further embodiment, the present invention provides a method for regenerating connective tissue at a wound site of a subject requiring regeneration of connective tissue at the wound site, comprising administering a therapeutically effective amount of an Npas2 expression inhibitor to the wound. In certain embodiments, the administration is by a route selected from topical, transdermal, and / or subcutaneous administration. In one embodiment, the wound is a cutaneous wound. In another embodiment, the cutaneous wound is a periodontal wound. In certain embodiments, the wound in the periodontal region includes degeneration of gingival connective tissue or resorption of alveolar bone. In various embodiments, the agent that inhibits Npas2 expression accelerates the migration of human cutaneous fibroblasts in a cell migration assay. In certain embodiments, the agent is selected from norepinephrine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants. In certain embodiments, the agent is reserpine. In some embodiments, the drugs are antimycin A, niflumic acid, morindone hydrochloride, and mefexamide hydrochloride. In one embodiment, the drugs are selected from econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolo, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R,S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium salt. In another embodiment, the agent is an Npas2 downregulator selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors. In one embodiment, the cytoskeleton / ECM inhibitor is brefelzin A, colchicine, podophyllotoxin, or 5175348.In another embodiment, the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952. In a particular embodiment, transdermal administration is the application to a wound of deformable nanoscale vesicles that encapsulate the drug. In one embodiment, transdermal administration is the application to a wound of a transdermal delivery system selected from the group consisting of drug-coated microneedles, a solid polymer matrix having an incorporated drug inside, a transdermal patch comprising a reservoir and semipermeable membrane for storing the drug, a transdermal gel containing a dissolved drug inside, and a transdermal spray containing a dissolved drug inside, and a quantitative transdermal spray containing a dissolved drug inside.

[0102] In another embodiment, the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene. In further embodiments, the siRNA is administered by a route selected from the group consisting of microneedle arrays, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods. In one embodiment, the siRNA is chemically modified in the 2' position of the ribose saccharide ring, the phosphate backbone, the nucleic acid base and ribose sugar, or a modification or conjugation at the 5' end. In another embodiment, the ribose saccharide ring is guanosine or uridine, and the modification at the 2' position is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE). In one embodiment, the phosphate backbone is modified with a phosphorodithioate, triazole dimer, amide, or boranophosphate. In another embodiment, the nucleic acid base and ribose sugar modifications are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphorodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine.In another embodiment, 5'-terminus modifications or conjugations include palmitic acid conjugation at the 5' end of siRNA, reverse thymidine (idT) coupling to the 3' end of siRNA, and topalmitic acid conjugation at the 5' end, conjugation of siRNA with cell-permeable peptides (CPPs), conjugation of siRNA with aromatic compounds selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with urea / thiourea-crosslinked aromatic compounds; polyethylene glycol (PEG) conjugation at the 3' end of the sense and antisense strands; and cholesterol conjugation of siRNA.

[0103] In certain embodiments, the connective tissue is one or more of collagen, dermal-like collagen fibers, or bone. In one embodiment, the wound site is a site of bone loss. In another embodiment, the bone loss is at a site of alveolar bone resorption induced by periodontitis. In a further embodiment, the wound site is a site of gingival connective tissue degeneration.

[0104] In another embodiment, the present invention provides a method for reducing wound area size, comprising topically administering an agent that inhibits Npas2 expression to an open wound site of a target. In certain embodiments, administration is by a route selected from topical, transdermal, and / or subcutaneous administration. In one embodiment, the wound is a cutaneous wound. In another embodiment, the cutaneous wound is a periodontal wound. In yet another embodiment, the periodontal wound includes degeneration of gingival connective tissue or resorption of alveolar bone. In one embodiment, the agent that inhibits Npas2 expression accelerates the migration of human cutaneous fibroblasts in a cell migration assay. In a specific embodiment, the agent is selected from norepinephrine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants. In another specific embodiment, the agent is reserpine. In one embodiment, the agents are antimycin A, niflumic acid, morindone hydrochloride, and mefexamide hydrochloride. In another embodiment, the drugs include econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolo, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R,S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium. The drug is selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors. In further embodiments, the cytoskeleton / ECM inhibitor is brefelzin A, colchicine, podophyllotoxin, or 5175348. In specific embodiments, the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952. In various embodiments, transdermal administration is the application of deformable nanoscale vesicles encapsulating the drug to the wound. In specific embodiments, transdermal administration is the application to a wound of a transdermal delivery system selected from the group consisting of drug-coated microneedles, a solid polymer matrix containing an incorporated drug, a transdermal patch comprising a reservoir and semipermeable membrane for storing the drug, a transdermal gel containing a dissolved drug, a transdermal spray containing a dissolved drug, and a quantitative transdermal spray containing a dissolved drug. In another embodiment, the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene. In specific embodiments, the siRNA is administered by a route selected from the group consisting of microneedle arrays, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods. In one embodiment, the siRNA is chemically modified in the 2' position of the ribose ring, the phosphate backbone, the nucleic acid base and ribose sugar, or in the modification or conjugation of the 5' end. In another embodiment, the ribose ring is guanosine or uridine, and the modification at the 2' position is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE).In further embodiments, the phosphate backbone is modified with a phosphorodithioate, triazole dimer, amide, or boranophosphate. In another embodiment, the nucleic acid base and ribose sugar modifications are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphorodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine. In certain embodiments, 5'-terminus modifications or conjugations include palmitic acid conjugation at the 5' end of siRNA, reverse thymidine (idT) coupling to the 3' end of siRNA, and topalmitic acid conjugation at the 5' end, conjugation of siRNA with cell-permeable peptides (CPPs), conjugation of siRNA with aromatic compounds selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with urea / thiourea-crosslinked aromatic compounds; polyethylene glycol (PEG) conjugation at the 3' end of the sense and antisense strands; and cholesterol conjugation of siRNA.

[0105] In another embodiment, the open wound site includes connective tissue selected from one or more of collagen, dermal-like collagen fibers, or bone. In yet another embodiment, the open wound site is a site of bone loss. In one embodiment, the bone loss is at a site of alveolar bone resorption induced by periodontitis. In some embodiments, the open wound site is a site of gingival connective tissue degeneration.

[0106] In one embodiment, a drug that inhibits Npas2 expression and / or a compound that downregulates Npas2 is formulated as a pharmaceutical composition for topical, transdermal, and / or subcutaneous administration. In a specific embodiment, the pharmaceutical composition is described herein. The pharmaceutical composition includes an agent that suppresses the expression of the clock gene Npas2 in a therapeutically effective amount. In certain embodiments, the pharmaceutical composition includes an agent that suppresses the expression of the clock gene Npas2 in a therapeutically effective amount, effective in regenerating alveolar bone at the site of bone loss, regenerating connective tissue at the wound site, and / or reducing the wound area size, in a wound site of a target where it is needed, particularly in an open wound site. In one embodiment, the pharmaceutical composition includes at least one agent that suppresses the expression of the clock gene Npas2. In another embodiment, the pharmaceutical composition includes a combination of agents that suppress the expression of the clock gene Npas2.

[0107] Peripheral circadian genes and wound healing The function and phenotype of connective tissue differ between skin and oral tissue. Skin fibroblasts, oral fibroblasts, and osteoblasts are connective tissue cells that maintain site-specific function and phenotype, contributing to homeostasis in health. Wounds in a broad sense affect connective tissue cells by altering their phenotype, leading to scarring or loss of function. The inventors hereby explain that the peripheral circadian clock plays a previously unrecognized role in wound healing.

[0108] Circadian clock genes have been reported to regulate physiological tissue regeneration in adult animals. The core circadian clock is strictly maintained in the suprachiasmatic nucleus (SCN) of the hypothalamus, which acts as the circadian pacemaker. Clock molecules, namely the circadian locomotor output cycle kaput (Clock), neuronal PAS domain 2 (Npas2), and aryl hydrocarbon receptor nuclear translocator-like transcription factors (Arntl, Bmal1), induce the expression of period (Per) and cryptochrome (Cry) genes, whose protein products then inhibit the transcriptional activity of Clock, Npas2, and Bmal1. Circadian rhythms are involved in a wide range of physiological homeostatic functions, and disruption of these rhythms is associated with chronic diseases and impaired tissue repair.

[0109] In addition to the core circadian clock of the skin cell network (SCN), peripheral tissues such as fibroblasts and osteoblasts possess autonomously functioning peripheral clocks, as described in Matsui MS, Biological Rhythms in the Skin. Int J Mol Sci. 2016;17(6), which is incorporated entirely herein by reference. Previous studies, such as Hoyle NP, et al., Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing. Sci Transl Med. 2017;9(415), which is incorporated entirely herein by reference, have reported from human burn databases that wounds sustained at night (rest time) heal more slowly than those sustained during the day (activity time). These results suggest a regulatory role of circadian rhythms in wound healing, although the mechanisms by which circadian rhythms contribute to skin wound healing remain unclear.

[0110] Mouse dermal fibroblasts have been reported to express Npas2, and as described in Landgraf D, et al., NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators. PLoS Genet. 2016;12(2):e1005882, which is incorporated herein by reference in its entirety, Npas2 may compensate for the lack of CLOCK expression. Npas2 was identified by microarray analysis among genes significantly upregulated in aged human skin, as described in Glass D, et al., Gene expression changes with age in skin, adipose tissue, blood and brain. Genome Biol. 2013;14(7):R75, which is incorporated herein by reference in its entirety. Recently, Sasaki H, et al., Neuronal PAS Doma As described in "in 2(Npas2)-Deficient Fibroblasts Accelerate Skin Wound Healing and Dermal Collagen Reconstruction" by Anat Rec (Hoboken), 2019, Npas2 has been observed to play a role in promoting skin wound healing.

[0111] Npas2- / - mice showed faster skin wound closure than other groups (Figures 1A and 1B). Cell proliferation, cell migration, and cell contraction of Npas2- / - fibroblasts were greater than those of WT fibroblasts (p<0.01) (Figures 3A, 3B, 3C, and 3D). Increased expression of type XII and XIV FAICT collagen and dermal-like collagen fiber formation were observed in vitro in Npas2 KO fibroblasts. Collagen fiber structure in granulation tissue regions was better reconstructed in Npas2- / - mice. These data suggest that circadian rhythms, particularly Npas2, may regulate skin wound healing. These observations provide a rationale for exploring new opportunities in therapeutic development.

[0112] As used above and throughout this disclosure, the following terms and abbreviations shall be understood to have the following meanings unless otherwise specified.

[0113] In this disclosure, the singular forms “a,” “an,” and “the” include plural references, and references to specific numerical values ​​include at least that specific value unless the context clearly indicates otherwise. Thus, for example, a reference to “compound” is a reference to one or more such compounds and their equivalents known to those skilled in the art. The term “plural” as used herein means two or more. Where a range of values ​​is expressed, another embodiment includes one specific value and / or up to another specific value. Similarly, where a value is expressed as an approximation, it is understood that by using the antecedent “about,” a specific value forms another embodiment. All ranges are inclusive and can be combined.

[0114] As used herein, the terms “component,” “composition,” “composition of compounds,” “compound,” “drug,” “pharmaceutical substance,” “agent,” “activator,” “therapeutic,” “treatment,” “procedure,” or “pharmaceutical” are interchangeable herein and refer to a compound or compound(s) or composition that, when administered to a subject (human or animal), induces a desired pharmaceutical and / or physiological effect through local and / or systemic action.

[0115] As used herein, the terms “treating” and “treatment” are interchangeable and mean (a) preventing a disease condition from occurring in a mammal, in particular, preventing the occurrence of such a disease condition if the mammal is susceptible to such a condition but has not yet been diagnosed with it; (b) inhibiting a disease condition, i.e., preventing its onset; and / or (c) alleviating a disease condition, i.e., causing its regression. As used herein, the term “treat” includes alleviating or reducing at least one adverse reaction or adverse effect or symptom of a symptom, disease, or disorder.

[0116] In one embodiment, “preventing” means, in particular, delaying the onset of symptoms, preventing relapses of the disease, reducing the number or frequency of relapsed episodes, increasing the latency period between symptomatic episodes, or a combination thereof. In one embodiment, “suppressing” or “inhibiting” means, in particular, reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the occurrence of disease-related symptoms, reducing the latency period of symptoms, relieving symptoms, and reducing secondary symptoms. This refers to reducing secondary infections, extending patient survival time, or a combination of these.

[0117] As used herein, the terms “administering,” “administer,” or “administration” refer to the delivery of one or more compounds or compositions to a subject parenterally, enterally, or topically. In one embodiment, the composition is applied topically. In another embodiment, the composition is applied systemically. Administration can be achieved in cells or tissue cultures, or in living organisms, such as humans. Exemplary examples of parenteral administration include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. Exemplary examples of enteral administration include, but are not limited to, oral, inhalation, intranasal, sublingual, and rectal administration. Exemplary examples of topical administration include, but are not limited to, transdermal and vaginal administration. In certain embodiments, the drug or composition is administered parenterally, optionally by intravenous or oral administration to the subject.

[0118] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to animals, such as humans, that are treated (including prophylactic treatment and inhibition of disease conditions and secondary infections) by agents and / or pharmaceutical compositions according to the present invention that suppress the expression of Npas2 as described herein. The term “subject” as used herein refers to humans and non-human animals. The terms “non-human animals” and “non-human mammals” are used interchangeably herein and refer to all vertebrates, such as non-human primates (especially higher primates), mammals such as sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and horses, as well as non-mammals such as reptiles, amphibians, chickens, and turkeys.

[0119] According to any of the methods of the present invention, in one embodiment, the subject described herein is a human. In another embodiment, the subject is a non-human. In one embodiment, the subject is a vertebrate. In another embodiment, the subject is a mammal. In another embodiment, the subject is a primate, and in one embodiment, a non-human primate. In another embodiment, the subject is a murine, which in one embodiment is a mouse and in another embodiment is a rat. In another embodiment, the subject is a dog, cat, cow, horse, goat, sheep, pig, monkey, bear, fox, or wolf. In one embodiment, the subject is a chicken or a fish.

[0120] In one embodiment, the composition of the present invention comprises a pharmaceutically acceptable composition. In one embodiment, the composition comprises an agent that suppresses the expression of a clock gene, the clock gene being neuronal PAS domain protein 2 (Npas2). In a particular embodiment, the agent that suppresses the expression of Npas2 is any one of the agents that suppress the expression of Npas2 described herein. In some embodiments, the “pharmaceutically acceptable composition” and the “pharmaceutical composition” are formulated for topical, transdermal, and / or subcutaneous administration. In one embodiment, the “pharmaceutically acceptable composition” and the “pharmaceutical composition” comprises a pharmaceutically acceptable carrier or excipient.

[0121] In one embodiment, the phrase “pharmaceutically acceptable” is used herein to refer to a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that is balanced by a reasonable benefit / risk ratio.

[0122] As used herein, “pharmaceutically acceptable carrier” or “excipient” means: Examples include physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. In one embodiment, the pharmaceutically acceptable carrier is suitable for topical, transdermal, and / or subcutaneous administration. Suitable topical formulations include gels, ointments, creams, lotions, and drops.

[0123] In one embodiment, the pharmaceutically acceptable carrier is suitable for parenteral administration.

[0124] Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular, sublingual, or oral administration. Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The use of such solvents and agents for pharmaceutically active substances is known in the art. Unless conventional solvents or agents are incompatible with the active compound, i.e., agents that inhibit Npas2 expression, their use in the pharmaceutical compositions described herein is intended in the present invention. Complementary active compounds may also be incorporated into the compositions.

[0125] Therapeutic pharmaceutical compositions are typically sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other modified structures suitable for high drug concentrations. Carriers can be, for example, solvents or dispersions containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size for dispersion, and the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Long-term absorption of injectable compositions can be achieved by including absorption-delaying agents, such as monostearates and gelatin, in the composition.

[0126] Furthermore, as described herein, agents that suppress Npas2 expression can be administered as sustained-release formulations, such as compositions containing sustained-release polymers. Agents that suppress Npas2 expression can be prepared using carriers that protect Npas2 from rapid release, such as sustained-release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid, polyglycolic acid copolymer (PLG) can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art.

[0127] Sterile injectable solutions can be prepared by incorporating an active compound, such as an Npas2 expression inhibitor as described herein, in the required amount into a suitable solvent containing, as necessary, one or a combination of the components listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound, along with a base dispersion medium and other necessary components from those listed above, into a sterile vehicle. In the case of sterile powders for preparing sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying, thereby obtaining any additional desired components from a previously sterile filtered solution in addition to the active ingredient powder. In some embodiments, as described herein, an Npas2 expression inhibitor may be formulated with one or more additional compounds that enhance its solubility.

[0128] In one embodiment, the composition of the present invention is administered in a therapeutically effective dose. In one embodiment, the "therapeutically effective dose" is used to improve or accelerate wound healing in a subject, to regenerate alveolar bone in the case of bone loss in a subject site, to regenerate connective tissue in a subject wound site, and / or to reduce the wound area size of an open wound site in a subject to which the drug or compound has been administered or administered. The present invention is intended to include an amount of the agent or compound alone, or an amount of the claimed agent or compound combination, or an amount of the agent or compound of the present invention in combination with other active ingredients effective to act as a suppressor, inhibitor or downregulator of clock gene expression, or as a neuronal PAS domain protein 2 (Npas2). In one embodiment, the “therapeutic effective dose” of the agent of the present invention that suppresses the expression of the clock gene Npas2 is an amount of the agent sufficient to provide a beneficial effect to the subject to whom the composition is administered.

[0129] The following examples are provided to more fully illustrate specific embodiments of the present invention. However, these examples should not be construed as limiting the broad scope of the present invention. [Examples]

[0130] Example 1 Npas2-deficient fibroblasts are a material and method for promoting skin wound healing and skin collagen reconstruction. Statement on animal ethics In this experiment, Npas2 knockout (KO) mice (B6.129S6-Npas2tm1Slm / J, Jackson Laboratory, Bar Harbor, ME) with a C57Bl / 6J background were used. Npas2 heterozygous mutant (Npas2+ / -) mice were generated from cryopreserved sperm samples, and a good breeding colony was established at UCLA. Both Npas2- / - and Npas2+ / - mice were used. C57Bl / 6J wild-type (WT) mice were used as the experimental group and as the control group. All experimental protocols involving animals were reviewed and approved by the UCLA Animal Research Committee (ARC#2003-009) and followed the guidelines of the Public Health Service Policy and the UCLA Animal Care and Use Training Manual. All animals were free to access food and water and were housed in a standard animal housing facility with a 12-hour light / dark cycle at the Division of Laboratory Animal Medicine, UCLA.

[0131] Mouse dorsal skin full-thickness excision wound model Mice weighing approximately 25g and aged 9-14 weeks (WT: 4 males and 4 females, Npas2+ / -: 7 males, Npas2- / -: 7 males) were used for full-thickness wound experiments on the dorsal skin. After anesthesia by isoflurane inhalation, a 5mm skin biopsy punch (INTEGRA, Integra Life Sciences, Plainsboro, NJ) was used to punch full-thickness skin wounds, passing through the tissue layers to simultaneously create identical skin wounds on the right and left sides of the dorsal skin. These surgeries were performed between 11:00 AM and 1:00 PM, and standardized photographs were taken on days 0, 2, 4, 6, and 12 during the wound healing process. The skin wound area was measured at each time point (NIH ImageJ ver. 1.51). Wound areas were compared daily using the Kruskal-Wallis test and Dunn's post-hoc test. These mice were euthanized on day 7 (n=4 in each group) and day 14 (WT: 4 mice, Npas2+ / -: 3 mice, and Npas2- / -: 3 mice) for histological analysis. The dorsal skin, including the wound area, was incised as a 1 cm square and immediately fixed with 10% neutral buffered formalin. Sections were stained with hematoxylin and eosin (HE) for histological evaluation.

[0132] Culture of dermal fibroblasts Primary fibroblasts obtained from the dorsal skin of mice of each of the three genotypes were cultured using the explant method. The cells were treated with 10% fetal bovine serum and 100 U penicillin / 0.1 mg / The cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing mL of streptomycin in a humidified incubator at 37°C and 5% CO2. Their genotypes were determined by polymerase chain reaction (PCR) targeting alleles of the WT and mutant Npas2 gene.

[0133] WST-1 Cell Proliferation Assay A cell proliferation assay was performed using the WST-1 reagent (Roche Applied Science, Indianapolis, IN). A total of 2,000 cells were seeded in a 96-well reading plate and cultured at predetermined time points (days 1, 3, 5, and 7). At each time point, the culture medium was changed to one containing 10% WST-1 reagent and incubated for 3 hours (n=4 at each time point). Absorption values ​​were measured using a 450 nm spectrophotometer equipped with a plate reader (SYHNERGY H1 plate reader, Biotek, Winooski, VT), compared using two-way ANOVA, and then Tukey's test was performed at each time point.

[0134] Circadian gene expression in dermal fibroblasts The steady-state mRNA expression levels of eight core circadian genes in dermal fibroblasts were determined by quantitative real-time PCR (RT-PCR) using the Taqman MGB probe (Thermo Fisher Scientific Inc., Waltham, MA). Fibroblasts were cultured in 24-well plates, incubated with 100 nM dexamethasone for 2 hours, and then washed with DMEM to synchronize to 80%–90% confluence (Nagoshi et al., 2004). Using the RNeasy kit (Qiagen, Valencia, CA), total RNA was extracted every 6 hours starting from 0–48 hours post-synchronization (n=4 per time point), and its quality and quantity were confirmed using NanoDrop (Thermo Fisher Scientific Inc.). RT-PCR was performed using the following commercially available primer / probe mixes (Thermo Fisher Scientific Inc.): Npas2 (Mm01239312_m1), Bmal1 (= Arntl:Mm00500223_m1), Clock (Mm00455950_m1), Per1 (Mm0050 1813_m1), Per2 (Mm00478099_m1), Per3 (Mm00478120_m1), Cry1 (Mm00514392_m1), and Cry2 (Mm01331539_m1). Gapdh was used as an internal control. In addition, LacZ reporter gene expression was measured. Statistical analysis was first performed by two-way ANOVA. Groups showing a significant correlation (P<0.05) and gene expression at each time point, as determined by two-way ANOVA, were further subjected to Turkey's test.

[0135] In vitro wound healing scratch plate assay Fibroblasts were seeded in 6-well plates and synchronized as described above. After 2 hours, scratch lines were created using a 20 μL plastic pipette and washed with culture medium (n=5 per group). These scratch areas were captured by slow-speed microscopy from 0 to 24 hours. The number of cells that migrated to the scratch areas was counted at 12 and 24 hours and compared using one-way ANOVA and post-hoc Holm test.

[0136] Suspended Collagen Gel Contraction Assay The suspension collagen gel contraction assay was performed using a previously established protocol with several modifications (Ngo et al., 2006). 500 μL aliquots of a collagen gel mixture (Collagen Type I, Corning, Manassas, VA) containing fibroblasts (50,000 cells) were applied to 24-well plates (n=5 in each group) and allowed to stand at room temperature for 20 minutes. The solidified gel was transferred to a 100 mm diameter dish and cultured (37°C, in a humidified incubator, 5% CO2). Images of the gel were taken at 0, 6, 12, and 24 The collagen gel was scanned at 48 and 72 hours. The area of ​​the collagen gel was measured at each time point (NIH ImageJ ver.1.51) and compared using two-way ANOVA followed by Tukey's test at each time point.

[0137] Evaluation of single-cell contraction Single-cell contraction was measured using a fluorescently labeled elastomer contractible surface (FLECS) (Forcyte Biotechnologies Inc., Los Angeles, CA) (Koziol-White et al., 2016). FLECS plates with a soft, film-like silicone elastomer at the bottom were micropatterned with a uniform "X" pattern (70 μm diagonally × 10 μm thick) of fluorescent fibrinogen. Approximately 30,000 cells were seeded into the wells of a 24-well FLECS plate. The plates were placed at room temperature for 40 minutes and then in an incubator (37°C, 5% CO2) for 30 minutes to allow the cells to adhere. After incubation for initial cell adhesion to the X pattern, suspension cells were removed by washing with culture medium, and the plates were incubated for a further 8 hours. Nuclear staining was performed with Hoechst 33,342 (1:10,000). Images of the fluorescent fibrinogen in the X pattern were captured using a fluorescence microscope equipped with a rhodamine filter. In the single-cell contraction evaluation, micropatterns associated with a single nucleus attached to the center of an X-shape were selected and classified as either non-contraction or contraction groups compared to cell-free patterns. The ratio of contraction patterns per captured image (containing approximately 1,000 X-shaped patterns) was compared among each genotype (n=5). Statistical analysis was performed by one-way ANOVA using the post-hoc Holm test.

[0138] Gene expression of actin, integrin, and collagen subunits As described above, total RNA samples were extracted from fibroblasts every 6 hours for 24-48 hours after synchronization. The RNA samples were analyzed by Taqman-based qRT-PCR for actin subunits - β-actin (Actb: Mm02619580_g1) and α-smooth muscle actin (α-SMA, Acta2: Mm00725412_s1) (Figure 3G), integrin subunits - integrin αV (ItgaV: Mm00434486_m1), integrin β3 (Itgb3: Mm00443980_m1), These were used to evaluate the gene expression of integrin β5 (Itgb5:Mm00439825_m1) (Figure 3H), and collagen subunits - type I (Col1a1:Mm00801666_g1 and Col1a2:Mm00483888_m1), type III (Col3a1:Mm00802300_m1), type XII (Col12a1:Mm01148576_m1), and type XIV (Col14a1:Mm00805269_m1) (Figure 4A). Statistical analysis was performed at each time point by two-way ANOVA and Tukey's test.

[0139] Evaluation of collagen synthesis in vitro using picrosilius red staining. Fibroblasts were seeded in 24-well plates and cultured in medium supplemented with ascorbic acid (50 μg / mL) at 80%–90% confluence for 1, 3, and 7 days. The cells were then fixed with 10% neutral buffered formalin and stained with picrosilius red (PolyScience, Niles, IL) to visualize collagen. Absorbance values ​​were measured using a 550 nm spectrophotometer equipped with a plate reader (SYHNERGY H1 plate reader) and compared by one-way ANOVA and post-hoc Holm test.

[0140] Collagen fiber structure of skin wound healing areas using picrosilius red staining and confocal laser scanning microscopy. Tissue sections from full-thickness dorsal skin wound experiments at 7 and 14 days post-surgery were stained with picrosilius red for collagen fibers during wound healing. Collagen fiber structures in granulation tissue (GT), wound closure area (WCA), and intact skin area (ISA) were examined using a confocal laser. - Evaluation was performed using a scanning microscope. The distance between the edges of tissue layers as the original wound width (a) and the distance between the edges of mature collagen in the skin punch area, measured as the GT width (b), were evaluated. The wound closure rate was calculated as (ab) / a and compared by the Kruskal-Wallis test and Dunn's post-hoc test.

[0141] result Closure of full-thickness dorsal skin wounds was accelerated in Npas2- / - mice. Full-thickness dorsal skin wounds continuously contracted from postoperative day 2 to postoperative day 12, and scar formation was observed by day 12 in all genotypes (Figure 1A). The relative wound area at day 12 in Npas2- / - mice was significantly smaller than in the other two genotypes (P<0.01) (Figure 1B). Histological observations revealed hyperkeratosis, residual blood clots, and an immune response in the GT region at postoperative day 7. Furthermore, the ends of the dermal connective tissue with hair follicles migrated toward the center of the wound by day 14. The epithelial layer of the wound area appeared similar to intact skin epithelium, and the immune response was reduced in all samples (Figure 1C). In this study, wounds were formed by full-thickness dorsal skin excision in mice during the daytime (11 a.m. to 1 p.m.). As described by Hoyle et al., 2017 Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing. Sci Transl Med 9:eaal2774, which is incorporated in its entirety herein by reference, skin burns occurring during human nighttime or rest periods have been reported to exhibit impaired healing. Daytime for nocturnal mice corresponds to nighttime for humans. Differences in wound healing between WT mice and Npas2 KO mice may become more apparent if the wound occurs during the mouse's dark / active period.

[0142] Effects of Npas2 KO mutations on dermal fibroblast proliferation and circadian rhythm gene expression. The genotype of each fibroblast sample was determined by PCR. Exon 2 of the mouse Npas2 allele was replaced with a LacZ expression reporter cassette (LacZ / Neo). Since exon 2 encodes a bHLH sequence, the resulting Npas2 molecule lacked DNA binding function. The amplified PCR product, which was larger than that of the wild (WT), recognized Npas2- / - fibroblasts, while both the mutant and wild PCR products recognized Npas2+ / - fibroblasts.

[0143] The WST-1 assay showed that both Npas2+ / - and Npas2- / - fibroblasts proliferated faster than WT fibroblasts (P<0.01) (Figure 2B).

[0144] Circadian expression of Npas2 was decreased in Npas2+ / - fibroblasts and undetectable in Npas2- / - fibroblasts. However, with the exception of Per2 expression, no effect of the Npas2 KO mutation on the expression patterns of other circadian genes was observed (Figure 2C). The reporter gene (LacZ expression) was detected only in Npas2 KO mice.

[0145] Promotion of in vitro wound healing of Npas2- / - dermal fibroblasts by scratch testing and suspension. In vitro evaluations of single-cell strength were performed using a wound healing scratch assay, a suspension collagen gel contraction assay, and FLECS. The number of migrating Npas2+ / - and Npas2- / - fibroblasts was greater than that of WT cells over 24 hours (video: https: / / players.brightcove.net / 656326989001 / default_default / index.html?videoId=6013197672001), which was statistically significant (P<0.05) (Figures 3A, 3B). However, there was no significant difference in cell migration velocity between Npas2+ / - and Npas2- / - fibroblasts (Figures 3A, 3B). The suspension collagen gel contraction assay showed that Npa We showed that s2- / - fibroblasts contracted faster than WT and Npas2+ / - fibroblasts (P<0.01) (Figure 3C, 3D).

[0146] Single-cell contraction and expression of α-SMA and integrins Evaluation of single-cell contraction using FLECS (Figure 3E) revealed that the proportion of contracted Npas2+ / - and Npas2- / - fibroblasts was higher than that of WT fibroblasts (P<0.01) (Figure 3F). Gene expression levels of β-actin (Actb), known to be involved in cell migration, and α-SMA (Acta2), known to upregulate the contractile activity of myofibroblasts, were evaluated by RT-PCR (Figure 3G). Expression of both actin subunits decreased over time; however, there were no significant differences among the three genotypes. Expression of integrin αV (ItgaV), integrin β3 (Itgb3), and integrin β5 (Itgb5) did not show a circadian rhythm in cutaneous fibroblasts. Npas2 KO mutations did not affect the steady-state levels of the analyzed integrin subunits (Figure 3H).

[0147] In Npas2- / - fibroblasts, dermal-like collagen synthesis increased in vitro. In this experiment, we analyzed the gene expression levels of type I (Col1a1, Col1a2), type III (Col3a1), type XII (Col12a1), and type XIV (Col14a1) collagen subunits (Figure 4A). Overall, no circadian patterns were observed in these collagen mRNAs. Col1a1 and Col1a2 were more highly expressed in Npas2- / - fibroblasts than in WT and Npas2+ / - fibroblasts, but the P-value for the interaction was significant only for Col1a2. No difference was observed in Col3a1 expression. Col12a1 expression was increased in Npas2+ / - and Npas2- / - fibroblasts. Surprisingly, the significantly increased expression of Col14a1 was observed in both Npas2+ / - and Npas2- / - fibroblasts compared to WT fibroblasts. Piclosilius red staining of fibroblasts cultured with ascorbic acid supplementation showed a strong positive reaction, indicating the formation and accumulation of collagen fibers in Npas2+ / - and Npas2- / - fibroblasts (Figure 4B), and their absorbance at 550 nm was significantly higher than that of WT fibroblasts on day 7 (P<0.01) (Figure 4C).

[0148] Reconstruction of dermal-like collagen fibers during skin wound healing in Npas2- / - mice. Tissue sections of full-thickness skin wound areas stained with Sirius Red were analyzed by confocal laser scanning microscopy (Figure 5A). There were no clear differences in the collagen fiber structure of the ISA, but collagen fibers in both the GT region and WCA appeared thicker in the Npas2+ / - and Npas2- / - samples than in the WT sample. In particular, the collagen fibers in the GT region of the Npas2- / - sample appeared more organized and partially resembled the collagen structure of intact skin. Histological measurements of wound closure were performed using sections stained with picrosirius Red (Figure 5B). The wound closure rate was higher in the Npas2+ / - and Npas2- / - samples than in the WT, but statistical significance was only obtained between the WT and Npas2- / - samples at day 14 (P<0.01) (Figure 5C).

[0149] Consideration Mammalian skin is a large barrier tissue composed of the epithelial layer (epidermis) and the underlying connective tissue (dermis). This study proposes a novel role for the circadian clock in dermal fibroblasts in skin wound healing, which may enable the reconstruction of dermal connective tissue collagen. Epithelial cells proliferate actively upon injury, migrating over the wound and leading to the rapid establishment of the barrier layer. In contrast, dermal fibroblasts proliferate and migrate to the wound area slowly. Furthermore, fibroblasts in the wound do not maintain the phenotype of dermal fibroblasts, but partially acquire a novel phenotype that contributes to GT formation and scarring. This study is incorporated herein by reference in its entirety by Kowalska et al., 201 3. NONO couples the circadian clock to the cell cycle. As described in Proc Natl Acad Sci USA 110:1592-1599, this demonstrates accelerated healing in an established full-thickness skin wound model and suggests the possibility of faster wound closure and / or less scarring in Npas2+ / - and Npas2- / - mice compared to WT mice (Figure 1). Thus, this study focused on the role of Npas2 KO mutations in the behavior of skin fibroblasts as a mechanistic analysis.

[0150] Npas2 is a core gene of the circadian rhythm that encodes a basic HLH transcription factor and is highly expressed in cutaneous fibroblasts. Npas2 is hypothesized to complement the role of Clock, which is relatively less expressed in fibroblasts (Figure 2C). In retinal cells, knockdown of the Clock gene reduced Npas2 mRNA and protein levels, but knockdown of Npas2 did not affect Clock mRNA or protein levels. The data in this study support the previous observation that Npas2 KO mutations did not significantly affect the expression of core circadian rhythm genes (Figure 2C). Therefore, the effects of Npas2 KO mutations may be mediated by mechanisms other than disruption of the circadian rhythm. Npas2 expression in SCNs peaks during the mouse dark / active phase. Wound responses in mice are expected to exhibit a circadian rhythm. However, this issue was not analyzed in this study.

[0151] We modeled tissue remodeling, wound contraction, and fibrosis using a three-dimensional collagen gel containing fibroblasts. The primary in vitro mechanism of contraction of the fibroblast-embedded gel is due to the fibroblast migration force. The tensile force of the cells is applied to the substrate ECM, contributing to the contraction of the collagen gel. Accelerated collagen gel contraction was demonstrated by Npas2+ / - and Npas2- / - fibroblasts (Figure 3C, 3D), suggesting increased fibroblast migration. Silencing Npas2 expression in human colorectal cancer cells has been reported to accelerate cell migration. In this study, we also demonstrated accelerated migration by Npas2+ / - and Npas2- / - fibroblasts in an in vitro scratch test (video: https: / / players.brightcove.net / 656326989001 / default_default / index.html?videoId=6013197672001, Figure 3A, 3B). It is known that phosphorylation-mediated activation of extracellular signal-regulated kinases (ERK) and phosphoinositide-3 kinase / protein kinase B (PI3K / AKT) modulates cell migration, collagen gel contraction, and skin wound healing. Activation of these signaling pathways has been suggested in the phenotypic transformation of fibroblasts to myofibroblasts, including increased α-SMA expression. In this study, the phenotypic transformation of fibroblasts was not suggested to be due to the Npas2 KO mutation (Figure 3G), and therefore, the involvement of the myofibroblast-like phenotype in the regulation of collagen gel contraction and fibroblast migration was excluded. However, it is important to characterize the effect of the Npas2 KO mutation on phosphorylation of the ERK / Akt / FAK pathway.

[0152] During migration, fibroblasts adhere to the extracellular matrix (ECM) via integrin molecules, generating a single-cell traction force. (References incorporated herein by whole: Koziol-White et al., 2016, Inhibition of PI3K promotes dilation of human small airways in a rho kinase-dependent manner. Br J Pharmacol 173:2726-2738; Pushkarsky et al., 2018 Elastomeric sensor surfaces for As described in "High-throughput single-cell force cytometry. Nat Biomed Eng 2:124-137," a recent development requires cell adhesion to fibronectin-printed FLECS. A newly developed single-cell contraction assay was used. The FLECS assay showed that mouse cutaneous fibroblasts exhibit increased cell contraction behavior due to the Npas2 KO mutation (Figure 3F). Wound-induced transformation of fibroblasts into myofibroblasts is hypothesized to play a pathological role in tissue contraction and fibrosis formation. Separately, increased expression of α and β integrins, which mediate cell adhesion to fibronectin, was considered important for the formation of wound fibrosis caused by cell contractility. For example, significantly elevated expression of integrin αVβ3 is hypothesized to cause idiopathic pulmonary fibrosis. In this study, the steady-state expression of the myofibroblast marker α-SMA, as well as integrin subunits αV, β3, and β5, was not altered by the Npas2 KO mutation in cutaneous fibroblasts (Figures 3G, 3H). Therefore, increased fibroblast contractility due to the Npas2 KO mutation is not considered to result in an abnormal wound healing phenotype of pathological wound contraction or fibrosis formation.

[0153] ECM molecules in connective tissue, particularly FACIT-class collagen, have been shown to influence cell migration and contraction mediated by integrin-mediated cell adhesion. FACIT-class collagen is hypothesized to modify the surface of collagen fibers. Externally exposed N-terminal globular domains, such as NC3 in type XII and XIV collagen, have been shown to be essential for fibroblast-mediated collagen gel contraction. Therefore, we hypothesize that increased expression of type XII and XIV collagen in Npas2 KO fibroblasts may influence migration and gel contraction behavior.

[0154] Downregulation of Npas2 expression has been reported to be associated with cell cycle progression and DNA repair capacity, but there are conflicting reports regarding the effect of Npas2 regulation on cell proliferation. This study showed that Npas2 KO mutations increased fibroblast proliferation (Figure 2B), which may have interfered with the cell migration assay. Evaluation of slow-speed microscopy (video: https: / / players.brightcove.net / 656326989001 / default_default / index.html?videoId=6013197672001) revealed no proliferating cells within the scratch region of any genotype, suggesting that the effects of Npas2 on cell proliferation and migration occur through mechanisms other than cell proliferation.

[0155] Previous studies have reported that titanium-based biomaterials increase Npas2 expression in bone marrow mesenchymal stromal cells (BMSCs) simultaneously with increased expression of cartilage collagen types II, IX, and X, suggesting that Npas2 may mediate BMSC differentiation by biomaterials. Therefore, as a first step in mechanical dissection, we decided to analyze the differentiation of dermal fibroblasts mediated by skin-related collagen. The dermal collagen ECM of skin is mainly composed of type I and type III collagens that form fibrils, which form thick collagen fibers. FACIT collagen types XII and XIV are observed in skin differentiation. Collagens of types XII and XIV are hypothesized to decorate the surface of collagen fibers and regulate the organization of the physiological ECM with tissue-specific functions. In contrast, wound fibroblasts synthesize a large amount of collagen ECM with different properties in GT. In this study, we revealed significant upregulation of FACIT collagen types XII and XIV by Npas2 KO fibroblasts (Figure 4A). In vitro collagen fiber formation, as demonstrated by picrosilius red staining, showed thick collagen fibers in cultures of Npas2+ / - and Npas2- / - fibroblasts (Figures 4B, 4C). Strongly increased FACIT expression may contribute to the reorganization of dermal-like collagen fibers in skin wounds. Indeed, Npas2- / - mice showed an increase in WCA containing mature dermal-like collagen structures (Figure 5A). Furthermore, GT of Npas2- / - mice showed thick collagen fibers partially resembling dermal-like collagen fiber structures. In summary, we suggest that fibroblasts with reduced Npas2 expression may contribute to the reorganization of muscle fibers We believe that these cells may differentiate into cutaneous fibroblasts rather than blast cells or GT fibroblasts, and that Npas2-suppressed fibroblasts may induce the ability to effectively reconstruct the collagen structure of the dermis, even if partial regeneration does not occur.

[0156] This example demonstrates that Npas2 suppression in peripheral skin fibroblasts modifies cell behavior, which is characterized by accelerated cell proliferation, cell migration, and cell contractility in vitro. Furthermore, Npas2 suppression resulted in increased synthesis of dermal FACIT collagen and formation of thick collagen fibers. These fibroblast phenotypes are thought to contribute to the improvement of skin wound healing and the potential for reconstruction of the dermal collagen structure. From the results of this study, the mechanism of circadian clock molecules such as Npas2 in skin wound healing may be considered to promote skin-specific cell differentiation. From these results, the inventors believe that Npas2 may be an attractive therapeutic target for improving skin wound healing.

[0157] Example 2 Screening of small molecule compounds that suppress Npas2 expression using high-throughput screening In Example 1, mouse skin fibroblasts with the developed and validated Npas2-LacZ reporter gene were used to confirm that the detection of LacZ coincides with Npas2 expression, as described by Sasaki H, et al., Neuronal PAS Domain 2 (Npas2)-Deficient Fibroblasts Accelerate Skin Wound Healing and Dermal Collagen Reconstruction. Anat Rec (Hoboken). 2019, which is incorporated herein by reference in its entirety.

[0158] The UCLA Academic Institute established a test platform for regenerative time therapy targeting Npas2: (1) high-throughput screening (HTS) of small molecule compounds, (2) in vitro biological verification, and (3) in vivo tests in a mouse periodontitis model. With this test platform, they successfully identified reserpine, a compound with regenerative activity, and additional compounds that suppress Npas2.

[0159] The inventors found that HTS is not very effective in identifying compounds that reduce the expression of target genes. Many compounds that suppress Npas2 were found to be false positives due to cytotoxicity that leads to cell death or inhibited proliferation.

[0160] The HTS protocol was improved to better identify Npas2 inhibitors. Mouse MSCs carrying the Npas2-LacZ reporter gene were immortalized using SV40 to accommodate large-scale HTS, as described by Hassan N, et al., PLoS One. 2017;12(8):e0183359, which is incorporated in its entirety herein by reference. Expression of the LacZ reporter gene in the immortalized primary MSCs was constant (data not shown). This cell line was used to screen an FDA-approved drug library (1120 compounds). Cell viability was also screened using the same library by calcein AM Hoechst 33342 staining. A high-throughput migration assay using human dermal fibroblasts was performed using a cell migration assay with an Oris™ Pro cell migration assay plate, as described by Joy ME et al., PLoS one. 2014;9(2):e88350, which is incorporated in its entirety herein by reference.

[0161] Hit compounds were identified using combined Z-scores (Table 1). A small number of compounds that inhibited Npas2 were identified (Table 1). The highest-hit compound for Npas2 expression inhibition. The inhibitor was Dwn1, i.e., reserpine. (Npas2 inhibition z-score: -2.57, and cell migration z-score: 4.19). Dwn1 was identical to the DwnC compound previously identified from the LOPAC library. The Dwn1 compound (reserpine) was selected for serial dilution analysis. The effective concentration (EC) ranged from 0.5 to 10 μM, and the inhibitory concentration (IC) was >12.5 μM (Figure 10). Dwn1 was used in the preliminary in vitro and in vivo studies described below. Additional compounds that inhibited Npas2 were identified (Table 2). [Table 1] [Table 2]

[0162] Example 3 In vitro phenotypic testing Reserpine was selected, and its effects on bone marrow stromal cells (BMSCs) were characterized in relation to osteogenic differentiation. Reserpine accelerated mineralization in vitro in a dose-dependent manner (Figure 7A). Expression of osteocracin (Ocn), a late-stage osteogenic differentiation marker, increased significantly on day 21, but the effect on osteopontin (Opn) was mild (Figure 7B).

[0163] To evaluate the mechanism of action involving Npas2, reserpine was applied to BMSCs of wild-type (WT), Npas2+ / -, and Npas2- / - mice, and in vitro osteogenic differentiation was assessed. The effect of reserpine was reproducibly observed in WT and Npas2+ / - BMSCs. However, it did not show a modulatory effect in Npas2- / - BMSCs, suggesting that the mechanism of action of reserpine supporting osteogenic differentiation in BMSCs (MOA) suppressed Npas2.

[0164] Example 4 In vivo skin wound healing test In vivo application studies were conducted using reserpine to evaluate the skin wound healing capacity of Npas2 inhibition. A prototype of a transdermal drug was fabricated using deformable nanoscale vesicles (DNVs) to encapsulate reserpine. The entire prototype is incorporated herein by reference, Subbiah N, et al., Deformable As shown in Nanovesicles Synthesized through an Adaptable Microfluidic Platform for Enhanced Localized Transdermal Drug Delivery. J Drug Deliv. 2017;2017:4759839, DNVs can penetrate the keratinized epidermis of mouse skin. Mouse dorsal skin punch wounds (5 mm in diameter) were formed and secured with custom-made rings, after which the wounds were applied daily with either a vehicle (MilliQ water), empty DNVs, or DNVs encapsulated with reserpine. On day 7, wounds treated with DNVs encapsulated with reserpine showed accelerated wound healing compared to the other application groups (Figure 8).

[0165] Example 5 Periodontal tissue regeneration in a mouse model of periodontitis A commonly used mouse model of ligature-induced periodontitis was used to analyze the pathological mechanisms. A ligature was placed around the maxillary second molar on day 0. Alveolar bone resorption due to periodontitis was observed (Figure 9A), and degeneration of gingival connective tissue was also observed (Figure 9C). Ligature placement induced severe inflammation, epithelial hyperplasia, and collagen disturbance in the connective tissue, consistent with periodontitis (Figure 9C). Next, the ligature was removed on day 7, and the teeth were allowed to heal for one week. This process mimicked routine dental scaling. Ligature removal significantly reduced the inflammatory response (inflammation), but epithelial and connective tissue abnormalities remained, and alveolar bone did not regenerate (Figure 9C). In this model, after ligature removal, reserpine incorporated into DNV (hereinafter referred to as "reserpine + DNV") was applied topically to the gingival tissue of the mice (Figure 9B). The reserpine + DNV treatment group showed rearrangement of gingival connective tissue and collagen, similar to the control group. Alveolar bone regeneration was also observed (Figure 9C).

[0166] Example 6 Efficacy of Npas2 inhibitory compounds in promoting osteogenic differentiation in vitro In Example 2, the small molecule compound Dwn1 (reserpine), which yielded the best combination z-score, was selected for the in vitro biological assay. Dwn1 supplementation induced bone formation. The culture medium dose-dependently increased the mineralization of MSCs in vitro (Figure 11A). BMP-2 was used as the gold standard for promoting osteogenic differentiation of MSCs (48, 49). The effect of Dwn1 (1 μM) was found to be equivalent to that of BMP-2 (100 ng / ml) supplementation (Figure 11A). Osteogenic differentiation by Dwn1 was also supported by the acceleration of osteocalcin (OCN) expression (Figure 11B). The expression of the core clock gene Bmal1 was not affected by Dwn1 (Figure 11C). Dwn1 increased the mineralization of Npas2+ / - MSCs in vitro, but not in Npas2- / - MSCs (Figure 11D), which means that the effect of Dwn1 (reserpine) was mainly promoted by Npas2 suppression.

[0167] Example 7 Rapid bone regeneration in tooth extraction sockets in Npas2 KO mice In C57Bl6J(B6) wild-type (WT) mice, the maxillary first molar was extracted, and wound healing was allowed in both the oral mucosa and alveolar bone (white arrows). Rapid bone regeneration was observed in the extraction socket of Npas2 KO mice (Figure 6A-6C). It has been previously demonstrated that bone formation in the extraction socket occurs as intramembrane ossification without cartilage precursor tissue. In wild-type mice, bone formation was limited to the lower half of the extraction socket. In contrast, Npas2 In KO mice, it was shown that 80% to 100% or more of the socket was filled with new bone. (The entire text is incorporated herein by reference: Coomes AM, et al., Buccal bone formation after flapless extraction: a randomized, controlled clinical trial comparing recombinant human bone) morphogenetic protein 2 / absorbable collagen carrier and collagen sponge alone.J As described in Periodontal. 2014;85(4):525-35, “osteogenesis” in the upper half of the extraction socket requires regenerative agents such as BMP-2. Since the upper half of the extraction socket is not normally filled with new bone, “osteogenesis” induced in the upper half of the extraction socket by regenerative agents such as BMP-2 was considered to be new bone formation or bone regeneration. The filling of the entire extraction socket in Npas2 KO mice can be interpreted as unprecedented bone regeneration activity. Mesenchymal stromal / stem cells (MSCs, also called BMSCs) derived from bone marrow of Npas2 KO mice also showed potent in vitro mineralization (Figure 6D) and increased BMP-2 expression (Figure 6E) when exposed to osteogenic medium, suggesting that circadian rhythms, particularly Npas2, may regulate bone regeneration. Npas2 KO MSCs also showed increased BMP receptor expression (data not shown). These observations provided a rationale for exploring new opportunities in therapeutic development.

[0168] The inventors believe that therapeutic inhibition of Npas2 enhances the ability to maintain the unmodified function and phenotype of connective tissue, such as dermal fibroblasts and alveolar bone regeneration, and that thereby inhibiting Npas2 enhances the ability of connective tissue regeneration and alveolar bone regeneration.

[0169] Example 8 Efficacy of Npas2 inhibitory compounds on alveolar bone regeneration in a mouse model of ligature-induced periodontitis. Dwn1 (reserpine), the selected top hit compound of Example 2, was applied with modifications to a well-established ligature-induced periodontitis model. After periodontitis was established on day 14, the ligature was removed from the molar teeth of mice that mimicked scaling and root planing (SRP), a non-invasive periodontal treatment (Figs. 12A - 12D). Dwn1 was formulated in ABR LLC's proprietary trans-epithelial deformable nanoscale vesicles (DNVs) (57) and locally administered to the palatal gingival tissue once a week (Fig. 12E). The experimental group showed normalized gingival epithelium (Figs. 12F, 12H). Regeneration of the alveolar bone was shown on the palatal side treated with Dwn 1 but not on the untreated buccal side (Fig. 12G). In the Dwn1 group, not only the alveolar bone but also the periodontal collagen including Sharpey's fibers seemed to be reconstructed (Fig. 12H). The in vivo regenerative effect of Dwn1 (reserpine) was clearly demonstrated.

[0170] Example 9 Screening of Npas2 Down-Regulating Compounds Femoral BMSCs derived from Npas2- / - mice are incorporated herein by reference in their entirety, Hassan, N., et al., (2017(Titanium biomaterials with complex surfaces induced aberrant peripheral circadian rhythms As described in *In bone marrow mesenchymal stromal cells. PLoS One 12, e0183359*, LacZ expression was previously characterized and used for high-throughput screening of LOPAC1280 (Figure 13A). Output data from the screening, analyzed for Z scores > 2.5 or <-2.5, yielded a total of 24 hits, namely 7 compounds upregulating Npas2 expression and 16 compounds downregulating Npas2 (Figure 13B). Validation tests identified a total of 14 compounds (Figure 13C), which were subjected to chemogenetic analysis. Compounds upregulating Npas2 were found to either reduce intracellular cAMP or stimulate α2-adrenergic receptors. In contrast, compounds downregulating Npas2 either stimulated or accumulated cAMP or induced activation of cAMP response element binding (CREB) (Table 3). [Table 3]

[0171] Example 10 High-throughput screening (HTS) for identification of small molecule compounds that regulate Npas2 expression The culture medium is dispensed into 384-well plates using the MultiDrop Combi system, and compounds from the selected library are applied using the automated BiomekFX system. Immortalized Npas2-LacZ MSCs (1,500 cells per well) are dispensed into each well and incubated at 37°C for 36 hours (the peak expression time for Npas2). Next, MSCs are incubated with LacZ detection reagents (Beta-Glo, Promega, Madison, WT), and β-galactosidase activity is measured by luminometry. The luminometer data is analyzed using the CDD Vault algorithm (Collaborative Drug Discovery Vault, Burlingame, CA), and those with a Z score < -2.5 are identified as the first inhibitor. Next, primary MSCs are incubated with the first hit compound, stained with calcein AM / Hoechst 33342, and examined using a high-throughput spinning disk confocal microscope (ImageExpress). Confocal Observation is performed using Molecular Devices (San Jose, CA). Cell viability is determined by the number and size of cells. Hit compounds are determined by the Npas2 Z-score (<-2.5) and the cell viability Z-score (>-2.5).

[0172] verification Hit compounds are dispensed into three 384-well plates, and primary MSCs containing the Npas2-LacZ reporter gene are added. After 36 hours of incubation, β-galactosidase activity is measured. The validated compounds must show statistically lower Npas2-LacZ expression than the untreated control, by Student's t-test (p<0.01).

[0173] Titration of EC and IC Validated hit compounds were titrated at 100 μM to 0.2 nM in 20 wells of three 384-well plates, and primary Npas2-LacZ MSCs (1,500 cells per well) were dispensed. After 36 hours of incubation, cell count and β-galactosidase activity were measured. The concentration ranges of the compound that resulted in significant downregulation of Npas2-LacZ and a decrease in cell count were defined as the EC range and IC range, respectively. 50 ≥10 × IC 50 The final hit compound with the minimum therapeutic index is identified.

[0174] Sex as a biological variable As described by Okubo N, et al., PLoS One. 2013;8(11):e78306, which is incorporated herein by reference in its entirety, no sex differences were observed in the behavior of the circadian clock in isolated skeletal cells. We propose using sex-separated MSCs in HTS (vertebrates).

[0175] Expected results From approximately 40,000 compounds, 40 (or 0.1%) hit compounds that downregulate Npas2 are expected to be identified, and approximately 100 compounds are expected to be validated. Hit compounds will be further eliminated by EC and IC indices. 20 candidate compounds are expected to be further analyzed.

[0176] Potential problems and alternative plans For HTS, we suggest using immortalized MSCs. PCR of the allele genome should be performed after 5 passages to ensure stability. While unlikely, genetic changes or other mutations may occur. If necessary, use a fresh batch of primary MSCs derived from Npas2-LacZ mice.

[0177] Example 11 Establishment of the efficacy of Npas2 inhibitory compounds in accelerating osteogenic differentiation in vitro. ALP expression assay and in vitro mineralization assay Wild-type male and female mouse MSCs are cultured at 37°C under 5% CO2 in DMEM supplemented with 10% FBS and 1% antibiotic. After reaching semi-confluence, the culture medium is changed. Replace the osteogenic medium (100 nM dexamethasone, 10 mg β-glycerophosphate, 50 μM ascorbic acid) with one of the hit compounds (0.1, 1, and 10 μM in 0.1% DMSO). Note that exposure to dexamethasone should be used to synchronize MSCs. On day 7 of culture, the enzyme ALP activity of the cell lysates is measured using a commercially available kit (Abcam). Measurements are performed using ab83369, etc. Colorimetric data are converted to ALP enzyme activity (n=3 per compound). Separately, on day 21, mineralization is evaluated in vitro according to a standard protocol (ARed-Q, Sciencell Research Laboratories, Carlsbad, CA, or QuantiChrome Calcium Assay Kit, BioAssay Systems, Heyward, CA). Mean values ​​(n=3) of Alizarin Red S concentration or Ca++ concentration are obtained.

[0178] Expression of genes related to bone formation and differentiation The "candidate" compounds will be used to characterize in vitro osteogenic differentiation over time. Using wild-type MSCs exposed to the "candidate" compounds identified from the above experiments at the optimal concentration for accelerated osteogenic differentiation, total RNA samples will be isolated at culture periods of 3, 7, 14, and 21 days. Real-time PCR will be performed against Runx2, Osx, Ocn, Bmp2, Bmpr2, Col1a1, Opn, and Gapdh as a control.

[0179] contrast As an untreated control, quantitative values ​​of ALP, in vitro mineralization, and osteogenic gene expression were obtained for MSCs treated with 0.1% DMSO. As a positive control, we propose supplementing the osteogenic medium with BMP-2 (100 ng / ml).

[0180] Data Analysis All assay data, including untreated and positive controls, are ranked on a scale of 1 to 1 (strongest). Next, compounds with a total rank exceeding that of the untreated MSCs are identified. From this list, the top five compounds with the strongest total ranks are selected as "candidate" compounds. These candidate compounds are expected to exhibit activity comparable to that of the BMP-2 treated positive controls.

[0181] Sex as a biological variable Bone volume and structure are secondary sexual characteristics, with male MSCs forming more mineralized nodules than female MSCs. Unique molecular differences between male and female MSCs have been suggested. We propose using male and female MSCs (vertebrates).

[0182] Expected results Hit compounds are expected to be favorably evaluated by (1) bone turnover marker, ALP expression, and (2) in vitro mineralization activity to demonstrate accelerated osteogenic differentiation. A ranking protocol should be able to identify candidate compounds. Most compounds are expected to produce statistically higher ALP and in vitro mineralization data than the untreated control. Top-ranked compounds may show levels comparable to BMP-2-derived osteogenic differentiation. Compounds with high rankings in osteogenic differentiation-related gene expression will be used. "Candidate" compounds should demonstrate accelerated in vitro osteogenic differentiation through coordinated time-dependent expression of osteogenic genes. The top 10 "candidate" compounds will be selected for in vivo testing to demonstrate the efficacy of Npas2 inhibitors for alveolar bone regeneration in a mouse ligature-induced periodontitis model.

[0183] Potential problems and alternative plans The effective dose may vary. If the EC range falls outside the proposed dose range of 0.1–10 μM for small molecule compounds identified as modulating Npas2 expression by HTS, the compound should be tested in a customized concentration range and medicinal chemical analysis as necessary. It should be noted that human and mouse MSCs exhibit different responses to BMP-2. A highly physiological bone formation and differentiation cocktail containing physiological concentrations of BMP-2 has been previously developed by a feedback control algorithm, as described by Honda Y, et al., Sci Rep. 2013;3:3420, which is incorporated herein by reference in its entirety. For the positive control, we suggest using conventional BMP-2 (100 ng / ml) medium, although alternative positive control cocktails may be used if necessary.

[0184] Example 12 Demonstration of the efficacy of Npas2 inhibitory compounds on alveolar bone regeneration in a mouse ligature-induced periodontitis model. Minimally invasive surgical debridement with and without recombinant growth factors showed similar radiographic bone grafting in small subosseous pockets. An intrinsic environment has been proposed to support alveolar bone regeneration. The hypothesized chronotherapy may not induce ectopic osteogenesis but supports the healing environment in the host. To test this hypothesis, the top five candidate compounds will be applied to a modified mouse ligature-induced periodontitis model to determine the efficacy of alveolar bone regeneration in vivo.

[0185] Formulation of DNV A water-soluble or lipid-soluble small molecule compound is dissolved in either an aqueous or lipid component. Using microfluidics, the aqueous and lipid components are mixed to produce a DNV as described in PCT / US2016062552, which is incorporated herein by reference in its entirety. The DNV is obtained as a lyophilized powder.

[0186] Mouse periodontitis Both male and female mice are used. 5.0 silk sutures are placed around the maxillary second molars to induce periodontitis. After 14 days, the sutures are removed by mimicking scaling and root planing (SRP), a non-surgical debridement treatment. Candidate compounds for DNV are purified, reconstituted with sterile water, and applied topically to the palatal gingiva (see Figure 12E).

[0187] Data Analysis On day 28, the maxilla of mice was harvested and subjected to microCT (n=20 / group), morphometric analysis by calcein injection (n=10 / group), and demineralization histology (H&E and picrosilius red staining: n=10 / group). Furthermore, serum levels of bone metabolism markers (Tracp5b, CTX, P1NP, and ALP) were measured.

[0188] Sample size Based on past microCT data (Figure 12G), we propose a sample size of 20 to achieve a force of 0.8.

[0189] Sex as a biological variable A higher prevalence of periodontitis in males as a secondary sexual characteristic has been reported, and this may be due to sex differences in innate immunity. To demonstrate the effectiveness of Npas2 inhibitory compounds for alveolar bone regeneration in a mouse ligature-induced periodontitis model, this example proposes the use of both male and female mice. (Vertebrates).

[0190] Expected results The main outcomes of alveolar bone regeneration are evaluated by microCT. Supporting evidence is obtained from histological measurements, histological analysis, and serum markers. Gingival / PDL connective tissue Exploratory results regarding regeneration will be evaluated using confocal laser scanning microscopy of picrosilius red-stained tissue. The top five compounds are expected to be identified for testing in Example "Phase II".

[0191] Potential problems and alternative plans It should be noted that detailed optimization is required for the formulation of candidate compounds. Within the scope of Phase I (the examples proposed herein), we propose using DNV oral epithelial drug formulations for all candidate compounds. The optimal formulation is described in Example 13 (Phase II).

[0192] Example 13 Phase II This example aims to determine (1) the final formulation, (2) its efficacy in canine periodontitis, and (3) the safety of small molecule compound-based chronotherapy for periodontal and alveolar bone regeneration.

[0193] We plan to conduct trials in large animals (dogs) as described by Kol A, et al., Companion animals: Translational scientist's new best friends. Sci Transl Med. 2015;7(308):308ps21 and Arzi B, et al., Craniomaxillofacial Disorders and Solutions in Humans and Animals. J Dent Res. 2018;97(4):364-70, which are incorporated herein by reference in their entirety.

[0194] Example 14 The role of Npas2 in craniofacial tissue regeneration Extensive injury and chronic inflammation often result in wound repair accompanied by fibrosis, which hinders tissue regeneration. Therefore, it was hypothesized that suppression of the "wound repair gene Npas2" could lead to wound healing toward tissue regeneration. Significant size defects in the cranial vault of Npas2 KO mice healed with robust regeneration of bone and bone marrow tissue in the presence of low doses of BMP-2 (325 ng or higher) (Figures 14A, 14B). Similar to cranial vault defects, bone formation in extracted sockets involves intramembranous ossification without cartilaginous progenitor tissue, but post-extraction bone regeneration is limited to the lower half of the alveolar bone socket. Currently, the only other way to fill the upper half of the extracted socket is to use regenerative agents such as BMP-2. Npas2 KO mice are unique in that they filled 80%–100% or more of the extracted socket with new bone without the application of exogenous BMP or stem cells (Figures 14C, 14D).

[0195] Example 15 Alveolar bone loss due to periodontitis was regenerated in Npas2 KO mice. Periodontitis is a chronic inflammation caused by abnormalities in the oral microbial flora, coupled with disharmonious oral barrier immunity. Placement of ligatures around the maxillary molars has been shown to induce periodontitis in mice, and this model was used to characterize oral microbial behavior, gingival barrier immune responses, and aggressive alveolar bone resorption (Figures 15A-15C). In this mouse model, Npas2 expression levels in the affected gingival tissue were found to gradually increase (Figure 15D). The current treatment involves mechanically removing plaque and calculus from periodontal pockets by scaling and root planing (SRP). SRP was mimicked by removing sutures on day 14 and monitoring healing for two weeks (D28). While gingival inflammation subsided (Figure 15E), lost alveolar bone height (Figure 15F) did not recover in WT mice (Figure 15G). When this periodontitis model was applied to Npas2 KO mice, alveolar bone loss at D14 was indistinguishable from that in WT mice (Figure 2F). However, after mimicking the "SRP" procedure, the alveolar bone height was comparable to that of Npas2 KO mice. Significant recovery was observed (Figure 2G).

[0196] Example 16 Small molecule compounds that inhibit Npas2 regenerated alveolar bone in mice with periodontitis. UCLA Molecular Screening Shared Resources (MSSR) is a paid core facility that supports high-throughput screening (HTS) of drug candidate compounds. MSSR is a unique facility for academic institutions and stores more than 200,000 compounds in various libraries, as well as genome-wide arrays of CRISPR, cDNA, shRNA, and siRNA (mssr.ucla.edu). The inventors performed multiple HTSs using mouse MSCs and fibroblasts carrying the Npas2-LacZ reporter system. The two objectives of the HTS were to [1] elucidate the mechanism of Npas2 regulation through chemogenomic analysis, and [2] determine whether compounds that suppress Npas2 can regenerate alveolar bone.

[0197] From the FDA-approved drug library (1120 compounds) and the LOPAC library (1280 compounds), we identified a list of compounds that modulate Npas2-LacZ expression. For the latter purpose, we selected one compound (Dwn1) that downregulates Npas2-LacZ expression and applied it to a mouse periodontitis / treatment model (Figures 15E-15G). Dwn1 was formulated for topical application to the palatine gingiva. Fourteen days after the induction of periodontitis, the ligature was removed, and Dwn1 was applied topically to the palatine gingiva once a week (Figure 16A). At D28, significant alveolar bone regeneration was observed only on the palatal side where Dwn1 was applied (Figures 16B, 16C). A further unexpected result was observed in which collagen fibers in the gingiva and periodontal ligament were reorganized along with the potential regeneration of Sharpey's fibers (Figure 16D). These results provided a basis for pursuing the clinical and transitional development of viable options for oral regeneration therapy.

[0198] Example 17 HTS - Chronobiological Hypotheses from Chemical Genomics Chemogenomic analysis of HTS data identified clusters of drugs targeting monoamine-related transporters and receptors (Figure 17). Monoamines (i.e., norepinephrine, serotonin, dopamine, and histamine) are broadly characterized as neurotransmitters, and numerous drugs targeting this axis have been developed. It should be noted that their receptors have been found in non-neuronal cells such as MSCs, and that monoamine-induced signaling is hypothesized to regulate osteoblast growth and differentiation. Furthermore, monoamine transporters have also been reported in non-neuronal cells, and all monoamine transporters were found to be expressed by MSCs (Figure 18).

[0199] In fact, Dwn1 is an inhibitor of a pan-monoamine transporter that downregulates Npas2 and exhibits tissue regeneration activity (Figures 16A-16D), suggesting that the regulatory axis of monoamine receptors and transporters may regulate Npas2 expression to induce regenerative capacity. Using HTS / chemogenomics data, we will conduct chronobiological analyses of tissue regeneration in studies focusing on the monoamine pathway axis and downstream Npas2 expression. This study should establish the proposed mechanism of action (MOA) of chronotherapy for periodontal tissue regeneration.

[0200] Example 18 Chronotherapy for periodontal tissue regeneration Synchronizing the circadian rhythm has an impact on many molecular, physiological, and biological processes. Dysregulation of circadian rhythms has been reported not only in neuropsychiatric disorders but also in metabolic diseases and cancer. For example, there are increasing reports suggesting that circadian clock molecules, such as Bmal1, may be therapeutic targets in cases of malignant pleural mesothelioma and Alzheimer's disease. The therapeutic potential of small molecules that modulate the circadian system is proposed as a new approach to "chronotherapy." This project proposes developing an innovative small molecule compound-based chronotherapy for dental tissue regeneration. To this end, the chemical space of the monoamine pathway axis will be examined in detail with respect to the regulation of Npas2 expression. The following approach will be used. Experiment 1. Construct a chemical space-specific HTS compound library and identify time-therapeutic compounds for modulating Npas2 (to be conducted by ABR LLC).

[0201] Rationale and Objectives. Preliminary HTS identified clusters of drugs targeting monoamine-related transporters and receptors that affect Npas2 expression in MSCs. The objective of this study is to perform a focused HTS using a customized compound library consisting of small molecule compounds selected in the chemical space of monoamine transporters and receptors. This study proposes to [1] construct a customized, focused compound library for the chemical space of the monoamine pathway, [2] perform a complete HTS, [3] verify Npas2 expression and osteogenic differentiation of MSCs, and [4] determine effective concentrations (EC) and inhibitory concentrations (IC).

[0202] ABR LLC will have an incubator space at the UCLA California NanoSystems Institute (CNSI), which will also house MSSR, UCLA's core facility for drug screening. ABR LLC will have full access to MSSR, where chemical space-specific libraries will be customized and High-Speed ​​Testing (HTS) will be conducted. ABR LLC is expected to complete the objectives of this project.

[0203] Experiment 1 (Chemical Space-Specific Library). Seven monoamine transporters exist (Figure 18). The MSSR contains a total of 51 compounds that target monoamine transporters and function as inhibitors. Separately, adrenergic receptors, dopamine receptors, serotonin receptors, muscarinic / nicotinic receptors, and H1 receptors are targeted by a total of 283 compounds. These compounds are either agonists or antagonists. As a result, a total of 334 compounds will be included in the monoamine chemical space-specific compound library. The compound library will be constructed in a 384-well plate containing a control well with only the vehicle for HTS.

[0204] Experiment 2 (HTS). Culture medium is dispensed into 384-well plates using the MultiDrop Combi system, and compounds from a chemically spatially specific library are applied using the automated Biomek FX system. Immortalized Npas2-LacZ MSCs (1,500 cells per well) are dispensed into each well and incubated at 37°C for 36 hours (peak expression time of Npas2). The MSCs are then scanned using a high-throughput spinning disk confocal microscope (ImageExpress) for cell viability measurement. Confocal Cells were stained with calcein AM / Hoechst 33342 for Molecular Devices (San Jose, CA) and incubated with LacZ detection agents (Beta-Glo, Promega, Madison, WT). Beta-galactosidase activity was measured by luminometry. The data were analyzed using the CDD Vault algorithm (Collaborative Drug Discovery Vault, Burlingame, CA) to measure Npas2 expression and cell viability, and the Z-score for each measurement was determined. Based on the above preliminary data, the Z-score thresholds for Npas2 are >2.5 and <-2.5, and the Z-score threshold for cell viability is -1.0 to +1.0.

[0205] Experiment 3 (Validation). Compounds effective in regulating Npas2 were dispensed into three 384-well plates, and MSCs containing the Npas2-LacZ reporter gene were added. 36 hours After incubation, β-galactosidase activity is measured. The validated compounds should show statistically regulated Npas2-LacZ expression compared to the untreated control, with Student's t-test showing p<0.01.

[0206] Figure 10 shows the titration assay. Dwn1 was serially diluted from 100 μM to 0.2 nM and applied to MSC Npas2-LacZ. EC was measured by LacZ expression, and IC was measured by cell viability using calcein AM / Hoechst33342 staining.

[0207] Experiment 4 (Titration of EC and IC). Hit compounds proven to inhibit Npas2 were titrated at 100 μM to 0.2 nM in 20 wells of three 384-well plates, and Npas2-LacZ MSCs (1,500 cells per well) were dispensed. After 36 hours of incubation, cell number and β-galactosidase activity were measured (Figure 10). The concentration ranges of the compounds that resulted in significant downregulation of Npas2-LacZ and a decrease in cell number were defined as the EC range and IC range, respectively. 50 ≥10 × IC 50 The final hit compound with the minimum therapeutic index is identified. This study also determines the optimal concentration of each hit compound.

[0208] Experiment 5 (Osteogenesis and Differentiation). An optimal concentration of an Npas2 inhibitory hit compound in 0.1% DMSO is added to conventionally known osteogenic culture media for MSCs of wild-type male and female mice. The degree of osteogenic differentiation is measured by ALP activity (e.g., Abcam ab83369) and in vitro mineralization (i.e., ARed-Q, Sciencell Research Laboratories, Carlsbad, CA) (Figure 19). In addition, total RNA samples are prepared for culture periods of 3, 7, 14, and 21 days. Real-time PCR is performed on Runx2, Osx, Ocn, Bmp2, Bmpr2, Col1a1, Opn, and Gapdh as a control. Control: ALP, in vitro mineralization, and osteogenic gene expression values ​​are measured in MSCs using 0.1% DMSO as an untreated control. Osteogenesis medium containing BMP2 (100 ng / ml) is used as a positive control.

[0209] Sex as a biological variable. No sex differences were observed in the circadian clock behavior of isolated skeletal cells. Experiments 1-4 suggest the use of male-female or female-specific MSCs. Bone volume and structure are secondary sexual characteristics, and male MSCs form more mineralized nodules than female MSCs. Intrinsic molecular differences between male and female MSCs are suggested. Experiment 5 uses both male and female MSCs.

[0210] Expected results. The novel chemical space-specific HTS-based chemogenomics analysis in this study should further elucidate the role of monoamine-related pathways in regulating Npas2 expression in MSCs at high resolution, which should lead to a mechanistic understanding of chronotherapy. The function of monoamine transporters in non-neuronal cells has recently been studied. Norepinephrine metabolism in perivascular adipose tissue (PVAT) has been studied. PVAT adipocytes were found to express monoamine transporters (vesicle monoamine transporters (VMAT1, VMAT2), plasma membrane monoamine transporter (PMAT), and norepinephrine transporter (NET)). Intracellular accumulation of norepinephrine analog fluorescence signals was significantly reduced in vitro by chemical inhibitors of VMAT, PMAT, and NET. Therefore, compounds targeting monoamine transporters are expected to regulate monoamine concentrations in the culture medium and potentially affect the function of monoamine receptors in MSCs.

[0211] Separately, monoamine receptor antagonists and agonists may directly modulate their function and influence downstream signaling pathways. Serotonin antagonists are associated with reduced hepatocyte regeneration and serotonin secreted from platelets and inflammatory cells. Serotonin plays a crucial role in skin wound healing. Recently, serotonin receptor agonists have been shown to promote skin wound healing. This study will measure the effects of agonists and antagonists on Npas2 expression.

[0212] Chemical genomics analysis should allow us to determine specific monoamine pathways that regulate Npas2 expression. In particular, we expect to obtain a list of compounds that reduce Npas2 expression. These compounds are expected to increase osteogenic differentiation, as assessed by statistically large ALP and in vitro mineralization, as well as coordinated time-dependent expression of osteogenic genes. Top-ranking compounds may exhibit levels of osteogenic differentiation comparable to those derived from BMP-2. These hit compounds with minimal therapeutic EC / IC indices will be identified in chronotherapy development in Phase II projects.

[0213] Potential problems and alternative plans. For HTS, we suggest using immortalized MSCs. PCR of the allele genome should be performed after 5 passages to ensure stability. Although unlikely, genetic changes or other mutations may occur. If necessary, use a fresh batch of primary MSCs derived from Npas2-LacZ mice.

[0214] Study 2. Determine the regulatory roles of monoamine receptors and transporters in Npas2 expression and bone formation differentiation in human MSCs (conducted by UCLA Research Institute). Theoretical basis and objectives. It has been revealed that Npas2 expression progresses to an increased level in gingival tissue affected by ligature-induced periodontitis (Figure 15D). Separately, it has been found that MSCs express monoamine transporters that were previously thought to be specific to nerve cells (Figure 18). The expression profile of adrenergic receptors in human MSCs has been shown to be sensitively regulated by the culture environment. Therefore, it is further hypothesized that environmental regulation of monoamine receptor and / or transporter expression affects the differentiation potential of MSCs via Npas2 expression. The objective of this study is to characterize the behavior of human MSCs due to the overexpression of monoamine-related molecules. [1] Expression plasmids of human monoamine receptors and transporters are prepared in lentiviral vectors. [2] Human MSCs are transduced to overexpress the monoamine receptor and transporter, respectively. [3] Npas2 expression and osteogenic differentiation are measured.

[0215] At the MPI UCLA lab, you will carry out all the projects proposed in Exam 2. It should be noted that the projects in Exam 1 and Exam 2 are complementary to the overall objective.

[0216] Experiment 1 (ORF expression plasmid). Based on chemical genomics analysis and literature review, seven monoamine transporters and seventeen monoamine receptors were selected for this project (Table 4). [Table 4]

[0217] A list of ORF expression vectors was obtained from a publicly available genome-scale lentiviral expression library (50) for human ORFs. Expression vectors for human monoamine-related molecules were constructed using conventionally known third-generation lentiviral vector construction protocols (51, 52).

[0218] Experiment 2 (Human MSCs overexpressing monoamine-related molecules). Human MSCs (iMSC3, Applied Biological Materials) are transduced using a lentiviral vector containing an antibiotic (blasticidin) resistance gene. Transduced cells are selected with blasticidin (5 μg / μl: from the mortality curve). Surviving MSCs are grown in growth medium, and overexpression of the transduced gene is confirmed. The following experiments are performed using transduced MSCs.

[0219] Experiment 3 (Npas2 expression). Npas2 expression was analyzed using RT-PCR in MSCs transduced with each monoamine-related molecule (53). Since Npas2 is a core gene of the circadian rhythm, the MSCs were synchronized with forskolin (10 μM) for 2 hours. RNA samples were prepared every 4 hours from 24 to 72 hours after synchronization.

[0220] Experiment 3 (Bone Formation and Differentiation). MSCs transduced with monoamine-related molecules are subjected to bone formation and differentiation. In vitro bone formation and differentiation are monitored by ALP enzyme activity, and in vitro mineralization is monitored by alizarin red staining. The time-dependent expression of bone formation and differentiation-related genes is monitored by qPCR.

[0221] Experiment 4 (Expression of stem cell markers). MSCs with the Npas2 KO mutation showed high pluripotency while maintaining high levels of the stem cell markers Nanog and Klf4 in the undifferentiated stage (Figures 20A-20C). MSCs, which are the origin of teeth, are said to play an important role in the regeneration of periodontal tissue. Therefore, we will analyze the effect of overexpression of monoamine-related molecules on the characteristics of stem cells. We will promote proliferation over time and the expression of the mesenchymal stem cell markers Nanog and Klf4.

[0222] Sex as a biological variable. Inherent molecular differences between male and female MSCs have been suggested. However, we propose using commercially available cloned MSCs, which will be provided without source information.

[0223] Expected results: Increased monoamine-related Npas2 expression is expected to reduce osteogenic differentiation in MSCs. In contrast, MSCs with reduced Npas2 expression are expected to behave similarly to Npas2 knockout MSCs (Figures 20A-20C). The main goal of Experiment 2 is to determine which monoamine-related molecules effectively regulate (increase or decrease) Npas2 expression. Interactions between serotonin and the circadian system have been reported in SCNs and mood disorders. Dopamine has been shown to affect circadian Npas2 expression in the retina. Upregulation of α-adrenergic receptors has been suggested to increase Npas2 expression in MSCs. Preliminary data here suggested the involvement of pan-monoamine transporters in the regulation of Npas2 expression (Figure 10). Therefore, it is likely that we will identify specific monoamine-related molecules that regulate Npas2 expression.

[0224] The identification of new monoamine-related molecules in this project should immediately suggest a novel molecular mechanism for the reduction of regenerative capacity in wound and chronic inflammation, namely, that the environmental regulation of monoamine-related molecules may potentially play a pathological role through downstream upregulation of Npas2. This would establish the proposed MOA of chronotherapy.

[0225] Potential problems and alternative plans. Npas2 is one of the core clock genes. Other clock genes may be regulated by monoamine-related molecules, and this should be characterized.

[0226] Serum components for cell culture contain monoamines at physiological concentrations. However, it has been shown that monoamines are supplemented in the culture medium.

[0227] The Phase I results, through complete in vitro characterization, efficiently identify monoamine compounds that suppress Npas2 in a chemical space-specific manner, thereby elucidating the chronobiological role of Npas2 in stem cell differentiation. The Study 1 (ABR) and Study 2 (UCLA) projects complementarily address the overarching objectives. MPI regularly exchanges progress and results to efficiently manage the proposed projects.

[0228] Phase II and commercialization plans may include: (1) optimal formulation of candidate small molecules suitable for clinical application; (2) evaluation of efficacy for alveolar bone regeneration in a mouse periodontitis model; (3) preclinical efficacy and safety studies using canine periodontitis; and (4) biocompatibility / safety evaluation in accordance with ISO 10993-1 for FDA application.

[0229] Example 19 Small molecule inhibitor of Npas2 for the prevention of surgical scarring Despite numerous efforts, effective treatments to minimize scarring have not been adequately developed. Globally, 100 million patients develop scarring each year solely from elective and traumatic surgery. In 40–70% of surgical cases, patients experience hypertrophic and raised scarring ("fibrosis") caused by excessive collagen deposition, characterized by the formation of dense collagen fibrous tissue (Figure 21). Current standard treatments for scar prevention include continuous corticosteroid injections, laser therapy, surgical excision, and / or radiotherapy.

[0230] Regarding burn wound healing, as shown in Figure 22, and in an in vitro scratch model of cutaneous fibroblast migration (the entire model is incorporated herein by reference, Hoyle et al.) As described in al., Sci. Trans Med 2017, circadian rhythms influence wound healing.

[0231] Whole-genome in vivo microarray analysis revealed that Npas2 is upregulated in rat femurs implanted with T-shaped titanium rods (Figure 23 left), and that Npas2 knockout (KO) (Npas2- / -) mice, compared to wild-type (WT) and Npas2+ / - mice, lack high-density collagen formation in the fibrous tissue on the titanium implant surface, as described by Mengatto et al, PlosOne, 2011 and Morinaga et al, Biomaterials, 2019, whose full contents are incorporated herein by reference (Figure 23 right). Npas2 KO improves wound healing in the mouse model, as described by Sasaki, et al., Anatomical Record, 2019, whose full contents are incorporated herein by reference.

[0232] In vitro wound healing assays demonstrated that Npas2 knockout dermal fibroblasts improved wound healing in an in vitro model (Figures 24-25 and 3C).

[0233] Example 2 described above illustrates a high-throughput screening of Npas2 inhibitors, identifying 10 compounds that downregulate Npas2 (shown in Figures 13A and 26).

[0234] As described herein, Dwn1, a small molecule inhibitor of neuronal PAS domain 2 (Npas2), improves wound healing and minimizes scarring. As shown in Figure 27, Dwn1 promoted (1) fibroblast migration in abrasion healing assays and (2) collagen gel contraction in vitro.

[0235] As shown in Figure 28, Dwn1 improved the healing of dehiscenced wounds with minimal scarring when administered to wounds using sutures, compared to wound closure with sutures alone and to both sutures and vehicle (10% DMSO) administration. Furthermore, administration of Dwn1 to sutured wounds reduced excessive collagen deposition on or around the wound compared to the use of sutures alone or sutures and vehicle (10% DMSO) administration, as shown in Figure 29.

[0236] Small molecule inhibitors of Npas2 are expected to promote wound closure and reduce scarring. Npas2 knockout did not cause embryonic or developmental pathologies. Thus, Npas chronotherapy, i.e., administration of Npas2 inhibitors to suppress Npas2 and modulate the circadian rhythm, is proposed as a therapeutic agent to reduce the formation of collagen-mediated "fibrosis" and reduce wound scarring.

[0237] While other target clock molecules for chronotherapy, such as Bmal1 and Clock, exist besides Npas2 (Figure 30), Npas2 has been shown to be a safe molecular target because knockout mutations of Bmal1 or Clock produce various pathological phenotypes and symptoms of immature aging (sarcopenia, cataracts, organ shrinkage) in peripheral bone tissue. However, Npas2 knockout mutations did not produce embryogenetic pathologies in the jawbone, vertebrae, or limb bones. Npas2 expression levels in SCNs are low and contribute little to the central circadian rhythm.

[0238] Reserpine primarily blocks vesicle monoamine transporters (VMATs) expressed in neuroendocrine cells and neurons. (Figure 31) Blocking neuronal VMATs by reserpine inhibits the uptake of monoamines such as norepinephrine, dopamine, serotonin, and histamine in neuronal synaptic vesicles, reducing neurotransmitter storage. The transcription of the monoamine oxidase A (MAOA) promoter is regulated by clock components BMAL1, Npas2, and PER2. In mice, mutations in Per2 lead to decreased MAOA activity.

[0239] Similar to VMAT, extraneuronal monoamine transporters (EMTs) exist that are expressed in various cells, including chondrocytes and smooth muscle cells (Figures 30 and 32A). When serotonin was administered in vitro (Figure 32B), the width of the scratch zone was reduced compared to the vehicle, as shown by Sadiq et al., Int J. Mol Sci 2028, which is incorporated herein by reference in its entirety. It is hypothesized that reserpine blocks EMTs in fibroblasts, leading to the accumulation of extracellular serotonin that accelerates fibroblast migration (Figure 32A).

[0240] Example 20 Mouse model for surgical scar healing / prevention Using a double-edged scalpel, vertical incisions (10 × 1.5 mm) were made on both the left and right sides of the back of mice. The center of the wound was ligated once with 5-0 nylon suture. (Figure 33A) Using the gross image of the wound / scar, the Visual Analog Scale (VAS) was recorded daily until postoperative day 7 (Figure 33A), and the postoperative gross image of the wound / scar was measured with a ruler. (Figure 33C) On postoperative day 7, histological analysis of the wound was performed using hematoxylin-eosin (HE) and Masson's trichrome (MT). (Figure 33D) The scar index was assessed using HE-stained slices. (Figure 33E) The area % of fibrous tissue was assessed using MT-stained slices. (Figure 33F)

[0241] We selected the candidate compound Dwn1 and evaluated its Npas2 suppression in dermal fibroblasts in vitro. A scatter plot of in vitro high-throughput drug screening assays using an FDA-approved compound library was created in UCLA's MSSR (Figure 34A). High absolute values ​​of the negative Npas2 Z-score indicate highly downregulated Npas2 expression (X-axis). High cell viability Z-scores indicate high fibroblast viability (Y-axis). Candidate compounds (Dwn1) were selected in descending order of absolute negative Npas2 Z-score and viability. Circadian Npas2 expression in mouse dermal fibroblasts treated with Dwn1 (1 μM or 10 μM) was evaluated and compared with the control (Figure 34B). Cell migration of mouse dermal fibroblasts treated with Dwn1 was evaluated (*p<0.05) (Figure 34C).

[0242] The effects of two different concentrations of Dwn1 (1 μM or 10 μM) on collagen synthesis in mouse dermal fibroblasts in vitro were analyzed. Mouse dermal fibroblasts were seeded in 24-well plates and added to a medium containing ascorbic acid (50 μg / mL), with concentrations ranging from 80% to 9%. Cells were cultured at 0% confluence for 1, 3, and 7 days. Next, cells were fixed in 10% neutral buffered formalin and stained with picrosilius red (PolyScience, Niles, IL) to visualize collagen. AA: l-ascorbic acid. OD: optical density. CTRL: cells treated in control medium without AA. Picrosilius red staining of fibroblasts cultured with ascorbic acid supplementation showed increased positive reactions, indicating collagen fiber formation and accumulation in fibroblasts at day 7 after treatment with 1 μM or 10 μM Dwn1, compared to control and 50 μg AA. (Figure 35A) Absorbance values ​​were measured with a 550 nm spectrophotometer equipped with a plate reader (SYHNERGY H1 plate reader) and compared by one-way ANOVA and post-hoc Holm test.

[0243] Gene expression of collagen types Col1a1, Col1a2, Col3a1, and Col14a1 was evaluated in granulation tissue and wound tissue after treatment with 1 μM or 10 μM Dwn1 or a control (vehicle). (Figure 35B) Gapdh was used as an internal control. In vivo immunohistochemical staining of αSMA was performed at 7 days postoperatively in wounds treated with vehicle or vehicle + Dwn1. Vehicle or Dwn1 + vehicle was applied every 24 hours postoperatively and evaluated at 3 and 7 days postoperatively, respectively.

[0244] The effect of Dwn1 on a linear wound / scar model on the dorsal side of a mouse was evaluated. Gross images are shown on day 0 (D0), day 2 (D2), day 5 (D5), and day 7 (D7) postoperatively and after the start of topical application of Dwn1 to the wound. Veh: Vehicle. The vehicle or Dwn1 + vehicle was applied every 24 hours postoperatively. (Figure 36A) The Visual Analog Score (VSA) scale was evaluated for wounds treated with the vehicle or vehicle + Dwn1. (Figure 36B) Tissue images of lateral wounds / scars treated with the vehicle or vehicle + Dwn1 on day 7 postoperatively are shown. (Figure 36C) The yellow dotted line indicates granulation tissue. The two on the left are stained with HE, and the two on the right are stained with MT. The scale bar is 1000 μm. The scar index was evaluated using HE-stained slices. (Figure 36D) The area of ​​fibrous tissue was evaluated using MT-stained slices. * indicates p < 0.05. (Figure 36E)

[0245] The molecular biological effects of Dwn1 on a linear dorsal wound / scar model in mice were evaluated. Typical laser capture microdissection (LCM) images were taken. (Figure 37A) Slides were briefly stained with hematoxylin and eosin before LCM. G: granulation tissue, W: wound tissue. Gene expression of Col1a1, Col1a2, Col3a1, Col14a1, Tgfβ1 and Acta2 was evaluated in (G) and wound tissue (W). (Figure 37B) Gapdh was used as an internal control. * indicates p<0.05. Immunohistochemical staining of αSMA in vivo was performed on wounds treated with vehicle or vehicle + Dwn1 at 7 days postoperatively. (Figure 37C) Yellow dotted lines indicate granulation tissue. Scale bar is 100 μm.

[0246] All patents, patent applications, and scientific publications cited herein are incorporated herein by reference in their entirety.

[0247] While certain features of the present invention have been illustrated and described herein, many modifications, substitutions, alterations, and equivalents will come to mind for those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and alterations so as to fall within the true spirit of the invention.

Claims

1. A method for improving or promoting wound healing, comprising administering a drug that suppresses the expression of a clock gene to the wound in need thereof, wherein the clock gene is neuron PAS domain protein 2 (Npas2).

2. The method according to claim 1, wherein the administration is performed via a route selected from local administration, transdermal administration, and subcutaneous administration.

3. The method according to claim 1, wherein the wound is a skin wound.

4. The method according to claim 3, wherein the skin wound is a periodontal wound.

5. The method according to claim 4, wherein the periodontal wound includes degeneration of gingival connective tissue or resorption of alveolar bone.

6. The method according to claim 1, wherein the agent that suppresses the expression of Npas2 accelerates the migration of human dermal fibroblasts in a cell migration assay.

7. The method according to claim 1, wherein the agent is selected from norepinephrine, dopamine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants.

8. The method according to claim 1, wherein the drug is reserpine.

9. The method according to claim 1, wherein the drug is antimycin A, diflumic acid, morindone hydrochloride, and mefexamide hydrochloride.

10. The method according to claim 1, wherein the drug is selected from econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolo, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R,S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium salt.

11. The method according to claim 1, wherein the agent is an Npas2 downregulator selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors.

12. The method according to claim 11, wherein the cytoskeleton / ECM inhibitor is brefeldin A, colchicine, podophyllotoxin, or 5175348.

13. The method according to claim 11, wherein the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952.

14. The transdermal administration involves the use of a deformable nanoscale vesicle that encapsulates the drug. The method according to claim 2, which is applied to wounds.

15. The method according to claim 2, wherein the transdermal administration is the application to the wound of a transdermal delivery system selected from the group consisting of a microneedle coated with the drug, a solid polymer matrix having the incorporated drug inside, a transdermal patch comprising a reservoir and a semipermeable membrane for storing the drug, a transdermal gel containing dissolved the drug inside, a transdermal spray containing dissolved the drug inside, and a quantitative transdermal spray containing dissolved the drug inside.

16. The method according to claim 1, wherein the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene.

17. The method according to claim 16, wherein the siRNA is administered by a route selected from the group consisting of a microneedle array, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods.

18. The method according to claim 16, wherein the siRNA is chemically modified in the 2' position of the ribose sugar ring, the phosphate backbone, the nucleic acid base and ribose sugar, and the modification or conjugation of the 5' end.

19. The method according to claim 18, wherein the ribose sugar ring is guanosine or uridine, and the modification at the 2' position is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE).

20. The method according to claim 18, wherein the phosphate skeleton is modified with a phosphorodithioate, a triazole dimer, an amide, or a boranophosphate.

21. The method according to claim 18, wherein the modifications of the nucleic acid base and ribose sugar are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine.

22. The method according to claim 18, wherein the 5' terminal modification or conjugation is palmitic acid conjugation at the 5' terminal of the siRNA, reverse thymidine (idT) coupling to the 3' terminal of the siRNA, and topalmitic acid conjugation at the 5' terminal, conjugation of the siRNA with a cell-permeable peptide (CPP), conjugation of the siRNA with an aromatic compound selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with an aromatic compound crosslinked with urea / thiourea; polyethylene glycol (PEG) conjugation at the 3' terminals of the sense and antisense strands; and cholesterol conjugation of the siRNA.

23. A method for regenerating alveolar bone, comprising administering a drug that suppresses the expression of Npas2 to a bone loss site in a target area where regeneration is needed.

24. The method according to claim 23, wherein the administration is by a route selected from local administration and transdermal administration.

25. The method according to claim 23, wherein the wound is a skin wound.

26. The method according to claim 25, wherein the skin wound is a periodontal wound.

27. The method according to claim 26, wherein the periodontal wound includes degeneration of gingival connective tissue or resorption of alveolar bone.

28. The method according to claim 23, wherein the agent that suppresses the expression of Npas2 accelerates the migration of human dermal fibroblasts in a cell migration assay.

29. The method according to claim 23, wherein the agent is selected from norepinephrine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants.

30. The method according to claim 23, wherein the drug is reserpine.

31. The method according to claim 23, wherein the drug is antimycin A, niflumic acid, morindone hydrochloride, and mefexamide hydrochloride.

32. The method according to claim 23, wherein the drug is selected from econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolo, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R, S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium salt.

33. The method according to claim 23, wherein the agent is an Npas2 downregulator selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors.

34. The method according to claim 33, wherein the cytoskeleton / ECM inhibitor is brefeldin A, colchicine, podophyllotoxin, or 5175348.

35. The method according to claim 33, wherein the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952.

36. The method according to claim 24, wherein the transdermal administration is by a deformable nanoscale vesicle that encapsulates the drug.

37. The transdermal administration comprises a transdermal patch comprising a microneedle coated with the drug, a solid polymer matrix having the incorporated drug inside, a reservoir for storing the drug and a semipermeable membrane, a transdermal gel containing the dissolved drug inside, a transdermal spray containing the dissolved drug inside, and a quantitative transdermal spray containing the dissolved drug inside. The method according to claim 24, wherein a transdermal delivery system selected from the group consisting of the above is applied to the wound.

38. The method according to claim 23, wherein the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene.

39. The method according to claim 38, wherein the siRNA is administered by a route selected from the group consisting of a microneedle array, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods.

40. The method according to claim 38, wherein the siRNA is chemically modified in the 2' position of the ribose sugar ring, the phosphate backbone, the nucleic acid base and ribose sugar, and the modification or conjugation of the 5' end.

41. The method according to claim 40, wherein the ribose sugar ring is guanosine or uridine, and the modification at the 2' position is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE).

42. The method according to claim 40, wherein the phosphate skeleton is modified with a phosphorodithioate, a triazole dimer, an amide, or a boranophosphate.

43. The method according to claim 40, wherein the modifications of the nucleic acid base and ribose sugar are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine.

44. The method according to claim 40, wherein the 5' terminal modification or conjugation is palmitic acid conjugation at the 5' terminal of the siRNA, reverse thymidine (idT) coupling to the 3' terminal of the siRNA, and topalmitic acid conjugation at the 5' terminal, conjugation of the siRNA with a cell-permeable peptide (CPP), conjugation of the siRNA with an aromatic compound selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with an aromatic compound crosslinked with urea / thiourea; polyethylene glycol (PEG) conjugation at the 3' terminals of the sense and antisense strands; and cholesterol conjugation of the siRNA.

45. A method for regenerating connective tissue in a wound site requiring the regeneration of connective tissue at the wound site, comprising administering a therapeutically effective amount of an Npas2 expression inhibitor to the wound.

46. The method according to claim 45, wherein the administration is by a route selected from local administration and transdermal administration.

47. The method according to claim 45, wherein the wound is a skin wound.

48. The method according to claim 47, wherein the skin wound is a periodontal wound.

49. The method according to claim 48, wherein the periodontal wound includes degeneration of gingival connective tissue or resorption of alveolar bone.

50. The method according to claim 45, wherein the agent that suppresses the expression of Npas2 accelerates the migration of human dermal fibroblasts in a cell migration assay.

51. The method according to claim 45, wherein the agent is selected from norepinephrine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants.

52. The method according to claim 45, wherein the drug is reserpine.

53. The method according to claim 45, wherein the drug is antimycin A, diflumic acid, morindone hydrochloride, and mefexamide hydrochloride.

54. The method according to claim 45, wherein the drug is selected from econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolo, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R, S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium salt.

55. The method according to claim 45, wherein the agent is an Npas2 downregulator selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors.

56. The method according to claim 55, wherein the cytoskeleton / ECM inhibitor is brefeldin A, colchicine, podophyllotoxin, or 5175348.

57. The method according to claim 55, wherein the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952.

58. The method according to claim 46, wherein the transdermal administration is the application of a deformable nanoscale vesicle that encapsulates the drug to the wound.

59. The method according to claim 46, wherein the transdermal administration is the application to the wound of a transdermal delivery system selected from the group consisting of a microneedle coated with the drug, a solid polymer matrix having the incorporated drug inside, a transdermal patch comprising a reservoir and a semipermeable membrane for storing the drug, a transdermal gel containing dissolved the drug inside, a transdermal spray containing dissolved the drug inside, and a quantitative transdermal spray containing dissolved the drug inside.

60. The method according to claim 45, wherein the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene.

61. The method according to claim 60, wherein the siRNA is administered by a route selected from the group consisting of a microneedle array, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods.

62. The method according to claim 60, wherein the siRNA is chemically modified in the 2' position of the ribose sugar ring, the phosphate backbone, the nucleic acid base and ribose sugar, and the modification or conjugation of the 5' end.

63. The method according to claim 62, wherein the ribose sugar ring is guanosine or uridine, and the modification at the 2' position is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE).

64. The method according to claim 62, wherein the phosphate skeleton is modified with a phosphorodithioate, a triazole dimer, an amide, or a boranophosphate.

65. The method according to claim 62, wherein the nucleic acid base and ribose sugar modification are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine.

66. The method according to claim 62, wherein the 5' terminal modification or conjugation is palmitic acid conjugation at the 5' terminal of the siRNA, reverse thymidine (idT) coupling to the 3' terminal of the siRNA, and topalmitic acid conjugation at the 5' terminal, conjugation of the siRNA with a cell-permeable peptide (CPP), conjugation of the siRNA with an aromatic compound selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with an aromatic compound crosslinked with urea / thiourea; polyethylene glycol (PEG) conjugation at the 3' terminals of the sense and antisense strands; and cholesterol conjugation of the siRNA.

67. The method according to claim 45, wherein the connective tissue is one or more of collagen, dermal-like collagen fibers, or bone.

68. The method according to claim 45, wherein the wound site is a site of bone loss.

69. The method according to claim 68, wherein the bone loss is in the area of ​​alveolar bone resorption induced by periodontitis.

70. The method according to claim 45, wherein the wound site is a site of gingival connective tissue degeneration.

71. A method for reducing the size of a wound area, comprising locally administering a drug that suppresses Npas2 expression to the target open wound site.

72. Claim 7, wherein the administration is by a route selected from local administration and transdermal administration. The method described in 1.

73. The method according to claim 71, wherein the wound is a skin wound.

74. The method according to claim 73, wherein the skin wound is a periodontal wound.

75. The method according to claim 74, wherein the periodontal wound includes degeneration of gingival connective tissue or resorption of alveolar bone.

76. The method according to claim 71, wherein the agent that suppresses the expression of Npas2 accelerates the migration of human dermal fibroblasts in a cell migration assay.

77. The method according to claim 71, wherein the agent is selected from norepinephrine and serotonin reuptake inhibitors, oxidative phosphorylation inhibitors, cyclooxygenase-2 inhibitors, dopamine antagonists, or central nervous system (CNS) stimulants.

78. The method according to claim 71, wherein the drug is reserpine.

79. The method according to claim 71, wherein the drug is antimycin A, niflumic acid, morindone hydrochloride, and mefexamide hydrochloride.

80. The method according to claim 71, wherein the drug is selected from econazole nitrate, aceclofenac, pravastatin, tyroxapol, isosorbide mononitrate, MS-1500387, (S)-(-)-atenolo, butenafine hydrochloride, acexidine hydrochloride, atropine sulfate monohydrate, trimetadione, chlorphensin carbamate, mafenide hydrochloride, nifenazone, alticaine hydrochloride, theobromine, nifloxazide, SAM001246626, dropropidine (R, S), diethylcarbamazine citrate, MS-1501214, drasetron mesylate, estrone, prednisolone, daunorubicin hydrochloride, cycloheximide, and monensin sodium salt.

81. The method according to claim 71, wherein the agent is an Npas2 downregulator selected from the group consisting of cytoskeleton / ECM inhibitors, hormone agonists, nitric oxide inhibitors, intracellular Ca++ release agents, kinase / phosphatase inhibitors, and kinase inhibitors.

82. The method according to claim 81, wherein the cytoskeleton / ECM inhibitor is brefeldin A, colchicine, podophyllotoxin, or 5175348.

83. The method according to claim 81, wherein the hormone agonist is AC-93253 iodide, the nitric oxide inhibitor is diphenyleneiodonium chloride, the intracellular Ca++ release agent is thapsigargin, the kinase / phosphatase inhibitor is PD-166285 hydrate, and the kinase inhibitor is PD-173952.

84. The method according to claim 72, wherein the transdermal administration is the application of a deformable nanoscale vesicle that encapsulates the drug to the wound.

85. The method according to claim 72, wherein the transdermal administration is the application to the wound of a transdermal delivery system selected from the group consisting of a microneedle coated with the drug, a solid polymer matrix having the incorporated drug inside, a transdermal patch comprising a reservoir and a semipermeable membrane for storing the drug, a transdermal gel containing dissolved drug inside, a transdermal spray containing dissolved drug inside, and a quantitative transdermal spray containing dissolved drug inside. Law.

86. The method according to claim 71, wherein the drug is a synthetic small interfering ribonucleic acid (siRNA) designed to target the mRNA of the Npas2 gene.

87. The method according to claim 86, wherein the siRNA is administered by a route selected from the group consisting of a microneedle array, electroporation, pressure, mechanical massage, cationic liposomes, cationic polymer-mediated delivery systems, ultrasound, conjugate delivery systems, microbubbles, liposome bubbles, ultrasound-sensitive nanobubbles, carbon nanotubes, lipid-based nanovectors, non-lipid organic-based nanovectors and inorganic nanovectors, gold nanoparticles, and gold nanorods.

88. The method according to claim 86, wherein the siRNA is chemically modified in the 2' position of the ribose sugar ring, the phosphate backbone, the nucleic acid base and ribose sugar, and the modification or conjugation of the 5' end.

89. The method according to claim 88, wherein the ribose sugar ring is guanosine or uridine, and the modification at the 2' position is selected from the group consisting of 2'-OMe, 2'-F, and 2'-O-methoxyethyl (2'-MOE).

90. The method according to claim 88, wherein the phosphate skeleton is modified with a phosphorodithioate, a triazole dimer, an amide, or a boranophosphate.

91. The method according to claim 88, wherein the nucleic acid base and ribose sugar modification are 5-fluoro-2'-deoxyuridine (FdU), 2'-O-methylphosphodithioate (2'O-MePS2), lipophilic boron cluster, 3-N-[(1,12-dicarba-closo-dodecacarboran-1-yl)propan-3-yl]thymidine (C2B10H11,CB), thymidine, and 5-bis(aminoethyl)-aminoethyl-2'-deoxyuridine.

92. The method according to claim 88, wherein the 5' terminal modification or conjugation is palmitic acid conjugation at the 5' terminal of the siRNA, reverse thymidine (idT) coupling to the 3' terminal of the siRNA, and topalmitic acid conjugation at the 5' terminal, conjugation of the siRNA with a cell-permeable peptide (CPP), conjugation of the siRNA with an aromatic compound selected from the group consisting of phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives; chemical modification of the 3' overhang region with an aromatic compound crosslinked with urea / thiourea; polyethylene glycol (PEG) conjugation at the 3' terminals of the sense and antisense strands; and cholesterol conjugation of the siRNA.

93. The method according to claim 71, wherein the open wound site includes connective tissue selected from one or more of collagen, dermal-like collagen fibers, or bone.

94. The method according to claim 71, wherein the open wound site is a site of bone loss.

95. The method according to claim 94, wherein the bone loss is in the area of ​​alveolar bone resorption induced by periodontitis.

96. The method according to claim 71, wherein the open wound site is a site of gingival connective tissue degeneration.

Images