阅读说明：本技术 唾液腺再生 (Salivary gland regeneration ) 是由 C·S·巴赫尼 S·诺克斯 E·阿尔斯伯格 O·乔恩 于 2019-12-10 设计创作，主要内容包括：本发明提供了一种用于促进有相应需求的受试者中唾液腺再生的方法,其包含向所述唾液腺的腺泡祖细胞施用胆碱能激动剂或毒蕈碱激动剂中的至少一种,以促进腺泡细胞再生。尤其是,可配制包含包封于海藻酸盐水凝胶中的毒蕈碱激动剂(例如西维美林)的制剂,用以局部施用于唾液腺并用于治疗口干症。(The present invention provides a method for promoting regeneration of salivary glands in a subject in need thereof, comprising administering at least one of a cholinergic agonist or a muscarinic agonist to acinar progenitor cells of the salivary glands to promote acinar cell regeneration. In particular, formulations comprising a muscarinic agonist (e.g., cevimeline) encapsulated in an alginate hydrogel can be formulated for topical administration to salivary glands and for the treatment of dry mouth.)

1. A composition comprising a muscarinic agonist encapsulated in a hydrogel formulated for topical administration to salivary glands for the treatment of dry mouth.

2. The composition of claim 1, wherein the muscarinic agonist is selective for the M1 and/or M3 muscarinic receptor subtypes.

3. The composition according to claim 2, wherein the muscarinic agonist is cevimeline.

4. The composition of claim 1, wherein the muscarinic agonist is pilocarpine.

5. The composition of any one of claims 1 to 4, wherein the hydrogel comprises alginate.

6. The composition of claim 5, wherein said alginate is ionically crosslinked.

7. The composition according to claim 6, wherein said alginate is ionically crosslinked by divalent calcium ions.

8. The composition of any one of claims 5 to 7, wherein the concentration of said alginate in said hydrogel ranges from about 2 wt% to about 10 wt% (weight percent).

9. The composition according to any one of claims 5 to 7, wherein said alginate is at least partially oxidized.

10. The composition of claim 8, wherein about 2% to about 10% of said alginate is oxidized.

11. The composition of claim 9, wherein about 2% of said alginate is oxidized and the concentration of said alginate in said hydrogel is about 5 wt%.

12. The composition of any one of claims 1 to 11, wherein the hydrogel delivers the muscarinic agonist continuously for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks after administration to the subject.

13. The composition of any one of claims 1 to 12, further comprising a pharmaceutically acceptable excipient.

14. The composition of any one of claims 1 to 13, further comprising a contrast agent.

15. A method of treating dry mouth in a subject, the method comprising topically administering to a salivary gland of the subject a therapeutically effective amount of the composition of any one of claims 1 to 14.

16. The composition of claim 15, wherein the composition is injected into or adjacent to the salivary gland.

17. The composition of claim 15 or 16, further comprising performing medical imaging or palpation to locate the salivary gland prior to injection.

18. The method of claim 17, wherein the medical imaging comprises performing ultrasound imaging.

19. The method of any one of claims 15-18, wherein multiple therapeutically effective doses of the composition are administered to the subject.

20. The method of any one of claims 15-19, wherein the dry mouth is caused by radiation or damage to the salivary glands caused by sjogren's syndrome.

21. The method of any one of claims 15 to 20, wherein the subject is a pet or livestock.

22. The method of any one of claims 15-21, wherein the subject is a mammal.

23. The method of claim 22, wherein the mammal is a dog, cat, horse, cow, goat, sheep, or pig.

24. The method of claim 22, wherein the mammal is a human.

25. A kit comprising the composition of any one of claims 1 to 14 and instructions for the treatment of dry mouth.

26. The kit of claim 25, further comprising a device for delivering the composition to the subject.

27. The kit of claim 25, further comprising a first syringe containing a composition comprising a muscarinic agonist encapsulated in an alginate hydrogel, a second syringe containing a solution comprising calcium chloride, and a luer lock, wherein the second syringe is connectable to the first syringe through the luer lock.

28. The kit of claim 27, wherein the first syringe containing the composition comprising the muscarinic agonist encapsulated in an alginate hydrogel is in a frozen state.

29. The kit of any one of claims 25 to 28, wherein the muscarinic agonist is cevimeline or pilocarpine.

30. A method of promoting salivary gland regeneration in a subject in need thereof, the method comprising: locally administering at least one of a cholinergic agonist or a muscarinic agonist to acinar progenitor cells and acinar cells of the salivary gland to promote proliferation of the acinar progenitor cells and the acinar cells, thereby increasing salivary secretion.

31. The method of claim 30, wherein the acinar progenitor cells are SOX2+ acinar progenitor cells.

32. The method of claim 30 or 31, wherein the muscarinic agonist is encapsulated in a hydrogel formulated for topical administration to the salivary gland.

33. The method of claim 32, wherein the hydrogel comprises alginate.

34. The method of claim 33, wherein said alginate is ionically crosslinked.

35. The method of claim 34, wherein said alginate is ionically crosslinked by divalent calcium ions.

36. The method of any one of claims 33-35, wherein the concentration of said alginate in said hydrogel ranges from 2 wt% to 10 wt% (weight percent).

37. The method of any one of claims 33 to 36, wherein said alginate is at least partially oxidized.

38. The method of claim 37, wherein about 2% to about 10% of said alginate is oxidized.

39. The method of claim 38, wherein about 2% of said alginate is oxidized and the concentration of said alginate in said hydrogel is about 5 wt%.

40. The method of any one of claims 30 to 39, wherein the muscarinic agonist is selective for the M1 and/or M3 muscarinic receptor subtypes.

41. The method of claim 40, wherein the muscarinic agonist is cevimeline.

42. The method of any one of claims 30 to 39, wherein the muscarinic agonist is pilocarpine.

43. The method of any one of claims 32-42, wherein the hydrogel continuously delivers the muscarinic agonist for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks after administration to the subject.

44. The method of any one of claims 30 to 43, wherein the acinar progenitor cells are Ki67+ or EdU⁺Acinar progenitor cells.

45. The method of any one of claims 30-44, wherein salivary flow is increased as a result of proliferation of the acinar cells in response to the muscarinic agonist.

46. The method of any one of claims 30-45, wherein the salivary gland is a sublingual gland.

47. The method of any one of claims 30-46, wherein the cholinergic agonist comprises at least one of an acetylcholine or an acetylcholine analog.

48. The method of claim 47, wherein the acetylcholine analog comprises carbachol.

49. The method of any one of claims 30 to 48, wherein the acinar progenitor cells are SOX2⁺/AQP5⁺/Ki67⁺A cell.

50. The method of any one of claims 30-49, wherein the acinar progenitor cells are Mucin (MUC)19^-A cell.

51. The method of any one of claims 30-50, further comprising isolating SOX2 from the salivary glands of the subject⁺Acinar progenitor cells and expansion of the SOX2⁺Acinar progenitor cells.

52. The method of claim 51, wherein the expanded cells are provided in an engineered tissue construct or a biocompatible matrix that provides controlled release of the at least one cholinergic agonist or muscarinic agonist in the expanded cells, the controlled release comprising at least one of a delayed release, a sustained release, a gradient release, a temporal release, a patterned release, or a spatial release.

53. The method according to claim 52, wherein the expanded cells and the at least one cholinergic agonist or muscarinic agonist are provided on and/or within a biodegradable natural polymer or macromer.

54. The method of claim 53, wherein the biodegradable natural polymer or macromer comprises a hydrogel.

55. The method of any one of claims 30 to 54, wherein the subject has received radiation therapy that causes dry mouth.

Background

Salivary Gland (SG) dysfunction severely impairs oral health and quality of life of patients: saliva can protect the oral mucosa, promote food digestion, aid pronunciation, and help remineralization of hard tissues of the teeth. Dry mouth or xerostomia may occur due to irreversible pathological damage caused by the autoimmune disease sjogren's syndrome (100-. Loss of salivary gland regeneration can abolish salivary secretion and significantly affect the quality of life of these patients. For salivary dysfunction, there is currently no regenerative therapy available, and new, implementable solutions are needed.

Currently, standard of care suggests palliative treatment, such as saliva substitutes or artificial saliva and/or systemic sialagogues. Cevimeline and pilocarpine are FDA approved two muscarinic agonists that promote transient salivation. Pilocarpine is a non-selective muscarinic agonist, whereas cevimeline has a higher affinity for the M1 and M3 muscarinic receptor subtypes, both of which are expressed in the submandibular and sublingual salivary glands. While these drugs are effective in promoting short-term salivation when taken 3-4 times per day, they cause undesirable parasympathomimetic side effects, including hyperhidrosis, diarrhea, headache, and blurred vision, resulting in decreased patient compliance. It has recently been reported that chronic oral administration of cevimeline or pilocarpine to patients improves salivary gland function (Barbe (2017) J. Dow.Dentist.17 (3): 268-) -270). Furthermore, continuous oral administration of pilocarpine to mice receiving gamma-ray treatment of the head and neck, as compared to the non-dosed control group, promotes salivary flow (Taniguchi et al, 2019) histochemical and cytochemical reports 52 (3): 45-58) indicating a muscarinic agonist effect or promoting salivary gland repair, except that the manner of implementation has not been determined.

Thus, there remains a need for better treatments for salivary dysfunction, particularly regenerative treatments that allow recovery of salivary acinar cells and salivary production.

Disclosure of Invention

The present invention provides compositions and methods for regenerating salivary glands by promoting acinar cell replacement. As a result, it was found that acinar progenitor cells (including SOX2) in adult salivary glands⁺Progenitor cells) are critical to the recruitment of acinar cells, with the unexpected ability to repopulate tissue following radiation-induced injury. It was also found that during homeostasis, cholinergic nerves play a crucial role in controlling acinar cell replacement, and that this neuronal effect can be replicated by adding cholinergic mimetics to acinar progenitor cells. Thus, direct targeting of acinar progenitor cells within the tissue with cholinergic and/or muscarinic agonists regenerates salivary gland secretory units, allowing functional salivary gland acini to be restored and treat oral diseases such as dry mouth following radiation therapy or dry mouth associated with sjogren's syndrome.

In one aspect, a composition comprising a muscarinic agonist encapsulated in a hydrogel formulated for topical administration to salivary glands for the treatment of dry mouth is provided. Such compositions are useful for treating dry mouth, for example, dry mouth caused by radiation or damage to the salivary glands caused by an autoimmune disease (e.g., sjogren's syndrome).

In certain embodiments, the muscarinic agonist is selective for the M1 and/or M3 muscarinic receptor subtypes. In one embodiment, the muscarinic agonist is cevimeline.

In certain embodiments, the muscarinic agonist is pilocarpine.

In certain embodiments, the hydrogel comprises alginate. The alginate may be ionically crosslinked with divalent cations. In some embodiments, the alginate is associated with divalent calcium cations (Ca)²⁺) And carrying out ionic crosslinking.

In certain embodiments, the concentration of the alginate in the hydrogel ranges from about 2 wt% to about 10 wt% (weight percent), including any wt% within this range, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 wt%.

In certain embodiments, the alginate is at least partially oxidized. In some embodiments, about 2% to about 10% of the alginate is oxidized, including any percentage within this range, such as 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In one embodiment, about 2% of the alginate in the hydrogel is oxidized and the concentration of the alginate is 5 wt%.

In certain embodiments, the hydrogel continuously delivers the muscarinic agonist for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks or more after administration to the subject. In some embodiments, the hydrogel delivers the muscarinic agonist continuously for up to 30 days.

In certain embodiments, the composition further comprises a contrast agent, e.g., to confirm the location of the composition relative to the salivary gland by medical imaging after administration. In some embodiments, the contrast agent is a microbubble (e.g., suitable for ultrasound imaging) or a radiopaque contrast agent (e.g., suitable for radiography).

In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

In another aspect, a kit is provided comprising a composition described herein comprising a muscarinic agonist encapsulated in a hydrogel and instructions for treatment of dry mouth. In some embodiments, the kit further comprises a device for delivering the composition to the subject. For example, the kit may comprise a first syringe containing a composition comprising a muscarinic agonist encapsulated in an alginate hydrogel, a second syringe containing a solution comprising calcium chloride, and a luer lock, wherein the second syringe may be connected to the first syringe through the luer lock. The first syringe containing the composition comprising the muscarinic agonist encapsulated in an alginate hydrogel may be stored frozen. In some embodiments, the muscarinic agonist in the kit is cevimeline or pilocarpine.

In another aspect, there is provided a method of treating dry mouth in a subject, the method comprising topically administering to the salivary glands of the subject a therapeutically effective amount of a composition comprising a muscarinic agonist encapsulated in a hydrogel.

In certain embodiments, the composition is injected into the salivary gland or a region adjacent to the salivary gland.

In certain embodiments, multiple therapeutically effective doses of the composition are administered to the subject.

In certain embodiments, the xerostomia is caused by radiation or damage to the salivary glands caused by sjogren's syndrome.

In certain embodiments, the method further comprises performing medical imaging (e.g., ultrasound imaging) or palpation prior to injection to locate the salivary gland.

In certain embodiments, there is provided a method of promoting salivary gland regeneration in a subject in need thereof, comprising: locally administering at least one of a cholinergic agonist or a muscarinic agonist to acinar progenitor cells and acinar cells of the salivary gland to promote proliferation of the acinar progenitor cells and the acinar cells, thereby increasing salivary secretion.

In certain embodiments, the cholinergic agonist comprises at least one of acetylcholine or an acetylcholine analog. In some embodiments, the acetylcholine analog comprises carbachol.

In certain embodiments, the acinar progenitor cell is SOX2⁺Acinar progenitor cells. In some embodiments, the SOX2⁺The acinar progenitor cell is AQP5⁺/Ki67⁺A cell. In other embodiments, SOX2 is included⁺/AQP5⁺/Ki67⁺SOX2 including acinar progenitor cells⁺The acinar progenitor cells are Mucin (MUC) 19-cells.

In some embodiments, the method further comprises isolating SOX2 from the salivary glands of the subject being treated⁺Acinar progenitor cells and expansion of the SOX2⁺Acinar progenitor cells, followed by implanting the expanded cells into the salivary gland of the subject.

In some embodiments, prior to implantation, the expanded cells are provided in an engineered tissue construct or biocompatible matrix that can controllably release the at least one cholinergic agonist or muscarinic agonist in the expanded cells. The controlled release may include at least one of delayed release, sustained release, gradient release, temporal release, patterned release, or spatial release. The engineered tissue construct or biocompatible matrix may comprise a biodegradable natural polymer or macromer, such as a biocompatible hydrogel.

In some embodiments, a subject treated by the methods described herein has an oral disease, such as a disease that affects saliva production. Examples of oral diseases include, but are not limited to, salivary gland tumors, cystic fibrosis, sjogren's syndrome, sialadenitis, mumps, siallitis, sialolithiasis, salivary gland stones, sialopholithiasis, mucocysts, sublingual cysts, hyposecretion, sialorrhoea, sialorrhea, xerostomia, benign lymphatic epithelial lesions of salivary glands, salivary gland dilatation, salivary gland enlargement, salivary duct stenosis, and salivary duct stenosis. In other embodiments, the subject may have previously received radiation therapy that can cause dry mouth. The methods described herein are useful for treating such oral diseases affecting saliva production (i.e., xerostomia) in a human subject. The methods described herein will also be useful in veterinary applications to treat xerostomia in domestic animals, including but not limited to pets (e.g., dogs and cats) and livestock (e.g., sheep, goats, pigs, horses, and cattle).

Other embodiments described herein relate to methods of promoting salivary gland regeneration in a subject in need thereof, the method comprising: isolating and expanding SOX2+ acinar progenitor cells in the subject's salivary gland prior to implanting the expanded cells into the subject's salivary gland. The amplified SOX2⁺The acinar progenitor cells may be AQP5⁺/Ki67⁺the/MUC 19-cell.

In some embodiments, at least one of a cholinergic agonist or a muscarinic agonist may be administered to the expanded cells before and/or after implantation of the expanded cells to promote acinar cell production.

In other embodiments, the expanded cells may be provided in an engineered tissue construct or a biocompatible matrix. The engineered tissue construct or biocompatible matrix may provide controlled release of the at least one cholinergic agonist or muscarinic agonist in the expanded cells, the controlled release comprising at least one of a delayed, sustained, gradient, temporal, patterned or spatial release.

Drawings

FIGS. 1A-1F show that SOX2 labels progenitor cells that form acini but do not form ductal cells in the salivary glands of an adult or adult animal. FIG. 1A adult Submandibular (SMG), Sublingual (SLG) and Parotid (PG) salivary glands (non-IR, 28-33 years old) SOX2, superiorRepresentative images of immunostaining of endothelial cells (E-cadherin; ECAD) or CD44 and nuclei. Single arrows indicate acinar cells expressing SOX 2. The scale bar is 20 m. FIG. 1B wild type mice SMG and SLG subjected to SOX2, ECAD and nuclear staining. Arrows indicate cells expressing SOX 2. The scale bar is 50 m. The yellow dotted line represents the boundary between SMG and SLG. FIG. 1C vs. Sox2^eGFPSublingual salivary glands (SLG) were immunostained with GFP and the differentiated acinar marker mucin 19(MUC 19). The white dotted line delineates Sox2^eGFP+The contour of MUC19(-) cells. Scale bar 20 m. FIG. 1D AQP5⁺SOX2⁺Cell occupancy Total AQP5⁺Percentage of acinar cells. FIG. 1E images of SOX2, Ki67 and E-cadherin (ECAD) immunostaining, an epithelial marker, on SLG. White arrows indicate proliferating Ki67⁺SOX2⁺A cell. The white lines outline individual cells and nuclei. Scale bar 10 m. Fig. 1F Sox2 pedigree traces representative images of SLG. Before immunostaining with SOX2, the acinar markers AQP5 and MUC19, and the ductal marker KRT8, at Sox2^CreERT2；Rosa26^mTmGRecombination was induced in mice and salivary glands and followed for 24 hours and 30 days, respectively. Denotes MUC19(-) Sox2^CreERT2GFP (+) cells. Scale bar 30 m. mT ═ membrane bound Tomato. Data information: (FIG. 1D) cells quantified were counted using three non-contiguous sections of SLG (n-5) from adult female animals. Data are presented as mean ± SD.

FIGS. 2A-2D show that SOX2 and SOX2⁺The cells are essential for the supplementation of salivary gland acinar cells. FIGS. 2A and 2B Sox2^CreERT2；Sox2^f1/f1；Rosa26^mTmG/+Mouse (FIG. 2A; see schematic) or Sox2^CreERT2Rosa26^DTA；Rosa26^mTmG/+Mouse (FIG. 2B; see schematic) Sox2 or SOx2 in SLG⁺And (4) cell ablation. Sections were subjected to AQP5, KRT8 or ECAD and nuclear immunostaining. Scale bar 50 m. The white dotted line outlines the catheter. n-3 (per genotype). FIG. 2C, 2D Sox2^CreERT2；Sox2^f1/f1(FIG. 2C) or Sox2^CreERT2Rosa26^DTA；Rosa26^mTmG/+Catheter area in SLG (FIG. 2D)Quantification of (expressed as a percentage of total epithelial area). In (FIG. 2C), right panel, Sox2^CreERT2；Sox2^f1/f1SOX2 in SLG⁺Number of cells (expressed as a percentage of total cell number). In the right panel (FIG. 2D), for wild type and Sox2^CreERT2Rosa26^DTA；Rosa26^mTmG/+KRT8 in mice⁺Catheter, AQP5⁺And SOX2⁺The total number of acinar cells was counted. Data information: the number of cells/area of vessels in three non-contiguous fluorescent sections per SLG taken from 3 mice/genotype were calculated. The data (n ═ 3) in (fig. 2C and 2D) are mean ± SD and have been analyzed by Student's t-test. In (fig. 2C), P is 0.011, P is 0.0041, and in (fig. 2D), left panel, P is 0.0015; right figure, P ═ 0.0007, P ═ 0.0018.

FIGS. 3A-3H show parasympathetic maintenance of SOX2⁺Cells and promotion of SOX 2-mediated acinar cell replacement. Figure 3A is a schematic showing the time course and tympanostomy location of tympanostomy (CT) denervation in adult mice. Figure 3B gene expression (qPCR) analysis of SLG intact (uninjured contralateral glands) and transected to nerves 7 and 30 days post-surgery (D7 or D30). Gene expression was normalized to Rsp18 and the full control for each time point. Figures 3C-3F denervation 7 days later, control and transected SLGs were immunostained for nerves (GFRa2), acinar cells (AQP5 and miss 1), ductal cells (KRT8), and Epithelial Cells (ECAD). SOX2 in SLG vs control and Cross-section⁺、AQP5⁺、MIST1⁺、KRT8⁺And KRT5⁺The number of cells was counted and expressed as a percentage of the number of cells in the control SLG (fig. 3F). Scale bar 25m in (fig. 3C, 3D and 3E). FIGS. 3G, 3H Prior to TUBB3 immunostaining at Sox2^CreERT2；Rosa^26mTmGRecombination was induced in mice (3 days after nerve transection) and SLG, followed for 11 days. GFP in control and transected glands⁺And mT⁺See figure 3G for the percentage of acinar cells. Scale bar 25m in (fig. 3H). Data information: the data (n-5) in (fig. 3B) are mean ± SEM and have been passed one-way anova and post hoc Dunnett's testAnd (6) carrying out analysis. Sox2(D7) × P ═ 0.0455, Tubb3(D7) × P ═ 0.0082, Tubb3(D30) × P ═ 0.0091, Vip (D7) ═ 0.0098, Vip (D30) ═ 0.0063, Vacht (D7) ═ P ═ 0.0071, Muc19(D7) × P ═ 0.0419, and Aqp5(D7) ═ P0.0468. The data in (fig. 3F and 3G) were calculated from the results associated with three non-contiguous fluorescent slices taken from each SLG of n-5 mice/group or genotype, are mean ± SD, and have been analyzed by Student's t-test. SOX2⁺*P＝0.0197，AQP5⁺*P＝0.0106，％GFP⁺***P＝0.0000096，％mT⁺***P＝0.0000096。

FIGS. 4A-4D show that muscarinic signaling promotes SOX2⁺And (5) cell proliferation. FIG. 4A shows SOX2, CHRM3 and nuclear immunostaining of SLG in adult animals. The images are 6m (left) and 1m (right) projections of 1m and 0.175m confocal slices. Scale bar 10 m. FIG. 4B flow cytometry on epithelial SOX2⁺Cell (CHRM 1)⁺Or CHRM3⁺) The percentage of the total EpCAM was counted and expressed as⁺SOX2⁺Percentage of cells. Figures 4C, 4D adult SLG from mice receiving pilocarpine or saline (control) administration were subjected to SOX2, Ki67 and nuclear immunostaining. White arrows indicate proliferating SOX2⁺(SOX2⁺Ki67⁺) Cells (fig. 4C). Scale bar 20 m. (FIG. 4D) SOX2 after receiving pilocarpine administration⁺And SOX2⁺Ki67⁺Fold change of cells (unit:%). Data information: (fig. 4B) SLG-related data were compiled from n-3 mice (10,000 events). The data in (fig. 4D) were calculated from the results obtained with respect to three non-consecutive fluorescence sections from each SLG of n-4 (saline) or n-5 (pilocarpine) mice, are mean ± SD, and have been analyzed by Student's t-test. P-0.0487.

Figures 5A-5E show that Sox2 is essential for SLG regeneration after radiation injury. FIGS. 5A-5D were taken from wild type (FIG. 5A) and Sox2^CreERT2；Rosa2^6mTmG(FIG. 5D) representative images of control (0 Gy; non-IR) and irradiated (10 Gy; IR) SLG of mice and analyzed after 1, 3 and 14 days. (FIGS. 5A-5C) nerves to SLG (TUBB3), SOX2, CyclinD1(CCND1)And nuclear staining (fig. 5A, 5C), and calculating the nerve density (fig. 5B). (FIG. 5C) quantifying SOX2⁺And CCND1⁺The number of cells. (FIG. 5D) following Irradiation (IR), Sox2 was observed with trace^CreERT2；Rosa26^mTmGMice were immunostained for 14 days with SOX2 (fig. 5D, red). White arrows indicate SOX2 negative progeny. The scale bar in (FIGS. 5A and 5D) is 50 m. FIG. 5E Sox2^CreERT2；Sox2^f1/f1Mice and wild type littermates received 10Gy of IR irradiation and SLG was analyzed 13 days later. SLG was subjected to SOX2, AQP5 and nuclear immunostaining. Scale bar 50 m. Data information: the data in (fig. 5B) are mean ± SD, where n is 3, and the values are plotted. The data in (fig. 5C) was calculated from the results of correlation of three non-consecutive fluorescence sections taken from each SLG of 3 mice/treatment protocol, is the mean ± SD, and has been analyzed by one-way anova and post hoc Dunnett's test. SOX2⁺(D1)*P＝0.0487，SOX2⁺CCND1⁺(D3)*P＝0.318，SOX2⁺CCND1⁺(D7)*P＝0.0291。

FIGS. 6A-6C show that SOX2⁺Progenitor cells can recruit acinar cells following radiation-induced injury in response to muscarinic stimulation. FIG. 6A Collection from Sox2 after a single 15Gy (dose) IR^CreERT2；Rosa26^mTmGSLG from mice, explants were cultured for 0-48 hours. The schematic shows the timing of recombination, culture and analysis. Figure 6B representative image of lineage tracing explants of nuclear immunostaining. The scale bar is 50 m. FIG. 6C GFP⁺Quantification of cell number. Data information: the data in (fig. 6C) were calculated from three random areas of n-3 immunostained explants per treatment protocol, and individual values were plotted and data analysis was performed by one-way anova and post hoc Dunnett's test. Error bars represent mean ± SD. non-IR and IR × P ═ 0.0022, IR and IR + CCh × P ═ 0.0058, IR and IR +4-DAMP × P ═ 0.0010.

Fig. 7A-7E show that acetylcholine/muscarinic signaling can maintain SOX2 and acinar lineages in human SG. FIG. 7A qPCR was performed on human salivary glands obtained from healthy individuals (non-IR; inframandibular) or patients receiving radiation therapy for head and neck cancer (IR). Figures 7B-7D human SMG explants were cultured for 7 days with murine parasympathetic ganglia (nerves) or E13 Mesenchyme (MES) at 13 days of embryonic age (E). Explants were analyzed by immunostaining with neural (fig. 7B, TUBB3) or cell proliferation (fig. 7C, Ki67) markers or by qPCR (fig. 7D). Scale bar 50m (fig. 7B, 7C). Figure 7E qPCR analysis of adult salivary gland (SMG or PG) explants from four different individuals cultured for 4 hours (± CCh, 200nM, n-4). See fig. 14D for individual data sets. Data information: the data n in (fig. 7A) is 11 (non-IR), and n is 7 (IR); 30-85 years old. The data were normalized to GAPDH and the individual values were plotted and analyzed by Student's t-test and set to a false discovery rate of 0.05. Error bars represent mean ± SD. AQP 3P is 0.017, MIST 1P is 0.086, AMY 1P is 0.005, SOX 2P is 0.079, CHRM 1P is 0.040, CHRM 3P is 0.011, EGFR P is 0.931, KRT 19P is 0.618. The data in (fig. 7D) are mean ± SD of 2 individuals, with solid bars representing individual 1 and dashed bars representing individual 2. Data were normalized to salivary glands (control, black dashed) from the same individuals cultured with murine mesenchyme. The data in (fig. 7E) is a boxplot of n-4 different individual correlation results showing the mean (horizontal line), upper and lower quartiles (boxes), and upper and lower limits (box whiskers). Data were normalized to untreated controls (black dashed line). n-5-8 explants per individual. Data analysis was performed by one-way analysis of variance and post hoc Dunnett's test. SOX 2P 0.00834, CHRM 3P 0.0449, CHRM 1P 0.0093, AQP 5P 0.0069, AQP 3P 0.0375, MIST 1P 0.0379, CD 44P 0.0485, KRT 19P 0.0461

FIGS. 8A-8B show that SOX2⁺Short-term ablation of cells results in reduced acinar cell replacement. (FIG. 8A) Sox2^CreERT2；Sox2^fl/^fl；Rosa26^mTmG/+SOX2 in mouse SLG⁺Cells were ablated for more than 3 days and SLG was analyzed on day 4 (see schematic). For Sox2^CreERT2；Sox2fl/fl；Rosa26^mTmG/+SLG sections were immunostained for SOX2 and myoepithelial cells (alpha-smooth muscle actin; SMA). Scale bar 50 μm (fig. 8B) Sox2^CreERT2Rosa26^DTA；Rosa26^mTmG/+SOX2 in mouse SLG⁺Cells were ablated for more than 4 days and SLG was analyzed on day 5 (see schematic). For Sox2^CreERT2Rosa26^DTASLG sections were immunostained with SOX2 and acinar cells (aquaporin 5; AQP5) or subjected to qPCR. Scale bar 50 μm. Data information: data in fig. 8B were normalized to Gapdh and wild-type controls. Data are mean ± s.d. (n-1, 3 technical replicates).

FIGS. 9A-9C show that 30 days post tympanostomy innervation allowed restoration of acinar lineages (FIGS. 9A-9C) SLG was immunostained with SOX2, TUBB3, AQP5, MIST1 and ECAD (FIG. 9A) and SOX2 was measured 30 days post-denervation⁺、AQP5⁺、MIST1⁺And KRT8⁺Cell number (fig. 9B) and acinar cell size (fig. 9C). n-5 mice/time point/condition. Cells were counted in 3-4 fields per animal. Data information: the data n in fig. 9B and 9C is 5. The data in fig. 9B are mean ± s.e.m. and have been analyzed by one-way anova, and the data in fig. 9C are boxplots of n-5 mouse-related results showing mean ± s.e.m. and have been analyzed by students t test. P is 0.0498.

Figures 10A-10F show that SOX2 marks a subset of acinar cells that complement acini. FIG. 10A wild type murine PG subjected to SOX2, ECAD and nuclear staining. The scale bar is 50 m. FIG. 10B treatment of acinus SOX2 using FACS⁺And SOX 2-cells (Ki 67)⁺) The percentage of total AQP5 was counted and expressed as⁺SOX2⁺Or AQP5⁺SOX2^-Percentage of cells. FIG. 10CSOX2⁺Acinar cells (CyclinD 1)⁺Or CyclinD1^-) Percentage (%) of the total amount of the components. FIG. 10D Rosa26^mTmGA schematic representation of Cre-mediated gene deletion (adapted from Muzumdar et al, 2007). Fig. 10E Sox2 pedigree traces representative images of SLG. Immunostaining by SOX2 at Sox2^CreERT2；Rosa26^mTmGCre-mediated recombination was induced in mice and SLG and analyzed 14 or 30 days later. Scale bar 25 m. FIG. 10F Kit lineage traces representative images of SLG and SMG. In Kit^CreERT2；Rosa26^mTmGCre-mediated recombination was induced in mice and SMG/SLG and analyzed after 14 days and 6 months, respectively. Tissues were stained with AQP5 to label acinar cells and KRT8 to label the inserted ductal cells. Scale bar 25 m. mT ═ membrane bound Tomato. Data information: (fig. 10B), SLG-related data were compiled from 2 mice (85,000 events). The data in (fig. 10C) were calculated from the results of correlation of three non-contiguous fluorescence slices taken from each SLG of 3 mice, and the values plotted.

Error bars represent mean ± SD.

FIGS. 11A-11D show that despite the presence of nerves, Sox2 or Sox2⁺Ablation of the cells still results in reduced acinar cell replacement. FIGS. 11A-11C Sox2^CreERT2；Sox2^fl/fl；Rosa26^mTmG/+Mouse (FIG. 2A; see schematic) or Sox2^CreERT2；Rosa26^DTA；Rosa26^mTmG/+Mouse (FIG. 2B; see schematic) Sox2 or SOx2 in SLG⁺And (4) cell ablation. (FIGS. 11A and 11B) for WT, Sox2^CreERT2；Sox2^fl/flAnd Sox2^CreERT2；Rosa26^DTASLG sections were subjected to SOX2 or TUBB3 and nuclear immunostaining. White arrows indicate SOX2⁺A cell. The white dashed square in the right image is enlarged to highlight the remaining SOX2 in the tissue⁺Fewer cells and non-nuclear (green) staining indicated the presence of debris. Scale bar 50 m. (FIG. 11C) raw integrated density of the nerves was calculated using ImageJ. FIG. 11D WT or Sox2 with cyclin D1(CCND1) and nuclear immunostaining^CreERT2；Sox2^fl/flAnd (7) SLG. Dotted line ═ catheter; arrow CCND1⁺Acinar cells. Scale bar 50 m. Data information: (fig. 11C), WT n is 4, Sox2^fl/fln is 4 and DTA n is 3. Individual values are plotted, expressed as mean ± SD, and data analysis has been performed by one-way anova and post hoc Dunnett's test. P ═ 0.0091. The data in (fig. 11D) are representative images from n-4 mice.

Figures 12A-12H show that tympanostomy allows acinar cells to be depleted within 7 days. (FIGS. 12A, 12C, 12D, 12G) 7 days after denervation, onControl and nerve transected SLG were subjected to tyrosine hydroxylase (TH; FIG. 12A), SOX2 and TUBB3 (FIG. 12C), KRT5 (FIG. 12D), caspase-3 (CASP 3; FIG. 12G), Epithelial Cell (ECAD) and nuclear immunostaining. Acinar cell size was quantified in adult Wild Type (WT) mouse SLGs with intact or transected tympanostomy (CT) 7 days after denervation (fig. 12B). The scale bar in fig. 12A, 12C, 12D, and 12G is 25 m. (FIGS. 12E, 12F) before TUBB3 immunostaining, Sox2^CreERT2；Rosa26^mTmGRecombination was induced in mice (24 hours before nerve transection) and SLG, followed for 15 days. GFP in control and transected glands⁺And mT⁺See figure 12F for the percentage of acinar cells. The scale bar in (fig. 12E) is 25 m. (H) Fold-change in gene expression involved in cell cycle and apoptosis 7 days after denervation compared to the intact control. The dashed line represents the complete control. Data information: data n in (fig. 12B, 12F, and 12H) is 5. The data in (fig. 12B) is a boxplot of n-5 mouse-related results showing mean (horizontal line), upper and lower quartiles (boxes), and upper and lower limits (boxlines), and has been analyzed by Student's t-test. P0.00000347. The data in (fig. 12F) are mean ± SD and have been analyzed by Student's t-test. % GFP⁺***P＝0.0000208，％mT⁺P0.0000208. Data in (fig. 12H) were normalized to Rsp18 and the full control (dashed line). Ccnd1 × P is 0.0477.

FIGS. 13A-13E show that IR can induce cell damage and nerve loss, and SOX2 and SOX2⁺Progenitor cells can recruit mouse acinar cells in response to cholinergic mimetics. FIGS. 13A, 13B transcriptional changes of murine SLG were analyzed by qPCR on days 0, 1, 3, and 7 after 10Gy IR. FIGS. 13C-13E were cultured ex vivo for 48 hours and subjected to smooth muscle actin (SMA, FIG. 13C), SOX2 (FIG. 13D) or Ki67 (FIG. 13E) and nuclear immunostaining with Sox2^CreERT2；Rosa26^mTmGMurine SLG explants. Recombination was induced 24 hours before harvesting SLG for culture. Scale bar 50 m. Data information: the data in (fig. 13A and 13B) were normalized to Rsp29 and day 0 controls. The data (n-3/time point) in (figures 13A and 13B) are mean ± SEM,and has been analyzed by one-way analysis of variance and post hoc Dunnett's test. Bax (D1) P ═ 0.00826, Bax (D3) P ═ 0.0871, Pmaip 1(D1) P ═ 0.0369, P1 (D1) P ═ 1, P1 (D1) P ═ 0.0337, 1(D1) P ═ 1, Sox 1(D1) P ═ 0.0072, Sox 1(D1) P ═ 0.0418, Aqp 1(D1) P ═ 1, mi3672 (D1) P ═ 1, Tubb 72 (D1) P ═ 36040.040, tubbp ═ 1(D1) P ═ 1, tubbp ═ 1 (1). The images in (fig. 13C, 13D and 13E) represent three experiments, with n being 3 SLG fragments per experiment.

Figures 14A-14D show that muscarinic activation is sufficient to increase SOX2 expression and acinar lineage in the salivary glands of human adults. FIG. 14A qPCR was performed on human salivary glands obtained from healthy individuals (non-IR; inframandibular) or patients receiving radiation therapy for head and neck cancer (IR). FIG. 14B representative images of adult (h) salivary glands (non-IR, 22-31 years old; SMG) immunostained for endogenous SOX10, MIST1, EGFR, CD44, KRT7, AQP3, KRT5, ECAD, and nuclei. Single arrows indicate acinar and ductal cells expressing SOX 10. Scale bar 50 m. FIG. 14C Ki67 in neural coculture⁺Quantification of cell number (representative image shown in fig. 7C). Figure 14D adult salivary gland explants (SMG or PG) from four individual patients (healthy, non-IR) were cultured for 4 hours (± 200 nMCCh). See fig. 7E for summary data. Data information: the data n in (fig. 14A) is 11 (non-IR), and n is 7 (IR); 30-85 years old. The data were normalized to GAPDH and the individual values were plotted and analyzed by Student's t-test and set to a false discovery rate of 0.05. Error bars represent mean ± SD. TUBB 3P ═ 0.346, VIP P ═ 0.461, GFRA P ═ 0.002, and TH P ═ 0.433. The data in (fig. 14C) are n-3 and mean ± SEM, and have been analyzed by Student's t-test. P ═ 0.0151. Data in (figure 14D) (n ═ 4 individual subjects) were normalized to GAPDH expression and salivary glands from the same subjects cultured with cultures without CCh and subjected to three replicates (control; black dashed line) and expressed as mean ± SD.

FIGS. 15A-15B show that intraglandular injection of cevimeline promotes the proliferation of acinar cells in the salivary glands of mice. Mice were injected intraglandular with Cevimeline (CV) or saline (S, control). Mice were sacrificed 18 hours later and proliferating cells taken from tissue sections (3 sections/gland, 3 mice/treatment regimen) were quantified. Scale bar 60 μm (representative image shown in fig. 15A; comparison graph of percentage (%) of acinar cells under the administration of cevimeline and the administration of physiological saline shown in fig. 15B).

FIGS. 16A-16D show the physical degradation of Oxidized Alginate (OA) hydrogels. (FIG. 16A) Effect of oxidation on physical degradation, wt% was kept constant at 5 wt%. (FIG. 16B) Effect of wt% on physical degradation, OA% was kept constant at 2% OA. (FIG. 16C) in vitro degradation of 2% OA at 5 wt% and in vivo degradation. (FIG. 16D) stability of 2% OA (5 wt%) under refrigerated (4 ℃) and frozen (-20 ℃) conditions.

A-17DA-17D demonstrates the release of cevimeline in an Oxidized Alginate (OA) hydrogel. (FIG. 17A) Effect of oxidation on the release of cevimeline, the wt% was kept constant at 5 wt%. (FIG. 17B) Effect of wt% on cevimeline, OA% remained unchanged at 2% OA. (FIG. 17C) Effect of increasing initial drug loading of cevimeline on the release kinetics of 2% OA hydrogel at 5 wt%. (FIG. 17D) Wevimeline release in 2% OA hydrogel (5 wt%) with different initial concentration of Wevimeline at a time.

FIGS. 18A-18C show that cevimeline release from most alginates promotes glandular cell proliferation for 3 days. Alginate (ALG, 5% WT: 2% OA) or alginate + cevimeline (ALG + CV) was injected immediately adjacent to the salivary glands (fig. 18A). The red circle highlights the alginate after injection. Mice were sacrificed on day 3 and proliferating Ki67+ and EdU + cells quantified (fig. 18B and 18C, 3 slices/gland, 3-4 mice/treatment regimen). Students t test compares + CV to ALG control, p < 0.05. Scale bar 40 μm.

Figures 19A-19E show that alginate (with or without cevimeline) promotes a pro-restorative inflammatory response 7 days after injection. Alginate (ALG, 5% WT: 2% OA) or alginate + cevimeline (ALG + CV) was injected next to the salivary glands. Mice were sacrificed on day 7. Salivary gland tissue sections were immunostained for CD3+ T cells (fig. 19A and 19B), CD68+ macrophages (fig. 19C and 19D), or CD206+ repair macrophages (fig. 19E) and nuclei. Macrophage density was quantified and compared to saline-injected animals of the same age (i.p.) or 22-month old mice (3 slices/gland, 3-4 mice/treatment regimen). One-way ANOVA assay compares + CV to ALG controls and controls, p < 0.05, p < 0.01, p < 0.00001. Scale bar 40 μm.

FIGS. 20A-20F show that cevimeline promotes acinar cell proliferation after radiation-induced injury. (FIG. 20A) schematic representation of radiation therapy. (FIG. 20B) citrate stimulated salivation decreased in C57/BL6 female mice at 6-8 weeks of age 13 days after receiving radiation therapy at a dose of 10 Gy. (FIGS. 20C-20F) after 14 days, animals were injected with saline or cevimeline 10mg/kg either intraperitoneally (FIGS. 20B and 20D) or intraglandularly (FIGS. 20C and 20D). At 18 hours post injection, animals were euthanized for tissue processing and immunofluorescence detection. Proliferation and SOX2+ cells were quantified in tissue sections (3 sections per animal).

Figures 21A-21D show that treatment of irradiated salivary glands with alginate + cevimeline promotes acinar cell proliferation and improves salivary flow. Figure 21A shows a timeline diagram of mice receiving dosing. Mice were injected with alginate or alginate + cevimeline 14 days after radiation treatment, and then sacrificed for proliferation analysis on day 3. Figure 21B shows that alginate + cevimeline resulted in an increase in the corresponding proliferation rate relative to the level of proliferation rate obtained with alginate alone. Figure 21C compares the percentage of epithelial cells treated with alginate + cevimeline to alginate alone. Figure 21D shows saliva related data measured 13 days after radiation treatment, 1 day before and 7 days after subcutaneous injection of saline, alginate or alginate + cevimeline. Two-way ANOVA, no RM, for time parameters, p ═ 0.0244.

Figure 22 shows that proliferation did not increase with increasing cevimeline. Alginate containing 10mg or 25mg/kg was delivered to mice and proliferating (Ki67+) acinar cells were quantified on day 3.

Figure 23 shows that alginate can promote acinar cell proliferation.

Figure 24 shows that alginate (with or without cevimeline) does not negatively impact mouse health.

FIG. 25 shows analysis of alginate oxidation efficiency¹³C Nuclear Magnetic Resonance (NMR) spectrum. D4 sodium 3- (trimethylsilyl) propionate (0.05 w/v%) was used as an internal standard.

Figure 26 shows a pair of syringes for producing an injectable hydrogel. A syringe containing the calcium cross-linked solution used to produce the hydrogel is connected by luer lock to a second syringe containing a solution of alginate and neurotransmitter mimics (e.g., cholinergic agonists and/or muscarinic agonists).

Figure 27 shows the location of canine and human salivary glands. 1.5ml to 2ml of alginate hydrogel can be injected directly into the submandibular gland of a human or canine subject.

Figure 28 shows that the salivary glands are similar in dogs and humans. The parotid and sublingual glands appear to be most similar in dogs and humans. The submandibular gland of a dog is similar to a human, as it is a mixed cell gland.

Figure 29 shows the injection of alginate hydrogel into the submandibular gland of dogs.

Detailed description of the preferred embodiments

The present invention provides compositions, methods and kits for regenerating salivary glands by promoting acinar cell replacement. The inventors have shown that progenitor cells in adult salivary glands (including SOX2)⁺Acinar progenitor cells) are critical for the recruitment of acinar cells, with the unexpected ability to repopulate tissue following radiation-induced injury (example 1). The inventors have also shown that cholinergic nerves play a crucial role in controlling acinar cell replacement during homeostasis, and that this neuronal effect can be replicated by adding cholinergic mimetics to acinar progenitor cells. Thus, direct targeting of progenitor cells within tissues with cholinergic and/or muscarinic agonists (including SOX2)⁺Acinar progenitor cells) that regenerate the secretory unit of the salivary gland, allowing functional salivary gland acinus to recover and treat oral diseases such as dry mouth following radiation therapy or dry mouth associated with sjogren's syndrome. In particular, formulations comprising a muscarinic agonist (e.g., cevimeline) encapsulated in an alginate hydrogel may be formulatedFor topical application to salivary glands and for the treatment of dry mouth (example 2).

Before the present compositions, methods, and kits are described, it is to be understood that this invention is not limited to the particular methodology or compositions described, as such may, of course, vary from practice. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the inventive concept, the scope of which will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no such intervening value, to the extent that there is provided a range of values, all of which is encompassed within the scope of the invention. Unless the context clearly dictates otherwise, each intermediate value should be as low as one tenth of the unit of the lower limit. The invention extends to each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described below. All publications mentioned in this patent are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be understood that, in case of conflict, the present disclosure should replace any disclosure in the cited publications.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and listed herein has layered components and features that may be readily separated or combined with the features of any of the other several embodiments without departing from the scope and spirit of the present disclosure. Any recited method may be implemented in the order of events recited or in any other order that is logically possible.

It must be noted that, as used in this patent and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and "the agonist" refers to one or more agonists and equivalents known to those skilled in the art, such as ligands or activators, and the like.

The publications discussed in this patent are provided solely for their disclosure prior to the filing date of the present patent. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definition of

The term "about," especially when referring to a given quantity, is intended to encompass deviations of plus or minus five percent.

The term "agent" refers to all substances that can be used in the preparation of pharmaceutical and diagnostic compositions, or can be all substances belonging to the class of compounds (e.g., small synthetic or naturally derived organic compounds), nucleic acids, polypeptides, antibodies, fragments, isoforms, variants, or other substances that can be used independently for these purposes, all of which are within the scope of the present invention.

The term "agonist" refers to a substance that binds to a specific receptor and triggers a cellular response. It mimics the action of an endogenous ligand (e.g., a hormone or neurotransmitter) that binds to the same receptor. A "full agonist" binds to and activates a (affinity) receptor and shows full efficacy at that receptor. One example of a drug that is a full agonist is isoproterenol, which mimics the effect of acetylcholine at beta adrenergic receptors. "partial agonists" (e.g., buspirone, aripiprazole, buprenorphine, or norclozapine) also bind to and activate a given receptor, but only produce partial efficacy at the receptor relative to full agonists.

"partial agonist" may also be considered to be a ligand that exhibits agonistic and antagonistic effects-when both full agonist and partial agonist are present, the partial agonist acts in effect as a competitive antagonist, competing with the full agonist for receptor occupancy, such that a net reduction in receptor activation is observed when the full agonist is used alone. "Co-agonists" together with other co-agonists produce the desired effect. Antagonists prevent the receptor from being activated by the agonist. Receptors can be activated or deactivated by endogenous (e.g., hormones and neurotransmitters) or exogenous (e.g., drugs) agonists and antagonists, thereby stimulating or inhibiting biological responses. Depending on the pathway of effect, the ligand may act as both an agonist and an antagonist of the same receptor.

The efficacy of an agonist is generally determined by its EC₅₀And (4) value limitation. For a given agonist, this can be calculated by determining the concentration of agonist required to elicit half of the maximum biological response of the agonist. EC (EC)₅₀The values can be used to compare the efficacy of drugs with similar efficacy to produce physiologically similar effects. EC (EC)₅₀The smaller the value, the greater the potency of the agonist, and the lower the concentration of drug required to elicit the maximum biological response.

As used herein, "biocompatible matrix" refers to a substance suitable for implantation in a subject onto which a population of cells can be deposited, which can be used to encapsulate one or more cholinergic and/or muscarinic agonists. Once implanted in a subject, the biocompatible matrix does not cause toxic or deleterious effects. In one embodiment, the biocompatible matrix is a polymer whose surface can be fashioned into a desired structure requiring repair or replacement. The polymer may also be molded as part of a structure that requires repair or replacement. In another embodiment, the biocompatible matrix is linearly deformed to fill the salivary gland and distribute throughout the gland. The biocompatible matrix may provide sustained release of one or more cholinergic and/or muscarinic agonists, and may also provide a supportive framework that allows cells to attach to and grow on it. In some embodiments, the cultured cell population is grown on a biocompatible substrate that provides the appropriate gap distance required for cell-cell interaction

The term "differentiation" or "transdifferentiation" as used herein generally refers to the process of differentiation of a precursor or progenitor cell into a particular cell type. The term may refer to the process by which acinar progenitor cells (e.g., acinar progenitor cells expressing SOX2) become differentiated acinar cells. Differentiated cells can be identified by their gene expression pattern and cell surface protein expression. The term "differentiated" as used herein refers to having a characteristic or function that is different from the original type of tissue or cell. Thus, "differentiation" refers to the process or behavior of differentiation.

The term "modulate" refers to the ability of a response to be up-regulated (i.e., activated or stimulated), down-regulated (i.e., inhibited or prevented), or a combination or separation of the two, e.g., altered by up-or down-regulating the activity of a protein, nucleic acid encoding a protein, pathway, protein within a pathway, etc.

The phrase "oral tissue cells" refers to any population of cells derived from the oral cavity. These include one or more different cell types that can be isolated from salivary glands, submandibular glands, sublingual glands, lingual glands, labial glands, buccal glands, palatine glands, striated ducts, drainage ducts, dental pulp tissue, dentin, periodontal tissue, bone, cementum, gingival submucosa, oral submucosa, tongue, and taste bud tissue. In a preferred embodiment, the oral tissue cells are derived from salivary glands. Examples of oral tissue cells include, but are not limited to, myoepithelial cells, epithelial cells, and the like.

The phrase "oral tissue" refers to any collection of cells that form a structure in the oral cavity. For example, oral tissue includes salivary glands, submandibular glands, sublingual glands, lingual glands, labial glands, buccal glands, palatine glands, striated ducts, drainage ducts, dental pulp tissue, dentin, periodontal tissue, bone, cementum, gingival submucosa, oral submucosa, tongue, and taste bud tissue. In a preferred embodiment, the oral tissue is salivary glands. The phrase also refers to a portion of the oral tissue, such as a portion of the salivary gland.

The phrase "oral tissue construct" refers to a substrate, preferably a biocompatible substrate that has been seeded with oral tissue cells, wherein the cells have attached and packed into the biocompatible substrate and grown, proliferated, and differentiated on the substrate. The phrase also refers to newly formed structures that represent early stages of oral tissue development.

The phrase "salivary gland construct" refers to a substrate, preferably a biocompatible substrate that has been seeded with salivary gland cells, wherein the cells have attached and packed into and grown, proliferated, and differentiated on the biocompatible substrate. The phrase also refers to newly formed structures that represent early stages of salivary gland development.

The phrase "oral disease" refers to a disease or condition affecting the oral cavity. Especially diseases or conditions affecting saliva production. Examples of oral diseases include, but are not limited to, salivary gland tumors, cystic fibrosis, sjogren's syndrome, sialadenitis, mumps, siallitis, sialolithiasis, salivary gland stones, sialopholithiasis, mucocysts, sublingual cysts, hyposecretion, sialorrhoea, sialorrhea, xerostomia, benign lymphatic epithelial lesions of salivary glands, salivary gland dilatation, salivary gland enlargement, salivary duct stenosis, and salivary duct stenosis.

The term "treatment" refers to therapeutic, prophylactic and preventative treatment, especially to the administration of a drug or the performance of a medical procedure to a patient to prophylactically treat (prevent) or cure or reduce the extent or likelihood of the occurrence of a disorder or disease or condition or event. Treatment regimens employing the agents described herein can be provided to stimulate or promote salivary gland regeneration and/or acinar cell replacement in the salivary glands.

The terms "individual", "subject" and "patient" are used interchangeably herein and refer to any member of the subfamily chordata, including but not limited to humans and other primates, including non-human primates, such as chimpanzees and other apes, and monkey species; livestock, such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs; birds, including poultry, wild birds and game birds, such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. In some cases, the methods of the invention can be used in laboratory animals, veterinary applications, and development of animal disease models, including but not limited to rodents, including mice, rats, and hamsters; primates and transgenic animals.

A "therapeutically effective dose" or "therapeutic dose" refers to an amount sufficient to achieve a desired clinical result (i.e., to achieve a therapeutic effect). A therapeutically effective dose may be administered in one or more administrations.

A "therapeutically effective dose or amount" of a composition comprising a cholinergic agonist (e.g., acetylcholine or carbachol) and/or a muscarinic agonist (e.g., cevimeline encapsulated in a hydrogel) is intended to produce a positive therapeutic response, e.g., improved recovery from dry mouth, when administered according to the methods described herein. Improving recovery can include improving salivary gland function and increasing salivary production and salivary flow. In addition, a therapeutically effective dose or amount can stimulate proliferation of acinar cells and acinar cell progenitors, resulting in re-proliferation of salivary glands and acinar cells. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug used, the mode of administration, and the like. Based on the information provided herein, one of ordinary skill in the art can determine an appropriate "effective" amount in any individual case by routine experimentation. A therapeutically effective dose may be administered in one or more administrations.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar untoward reactions (e.g., gastrointestinal upset, dizziness, etc.) when administered to humans (or, in the case of veterinary use, non-human animals). Preferably, the term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Aqueous or aqueous solutions saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in e.w. martin, leimington pharmaceutical university.

"pharmaceutically acceptable excipient" refers to an excipient that can be used to prepare safe, non-toxic, intended pharmaceutical compositions, including excipients acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or in the case of aerosol compositions, gaseous.

"pharmaceutically acceptable salts" include, but are not limited to, amino acids, salts prepared with inorganic acids (e.g., chloride, sulfate, phosphate, diphosphate, bromide, and nitrate), or salts prepared with the corresponding inorganic acid forms of any of the foregoing (e.g., hydrochloride, and the like), or salts prepared with organic acids (e.g., malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, p-toluenesulfonate, pamoate, salicylate, and stearate, as well as propionate laureate, glucoheptonate, and lactobionate). Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

The term "contrast agent" as used herein refers to a substance used in medical imaging to enhance the contrast of a target tissue, fluid or structure (e.g., salivary glands) in a subject. Exemplary contrast agents include, but are not limited to, ultrasound contrast agents (e.g., SonoVue microbubbles comprising sulfur hexafluoride, Optison microbubbles comprising an albumin shell and an octafluoropropane gas core, Levovist microbubbles comprising a lipid/galactose shell and a gas core, perflex lipid microspheres comprising perfluorocarbon microbubbles, Perflutren lipid microspheres comprising octafluoropropane encapsulated in a lipid shell), Magnetic Resonance Imaging (MRI) contrast agents (e.g., gadodiamide, gadobenic acid, gadopentetic acid, gadoteridol, gadofovir, gadofosaride, gadoxerucic acid), and radiological contrast agents, e.g., contrast agents for Computed Tomography (CT), radiography, or fluoroscopy (e.g., diatrizoic acid, metrizoate, iodoxamide, iothalate, ioglatiramerate, ioxydizonic acid, ioxybenzoic acid, iocatylic acid, iodomesulfone, ioxovone, metrizamide, iohexol, ioxaglicol, ioxol, ioxofenac, iod, Iopromide, iotrolon, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iosalic acid, iotoxicam acid, iodicacid, iodipamoic acid, iobenzamic acid, iopanoic acid, iosetamic acid, sodium iophosphate, iodobenzylbutyric acid, and calcium iophosphate).

Regeneration of salivary glands by promoting acinar cell replacement

The present invention provides compositions and methods for regenerating salivary glands by promoting acinar cell replacement. Salivary gland regeneration or possible permanent relief of dry mouth. The autonomic nervous system innervates salivary gland acinus, myoepithelial cells, vascular cells and inserted ductal cells. Salivary gland acinar cells secrete most of the fluids, electrolytes and proteins in saliva. Neurotransmitter receptors are present on the basolateral membrane of acinar cells, which have alpha-adrenergic, beta-adrenergic, M3 muscarinic and cholinergic substance P receptors. The major pathway of protein exocytosis occurs through activation of adrenergic receptors (sympathetic pathways), while the major stimulation of fluid secretion occurs through activation of M3 muscarinic receptors (parasympathetic pathways).

As a result, it was found that progenitor cells (including SOX2) in adult salivary glands⁺Acinar progenitor cells) are critical for acinar recruitment, with the unexpected ability to repopulate tissue following radiation-induced injury. It was also found that during homeostasis, cholinergic nerves play a crucial role in controlling acinar cell replacement, and that this neuronal effect can be replicated by adding cholinergic mimetics to acinar progenitor cells. Thus, direct targeting of acinar progenitor cells within tissue with cholinergic and/or muscarinic agonists, or cholinergic agonistsAnd/or the isolation and expansion of acinar progenitor cells by muscarinic agonists for transplantation and activation, allows regeneration of salivary gland secretory units, thereby allowing functional salivary gland acinus to recover and treat oral diseases, such as dry mouth following radiation therapy or dry mouth associated with sjogren's syndrome. Given that organs such as the gut, glandular stomach, trachea and taste buds expressing SOX2 are heavily innervated by the autonomic nervous system and damaged by therapeutic radiotherapy used to eliminate cancer, such strategies may be applicable to repair multiple organ systems.

In some embodiments, a method for promoting regeneration of salivary glands in a subject in need thereof comprises administering at least one of a cholinergic agonist or a muscarinic agonist to acinar progenitor cells of the salivary glands to promote acinar cell regeneration. The cholinergic agonist and/or muscarinic agonist may mimic parasympathetic acetylcholine/muscarinic signaling demonstrated to maintain acini in human salivary glands. In some embodiments, one or more cholinergic agonists and/or muscarinic agonists are administered topically to the salivary glands to promote acinar cell proliferation and increase salivary secretion.

In some embodiments, the acinar progenitor cells to which one or more cholinergic and/or muscarinic agonists are administered comprise SOX2⁺Acinar progenitor cells. The SOX2⁺The acinar progenitor cells may comprise AQP5⁺/Ki67⁺Cells and/or Mucin (MUC) 19-cells. Exposure of endogenous acinar progenitor cells to cholinergic and/or muscarinic agonists causes the acinar progenitor cells to proliferate and develop into secretory acinar cells. Such treatment regimens can be used to repopulate salivary glands with functional secretory acinar cells, e.g., by restoring salivary flow to treat subjects having oral diseases (e.g., salivary gland diseases). The subject can be monitored for improvement in salivary gland disease and saliva production and secretion following treatment.

In some embodiments, the cholinergic agonist may be selected from the group consisting of acetylcholine, bethanechol, carbachol, methacholine, arecoline, nicotine, galantamine, cevimeline, levamisole, muscarinic, pilocarpine, donepezil, efenolammonium chloride, neostigmine, physostigmine, pirstine, rivastigmine, tacrine, caffeine, huperzine, diethylphosphonothiocholine, isoflurophosphorus, cisapride, droperidol, domperidone, metoclopramide, risperidone, and paliperidone. In other embodiments, the cholinergic agonist may include at least one of acetylcholine or an acetylcholine analog. For example, the acetylcholine analog may include carbachol.

In some embodiments, the muscarinic agonist may comprise an agonist that activates a muscarinic acetylcholine receptor ("muscarinic receptor"). Muscarinic receptors are divided into five subtypes, known as M1-M5. Muscarinic agonists may include, but are not limited to, pilocarpine, aceclidine, xanomeline, tasimeltidine, sabcomeline, cevimeline, acrameline, arecoline, melameiline, SDZ-210-086, YM-796, RS-86, CDD-0102A (5- [ 3-ethyl-1, 2, 4-oxadiazol-5-yl ] -1, 4, 5, 6-tetrahydropyrimidine hydrochloride), N-arylurea substituted 3-morpholinarecoline, VUO255-035(N- [ 3-oxo-3- [4- (4-pyridyl) -1-piperazinyl ] propyl ] -2, 1, 3-benzothiadiazole-4-sulfonamide), benzylquinolonecarboxylic acid (BQCA), WAY-132983, AFB267B (NGX267), AC-42, AC-260584, chloropyrazine (including but not limited to L-687, 306, L-689-. In some embodiments, muscarinic agonists that are selective for M1 and/or M3 muscarinic receptor subtypes (e.g., cevimeline) are used.

The cholinergic agonist and/or muscarinic agonist may be provided in the form of a pharmaceutical composition that may be administered in vivo or ex vivo to acinar progenitor cells (including SOX2)⁺Acinar progenitor cells). The pharmaceutical composition may be formulated into various dosage forms. The dose can be a pharmaceutically or therapeutically effective amount to promote acinar cell replacement and/or acinar progenitor cell maintenance and survival.

In various embodiments, therapeutically effective doses of the cholinergic agonist and/or muscarinic agonist described herein may be present in varying amounts. For example, in some embodiments, the therapeutically effective amount of the cholinergic agonist and/or muscarinic agonist can be in the range of about 10-1000mg (e.g., about 20mg-1,000mg, 30mg-1,000mg, 40mg-1,000mg, 50mg-1,000mg, 60mg-1,000mg, 70mg-1,000mg, 80mg-1,000mg, 90mg-1,000mg, 10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-600mg, 100-500mg, 100-900mg, 200-800mg, 200-700mg, 200-600mg, 200-500mg, 200-400mg, 200-800mg, 200-700mg, 200-600mg, 1000mg of 300-. In some embodiments, the 15-PGDH inhibitor is present in an amount equal to or greater than about 10mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800 mg. In some embodiments, the 15-PGDH inhibitor is present in an amount equal to or less than about 1000mg, 950mg, 900mg, 850mg, 800mg, 750mg, 700mg, 650mg, 600mg, 550mg, 500mg, 450mg, 400mg, 350mg, 300mg, 250mg, 200mg, 150mg, or 100 mg.

In other embodiments, a therapeutically effective dose of the cholinergic agonist and/or muscarinic agonist may be, for example, about 0.001mg/kg body weight to 500mg/kg body weight, e.g., about 0.001mg/kg body weight to 400mg/kg body weight, about 0.001mg/kg body weight to 300mg/kg body weight, about 0.001mg/kg body weight to 200mg/kg body weight, about 0.001mg/kg body weight to 100mg/kg body weight, about 0.001mg/kg body weight to 90mg/kg body weight, about 0.001mg/kg body weight to 80mg/kg body weight, about 0.001mg/kg body weight to 70mg/kg body weight, about 0.001mg/kg body weight to 60mg/kg body weight, about 0.001mg/kg body weight to 50mg/kg body weight, about 0.001mg/kg to 40mg/kg body weight, about 0.001mg/kg to 30mg/kg body weight, About 0.001mg/kg body weight to 25mg/kg body weight, about 0.001mg/kg body weight to 20mg/kg body weight, about 0.001mg/kg body weight to 15mg/kg body weight, about 0.001mg/kg body weight to 10mg/kg body weight.

In yet other embodiments, a therapeutically effective dose of the cholinergic agonist and/or muscarinic agonist can be, e.g., about 0.0001 to 0.1mg/kg body weight, e.g., about 0.0001 to 0.09mg/kg body weight, about 0.0001 to 0.08mg/kg body weight, about 0.0001 to 0.07mg/kg body weight, about 0.0001 to 0.06mg/kg body weight, about 0.0001 to 0.05mg/kg body weight, about 0.0001 to 0.04mg/kg body weight, about 0.0001 to 0.03mg/kg body weight, about 0.0001 to 0.02mg/kg body weight, about 0.0001 to 0.019mg/kg body weight, about 0.0001 to 0.017mg/kg body weight, About 0.0001mg/kg body weight to 0.016mg/kg body weight, about 0.0001mg/kg body weight to 0.015mg/kg body weight, about 0.0001mg/kg body weight to 0.014mg/kg body weight, about 0.0001mg/kg body weight to 0.013mg/kg body weight, about 0.0001mg/kg body weight to 0.012mg/kg body weight, about 0.0001mg/kg body weight to 0.011mg/kg body weight, about 0.0001mg/kg body weight to 0.01mg/kg body weight, about 0.0001mg/kg body weight to 0.009mg/kg body weight, about 0.0001mg/kg body weight to 0.008mg/kg body weight, about 0.0001mg/kg body weight to 0.007mg/kg body weight, about 0.0001mg/kg body weight to 0.006mg/kg body weight, about 0.0001mg/kg to 0.005mg/kg body weight, about 0.0001mg/kg body weight to 0.005mg/kg body weight, about 0.004mg/kg body weight to 0.003mg/kg body weight, About 0.0001mg/kg body weight to 0.002mg/kg body weight.

In some embodiments, the therapeutically effective dose can be 0.0001mg/kg body weight, 0.0002mg/kg body weight, 0.0003mg/kg body weight, 0.0004mg/kg body weight, 0.0005mg/kg body weight, 0.0006mg/kg body weight, 0.0007mg/kg body weight, 0.0008mg/kg body weight, 0.0009mg/kg body weight, 0.001mg/kg body weight, 0.002mg/kg body weight, 0.003mg/kg body weight, 0.004mg/kg body weight, 0.005mg/kg body weight, 0.006mg/kg body weight, 0.007mg/kg body weight, 0.008mg/kg body weight, 0.009mg/kg body weight, 0.01mg/kg body weight, 0.02mg/kg body weight, 0.03mg/kg body weight, 0.04mg/kg body weight, 0.05mg/kg body weight, 0.06mg/kg body weight, 0.07mg/kg body weight, 0.08mg/kg body weight, 0.09mg/kg body weight, or 0.1mg/kg body weight. The effective dose for a particular individual may vary (e.g., increase or decrease) over time, depending on the individual's needs.

In some embodiments, the therapeutically effective dose of the cholinergic agonist and/or muscarinic agonist may be 10 picograms/kg/day, 50 picograms/kg/day, 100 picograms/kg/day, 250 picograms/kg/day, 500 picograms/kg/day, 1000 picograms/kg/day, or more. In various embodiments, the amount of the cholinergic agonist and/or muscarinic agonist is sufficient to provide a dose in the range of 0.01Pg/kg to 10Pg/kg, 0.1Pg/kg to 5Pg/kg, 0.1Pg/kg to 1000Pg/kg, 0.1Pg/kg to 900Pg/kg, 0.1Pg/kg to 800Pg/kg, 0.1Pg/kg to 700Pg/kg, 0.1Pg/kg to 600Pg/kg, 0.1Pg/kg to 500Pg/kg, or 0.1Pg/kg to 400Pg/kg to the patient.

Various embodiments may include different dosing regimens. In some embodiments, the cholinergic agonist and/or muscarinic agonist may be administered once every two months, once a month, twice a month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, once a day, twice a day, or according to another clinically desirable dosing regimen. The dosage regimen for an individual subject need not be at regular intervals, but may vary over time, depending on the needs of the subject.

Based on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prophylactic, preventative, therapeutic); the compositions were formulated in a manner consistent with these parameters. For a summary of such techniques, see latest edition Remington pharmaceutical university (Mack Publishing Co., Iston, Pa.).

The preparation of therapeutic compositions containing small organic molecule polypeptides, analogs or active fragments as the active ingredient is well known in the art. The compositions of the present invention may be administered parenterally, topically or via an implanted reservoir. Such compositions may be prepared as injections, which may be liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid prior to injection may also be prepared. The formulation may also be emulsified. The active therapeutic ingredient is typically mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, physiological saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. The formulations typically include diluents, and in some cases, adjuvants, buffers, preservatives, and the like. Furthermore, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, which may enhance the efficacy of the active ingredient.

The composition may also be administered topically to the site of the subject using a variety of techniques known to those skilled in the art. For example, these may include sprays, lotions, gels or other vehicles such as alcohols, polyethylene glycols, esters, oils and silicones. The compositions described herein can be administered with pharmacokinetic and pharmacodynamic control by calibrating various administration parameters, including frequency, dose, duration pattern and route of administration. Variations in the dosage, duration and mode of administration may also be manipulated to produce the desired activity. The therapeutic compositions are typically administered in unit dosage forms, e.g., intravenous unit dosages. The term "unit dose" when used in reference to a therapeutic composition of the present invention, means physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent (i.e., carrier) or vehicle.

The composition may be administered in a therapeutically effective amount in a manner compatible with the agent, dosage form, selected for treating the subject. If desired to achieve the desired effect in vitro, the effective amount of the cholinergic and/or muscarinic agonist may range from about 0.1nM to about 10M, more preferably from about 0.1nM to about 5M, and most preferably from about 0.1nM to about 1M. The desired effect refers to the effect of the agent on promoting acinar cell replacement and/or acinar progenitor cell maintenance and survival. The precise amount of the active ingredient to be administered depends on the judgment of the practitioner and will vary from person to person.

The agents described herein may be modified or formulated for administration to a pathological site. Such modifications may include, for example, formulations that improve or extend the half-life of the compound or composition, particularly in the environmentAnd (4) preparing the preparation. In addition, such modifications may include formulating a compound or composition to include a targeting protein or sequence that may facilitate or enhance the uptake of the acinar progenitor cells by the compound/composition. In a particular embodiment, such improvements can cause the compounds to preferentially target SOX2 in the salivary glands relative to other locations or cells⁺Acinar progenitor cells.

The sterile injectable form of the composition may be an aqueous or oleaginous suspension. The suspensions may be formulated according to the techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be used include water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids (e.g., oleic acid and its glyceride derivatives) are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils (e.g., olive oil or castor oil, especially in their polyoxyethylated versions). These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant (e.g., carboxymethyl cellulose or similar dispersing agents) which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants (e.g., Tweens, Spans, and other emulsifiers or bioavailability enhancers) commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used in formulating the formulation.

The parenteral formulation may be a single bolus dose, infusion or loading bolus dose, followed by administration of a maintenance dose. These compositions may be administered once daily or "on demand". The pharmaceutical composition may be administered orally in any orally acceptable dosage form, including capsules, tablets, aqueous suspensions or solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral administration, the active ingredient is used in combination with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the pharmaceutical compositions may be administered rectally in the form of suppositories. These can be prepared by mixing the medicament with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. These materials include cocoa butter, beeswax and polyethylene glycols. The pharmaceutical compositions of the present invention may also be administered topically. Topical administration may be in the form of rectal suppository formulations (see above) or suitable enema formulations. Topical transdermal patches may also be used. For topical administration, the pharmaceutical compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

In some embodiments, the cholinergic agonist and/or muscarinic agonist may be formulated to release the cholinergic agonist and/or muscarinic agonist to the endogenous salivary gland acinar progenitor cells for controlled release. The controlled release may include at least one of delayed release, sustained release, gradient release, temporal release, patterned release, or spatial release. Delayed, sustained, gradient, temporal, patterned or spatial release is one mechanism used in medicine that allows the release of the active ingredient over time. The advantage of controlled release formulations is that they are delivered less frequently and in a defined release pattern than immediate release formulations of the same active compound.

Controlled release formulations can be designed to release the active agent at a predetermined rate to maintain a constant level of agent over a specified extended period of time, for example up to about 1 hour, about 12 hours, about 24 hours, about 2 days, about 3 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or more after administration or after a lag phase associated with delayed release of the agent. In certain embodiments, the active agent is released over a period of time ranging from about 1 week to about 2 weeks or more. Alternatively, the active agent may be released for at least 1 week, at least 2 weeks, at least 3 weeks, or up to 14 days, up to 20 days, up to 30 days, or longer. In other embodiments, the active agent is released over a period of about 1 week to about 3 weeks or more after administration.

In other embodiments, the cholinergic agonist or muscarinic agonist is provided in a biocompatible carrier, matrix, or scaffold that can be administered to the subject. For example, the biocompatible matrix may comprise a polymeric macro-or micro-scaffold and at least one carrier material bound on or within the polymeric macro-or micro-scaffold. The at least one carrier material, matrix or scaffold may comprise a material capable of carrying and releasing in a differential and/or controlled manner at least one cholinergic or muscarinic agonist to endogenous salivary gland acinar progenitor cells.

The carrier, matrix, or scaffold can be any material that allows for the incorporation of cholinergic and/or muscarinic agonists, and can be associated with expanded acinar progenitor cells (e.g., SOX2)⁺Cells) or in the presence of said cells. The carrier, matrix or scaffold is primarily non-immunogenic and biodegradable. Examples of biodegradable materials include, but are not limited to, alginate, polyglycolic acid (PGA), polylactic acid (PLA), hyaluronic acid, suture material, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin, cotton, or other naturally occurring biodegradable materials. It may be preferred to sterilize the matrix or scaffold material prior to application or implantation, for example by ethylene oxide treatment or gamma or electron beam irradiationAnd (5) line processing. In addition, many other materials may be used to form the stent or framework structure, including but not limited to: nylon (polyamide), dacron (polyester), polystyrene, polypropylene, polyacrylates, polyvinyls (e.g., polyvinyl chloride), Polycarbonates (PVC), polytetrafluoroethylene (PTFE, teflon), thermomanox (tpx), hydroxy acid polymers (e.g., polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-glycolic acid (PLGA)), polyorthoesters, polyanhydrides, polyphosphazenes, and various polyhydroxyalkanoates and combinations thereof.

Suitable matrices include polymer meshes or sponges and polymer hydrogels. Hydrogels are defined as substances formed when organic polymers (natural or synthetic) are cross-linked by covalent, ionic or hydrogen bonds to form a three-dimensional open lattice structure to trap water molecules to form a gel. Typically, these polymers are at least partially soluble in aqueous solutions (e.g., water, buffered salt solutions, or aqueous solutions of alcohols having pendant charged groups) or their monovalent ion salts.

In some embodiments, the matrix may achieve biodegradation over a period of less than one year, more preferably less than six months, most preferably two to ten weeks. The polymer compositions and methods of manufacture can be used to determine degradation rates. For example, mixing a greater amount of polylactic acid with polyglycolic acid can shorten the degradation time. Polyglycolic acid webs that may be used are commercially available, for example, from surgical supply companies (e.g., Ethicon, new jersey).

For example, the muscarinic agonist may be encapsulated in an alginate hydrogel. In some embodiments, the encapsulated muscarinic agonist is selective for the M1 and/or M3 muscarinic receptor subtypes (e.g., cevimeline). In other embodiments, the muscarinic agonist is a non-selective muscarinic agonist (e.g., pilocarpine). The alginate in the hydrogel can be ionically crosslinked with divalent cations (see, e.g., example 2 for an understanding of the description of biodegradable crosslinked calcium alginate hydrogels encapsulating cevimeline). In some embodiments, the concentration of the alginate in the hydrogel ranges from about 2 wt% to about 10 wt% (weight percent), including any wt% within this range, such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 wt%. In some embodiments, the alginate is partially oxidized. For example, from about 2% to about 10% of the alginate may be oxidized, including any percentage within this range, such as 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In one embodiment, about 2% of the alginate in the hydrogel is oxidized and the concentration of the alginate is 5 wt%. Such alginate hydrogels are capable of sustained delivery of muscarinic agonists for at least 7 days following administration to a subject (see, e.g., example 2)

A composition comprising an encapsulated cholinergic agonist and/or muscarinic agonist suitable for local delivery to the salivary glands. The physician may locate the salivary glands to be injected, for example, by palpation or medical imaging (e.g., ultrasound, radiography, or MRI). In some embodiments, a contrast agent is included in the composition comprising the encapsulated cholinergic agonist and/or muscarinic agonist, such that the location of the composition relative to the salivary glands is confirmed by medical imaging after administration. In some embodiments, the contrast agent is a microbubble (e.g., suitable for ultrasound imaging) or a radiopaque contrast agent (e.g., suitable for radiography). The contrast agent may be included in the same composition or a different composition with the cholinergic agonist and/or muscarinic agonist and used either before or after administration of the cholinergic agonist and/or muscarinic agonist.

In certain embodiments, the composition is injected into the salivary gland or a region adjacent to the salivary gland. Salivary glands that can be treated by the subject methods include, but are not limited to, parotid, submandibular and sublingual glands and minor salivary glands, including serous, mucoid or serous salivary glands.

In some embodiments, the subject receiving treatment has an oral disease, such as a disease that affects saliva production. Examples of oral diseases include, but are not limited to, salivary gland tumors, cystic fibrosis, sjogren's syndrome, sialadenitis, mumps, siallitis, sialolithiasis, salivary gland stones, sialopholithiasis, mucocysts, sublingual cysts, hyposecretion, sialorrhoea, sialorrhea, xerostomia, benign lymphatic epithelial lesions of salivary glands, salivary gland dilatation, salivary gland enlargement, salivary duct stenosis, and salivary duct stenosis. In other embodiments, the subject may have previously received radiation therapy that can cause dry mouth. The methods described herein are useful for treating such oral diseases affecting saliva production (i.e., xerostomia) in a human subject. The methods described herein will also be useful in veterinary applications to treat xerostomia such as that experienced by domestic animals, including but not limited to pets (e.g., dogs and cats) and livestock (e.g., sheep, goats, pigs, horses and cattle).

Reagent kit

The invention also provides a kit for treating dry mouth in a subject with at least one of a cholinergic agonist or a muscarinic agonist to promote acinar cell production. The cholinergic agonist and/or the muscarinic agonist may be comprised in separate compositions or in the same composition. The kit may include a unit dose of a formulation comprising a cholinergic agonist and/or a muscarinic agonist suitable for use in the methods of treatment described herein, e.g., an injectable dose. In such kits, in addition to the containers containing the unit doses, there are pharmaceutical instructions describing the use and concomitant benefits of dry mouth treatment. The kit can include, for example, a dosing regimen of a cholinergic agonist and/or a muscarinic agonist included in the kit.

Of particular interest are formulations suitable for topical administration to the salivary gland, and in such embodiments, the kit may further comprise one or more syringes or other devices to accomplish such administration. In some embodiments, the kit comprises a first syringe or device pre-loaded with the composition (e.g., the muscarinic agonist, e.g., encapsulated in an alginate hydrogel, which can be stored frozen). The kit may further comprise a second syringe containing calcium chloride, which may be connected to the first syringe by a luer lock. For example, the first injection may be thawed prior to injection into the salivary glandMixing the contents of the syringe (e.g., cevimeline encapsulated in alginate hydrogel) with the contents of the second syringe (e.g., CaCl)₂Which produces a cross-linked calcium alginate hydrogel encapsulating cevimeline) to use the kit. The physician can locate the salivary gland to be injected, for example, by palpation or ultrasound imaging.

In some embodiments, the kit further comprises a contrast agent to confirm the location of the composition comprising the muscarinic agonist (e.g., cevimeline encapsulated in an alginate hydrogel) relative to the salivary glands by medical imaging after administration. In some embodiments, the contrast agent is a microbubble (e.g., suitable for ultrasound imaging) or a radiopaque contrast agent (e.g., suitable for radiography). The contrast agent may be included in the same composition or a different composition with the muscarinic agonist and added before or after administration of the muscarinic agonist.

In addition to the components described above, the subject kits may further include (in certain embodiments) instructions for performing the subject methods. These instructions may be present in the subject kits in various forms, one or more of which may be present in the kit. One form in which these instructions may exist is printed information printed on a suitable medium or substrate (e.g., a sheet or sheets of paper with information printed thereon), reagent kit packaging, package instructions, and the like. Another form in which these instructions may exist is a computer-readable medium, such as a floppy disk, Compact Disk (CD), portable flash drive, etc., having information recorded thereon. Another form in which these instructions may exist is a web site, whereby information on a remote web site may be accessed via the internet.

Other embodiments of the invention described herein will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All U.S. patents and other references mentioned herein are expressly incorporated herein by reference, for whatever reason. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Experiment of

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of methods of making and using the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The present invention has been described in terms of specific examples discovered or suggested by the inventors to include the preferred embodiments of the invention. It will be understood by those skilled in the art in light of the present disclosure that various modifications and changes may be made in the specific embodiments illustrated without departing from the intended scope of the invention. For example, potential DNA sequences can be altered without affecting the protein sequence due to codon redundancy. Furthermore, the structure of the protein can be changed without affecting the kind or amount of biological action in consideration of the biological functional equivalence. All such modifications are intended to be included within the scope of the appended claims.

Example 1

Salivary gland acini are regenerated after radiation therapy in response to cholinergic activation through a progenitor-dependent mechanism

Introduction to

Therapeutic radiation remains a life-saving treatment for cancer patients, being used in a range of malignancy treatments including head and neck tumors. In fact, the vast majority of patients with head and neck cancer receive radiation therapy in addition to chemotherapy and surgery (about 60,000 new patients per year in the United states; Siegel et al 2015). While this combination therapy is very effective in eliminating tumors, it can cause serious side effects that damage and/or destroy healthy tissue located in the irradiated area. These organs, including salivary glands, exhibit tissue dysfunction even after low dose radiation therapy (Grundmann et al, 2009). At higher doses (60Gy) routinely given to patients, off-target radiation destroys salivary synthesizing acinar cells (Sullivan et al, 2005; Redman, 2008) and leads to lifelong dry mouth and complications (e.g., caries, oral infections, poor wound healing (Brown et al, 1975; Dreizen et al, 1977; Dusek et al, 1996)). Although intensity modulated radiation therapy has been successful in avoiding destruction of one of the three major salivary glands (parotid gland), the close proximity of the glands to the tumor site often prevents the use of this technique, resulting in dry mouth syndrome in 80% of patients with head and neck cancer (Lee & Le, 2008).

As with all other organs damaged by radiation therapy, including the lungs, heart and bladder (Emami et al, 1991), there are few available treatment regimens to improve or restore tissue function. Current treatment regimens for cancer survivors suffering from radiation-induced salivary gland dysfunction and degeneration focus on short-term remission, but no long-term restorative therapy. Regeneration strategies have been proposed, such as reactivation of endogenous stem cells or transplantation of unirradiated stem cells (Lombaert et al, 2008; Ogawa et al, 2013; Pringle et al, 2016). However, these applications are limited by the lack of knowledge of the properties of adult salivary gland progenitors that produce acini under homeostatic or damaging conditions. Although it has recently been proposed that acinar cells are produced by self-replicating rather than defined progenitor cells (Aure et al, 2015), no analysis of progenitor-like activity of these cell subsets has been performed. In this regard, it is also desirable to determine whether acinar cells (whether by self-replication or by progenitor cell expansion) can be repopulated with cells following genotoxic insult. Although there have been a number of studies using irradiated salivary glands as a model of degeneration (Zeilstra et al, 2000; Coppes et al, 2001, 2002), the ability of adult salivary gland cells to regenerate in vivo, damaged by radiation therapy, has not been investigated.

Little is known about how acinar cells are displaced during salivary homeostasis. Studies of adult organs over the past 150 years clearly show that peripheral nerves are critical for maintaining organ and tissue integrity (Erb, 1868). Skeletal muscle atrophy in the absence of motor neuron stimulation (Fu & Gordon, 1995; Batt & Bain, 2013), epithelial organs such as mushy taste buds (Von Vintschau & Honigschmied, 1877), prostate (Wang et al, 1991; Lujan et al, 1998), and degradation of salivary glands following sensory and/or autonomic nerve ablation (Schneyer & Hall, 1967; Mandour et al, 1977; Kang et al, 2010). Although it is not clear how nerves control tissue homeostasis of these organs, skin studies indicate that sensory nerves promote self-renewal of adult epithelial stem cells through sonic hedgehog secretion, thereby maintaining the downstream cell lineage, i.e., dome cells in the skin (Peterson et al, 2015; Xiao et al, 2015). Furthermore, studies with salamanders (Wallace, 1972) and embryonic salivary glands (Knox et al, 2013) indicate that peripheral nerves have the ability to regenerate tissues by activating pluripotent stem cells, but lack corresponding evidence in adult mammalian systems.

In combination with mouse genetics, ex vivo culture and human tissue explants, we have unexpectedly found that salivary gland acini can regenerate after receiving radiation therapy by responding to cholinergic activation by a progenitor-dependent mechanism. We demonstrated that SOX2 can label only progenitors of the acinar lineage that are able to displace acinar cells during homeostasis and after radiation-induced injury, suggesting that salivary gland progenitors can withstand genotoxic shock at least for a short period of time. Importantly, treatment of healthy and irradiated tissue with cholinergic mimetics stimulates acinar cell recruitment. Thus, our data indicate that tissues have extensive regenerative capacity even under genotoxic shock and indicate targeting SOX2⁺Cells may be a treatment for tissue regeneration damaged by radiotherapy.

Results

SOX2 labels progenitor cells that form acini but not ductal cells during salivary gland homeostasis

SOX2 has been identified as subglottic salivary glands and sublingual saliva in fetal ratsProgenitor cell markers in glands, but SOX2 in adult tissues is not currently known⁺Whether the cells also produce acinar and ductal cells (Arnold et al, 2011; Emmerson et al, 2017). Furthermore, it is not clear whether these cells are also present in adult salivary glands. We found that SOX2 was expressed by a subset of acinar cells in all three major adult salivary glands [ fig. 1A, submandibular gland (SMG), sublingual gland (SLG), Parotid Gland (PG)]. In mice, the SOX2 protein was restricted to adult murine SLG (not found in SMG and PG, fig. 1B and 10A), which is composed of undifferentiated Aquaporin (AQP)5 positive, Mucin (MUC)19 negative acinar cells (accounting for all AQP5)⁺21 ± 4% of acinar cells; FIGS. 1C and 1D). Consistent with its potential role as a progenitor cell, about 6% SOX2⁺AQP5⁺Cells co-expressed Ki67 (FIGS. 1E and 10B), while 19 + -4% of the cells were in the cell cycle (cyclinD 1)⁺(ii) a Fig. 10C). To determine SOX2⁺Whether cells exert effects on the acinar and ductal lineages, we used Rosa26^mTmGHybrid Sox2 reporter Gene strains^CreERT2Mice (Arnold et al, 2011) were subject to genetic lineage tracing. Rosa26^mTmGThe mouse is a dual fluorescent reporter mouse that, when crossed with the Cre strain, expresses membrane-targeted tandem dimers tomato (mt) prior to Cre-mediated excision and green fluorescent protein (mG) after excision (Muzumdar et al, 2007; fig. 10D). Thus, lineage-tracking cells will express mG. As shown in FIG. 1F, SOX2⁺Cells self-renew after 14 or 30 days and produce differentiated acinar cells labeled by AQP5 and MUC19, but not KRT8⁺Ductal cells (fig. 1F and EV 1E). Thus, our pedigree tracing results show that SOX2⁺The cells are lineage-restricted progenitor cells that give rise to differentiated progeny, similar to that observed in the epidermis, intestine and incisors (Owens)&Watt, 2003; barker, 2014; seidel et al, 2017).

In view of KIT⁺Cells are mainly present in the insertion catheters for SLGs and SMGs (Andreadis et al, 2006; Nelson et al, 2013), which have been proposed in the past to produce acinar cells in adult tissues (lombart et al, 2008; Nanduri et al, 2013, 2014; Pringle et al, 2016),therefore, we used Rosa26 at 6 weeks of age^mTmGKit for reporter gene hybridization^CreERT2Promoters, which allow genetic tracking of these cells. However, there was no overt KIT in SLG or SMG at 14 days or 6 months post-induction⁺Cell-derived acinar cells (i.e., AQP5 and mG were double positive) (fig. 10F). In contrast, KIT⁺Cells only functioned with the insert catheter in SLG (which was observed by co-staining for the insert catheter marker KRT8) and the insert catheter and larger catheters in SMG. Thus, these data indicate that KIT⁺The cells are ducts of the acinar lineage and SOX2⁺A progenitor cell of a cell.

SOX2 and SOX2⁺Cells are essential for the production of secretory acini

Our lineage tracing analysis confirmed SOX2⁺The cells produce acini but not ductal cells. However, as we observed otherwise Ki67⁺SOX 2-acinar cells (about 6% SOX2)⁺Ki67⁺And 16.5% SOX2^-Ki67⁺Cells, FIG. 10B) Presence-representing alternative progenitor cells or transit amplifying cells of acinar lineage, we used Sox2^CreERT2；Sox2^f1/f1Mice (FIGS. 2A and 2C) were genetically deprived of SOX2⁺Sox2 in cells, or using the inducible Sox2 promoter (Sox 2)^CreERT2；Rosa26^DTA(ii) a Fig. 2B and 2D) Diphtheria Toxin (DTA) ablation SOX2 expressed under control⁺Cells to study maintenance and repair of SOX2 and SOX2 in SLG⁺The requirement of the cell. In the latter assay, SOX2⁺Cell death occurs in response to intracellular DTA production. Ablation SOX2⁺Sox2 in cells or SOx2 elimination by DTA⁺Cells make SOX2⁺And AQP5⁺Cell (instead of KRT8)⁺Catheter cells) were severely depleted, indicating Sox2 and Sox2⁺Cells are essential for maintaining functional acini (FIGS. 2A-2D; Sox2 or SOx2)⁺Efficiency of cell ablation see fig. 11A). In the absence of Sox2, acinar cells, but not ductal cells, exit the cell cycle, which can be detected from cyclin D1(CCND1)⁺Seen in the decrease of acinar cells (FIG. 11D; arrows indicate CCND)1⁺Cells, white dashed line highlighting ductal cells). Further, SOX2⁺Ablation of the cells resulted in almost no acinar remaining at day 8 (figures 2B and 2D and 11A), which may be completely free of AQP5 from a larger area of the catheter network⁺This is seen in the fact that the cells (the catheter is marked with a dashed line or KRT8, FIG. 2B). To rule out the possibility that tissue degeneration is entirely due to tissue instability rather than loss of acinar cell replacement, we examined SLG after short-term ablation. As shown in FIG. 8, on day 4 or day 5 (3 or 4 days after tamoxifen treatment), Sox2^CreERT2；Sox2^fl/flAnd Sox2^CreERT2；Rosa26^pTAOnly a small amount of SOX2 was present in the glands of SLG⁺In cells (fig. 8A and 8B), Sox2 transcript was significantly reduced (fig. 8B). The acini are disorganized in appearance and are in an atrophied state. Furthermore, we did not observe SOX2⁺Increased number of cells (or Sox2 transcripts), indicating that Sox2 is not ectopically expressed in acinar cells in response to tissue injury. We also determined whether changes in tissue composition are due to a decrease in the level of innervation (an important regulator of tissue function). However, we are at Sox2^CreERT2；Sox2^fl/flA similar degree of innervation as the wild-type control was measured in SLG, and Sox2 was measured^CreERT2；Rosa26^DTAThere was a significant increase in axonal bundles in SLG (fig. 11B and 11C). The latter finding suggests that cellular ablation triggers the release of factors that promote innervation, but even with elevated innervation levels, cannot regenerate without SOX2+ cells. Taken together, these results show that SOX2⁺The cells, at least under the test conditions, are the only acinar progenitor cells in SLG, and the acini are not produced by self-replication of fully differentiated acinar cells as previously described (Aure et al, 2015). Study with epidermis, intestine and incisors (Owens)&Watt, 2003; barker, 2014; seidel et al, 2017), our data also indicates the presence of SOX2 from⁺A transient expanded population of cells, which may involve rapid re-filling of the acinar compartment.

Parasympathetic protection SOX2⁺Progenitor cells and promotion of SOX 2-mediated acinar cell replacement

Eliminate the side intersectionAfter sensory neural activity, the salivary glands in adult mice and humans atrophy. However, the effect of denervation on acinar cell replacement and progenitor cells has not been studied (Garrett et al, 1999; Raz et al, 2013). To this end, we denervated one of the two pairs of murine SLGs by transecting the tympanum (FIG. 3A; contralateral glands were used as internal controls). After 7 days, neuronal genes Tubb3, Vip and Vacht (FIG. 3B, red bars) and GFRa2⁺Or TUBB3⁺The transcript levels of the nerves (FIGS. 3C and 12C) were severely reduced, indicating successful denervation. We did not observe simultaneous loss of the cholinergic muscarinic receptor Chrm1 and Chrm3 transcripts (fig. 3B, red bars); however, in the absence of parasympathetic innervation, a compensatory mechanism or possibility maintains transcription of Chrml and Chrm 3. Although SLG is predominantly innervated by parasympathetic branches, which are less innervated by sympathetic nerves than by contrast, (Emmelin et al, 1965), we did observe a decrease in the level of sympathetic innervation following tympanum transection (fig. 12A). Thus, although sympathetic levels are low, we cannot rule out the possibility that some of the effects of innervation are due to loss of sympathetic input.

Similar to the effect of radiotherapy on tissue architecture (Sullivan et al, 2005; Redman, 2008), adult acinar cells and SOX2⁺Progenitor cells are more sensitive to loss of innervation than catheters. Denervation leading to a reduction in acinar cell size (as observed previously; Patterson et al, 1975; fig. 12B) resulted in a reduction in AQP5 protein and transcript levels of the differentiated acinar cell marker Muc19 (fig. 3B and 3D). Interestingly, transcript and protein levels of MIST1 were unchanged after denervation (fig. 3B, 3E and 3F), indicating that although functional markers of acinar cells were disrupted in the absence of innervation, the properties of acinar cells were not adversely affected. Surprisingly, SOX2⁺Cells lost Sox2 expression ability (using Sox2)^eGFPMice demonstrated), and the levels of SOX2 protein and transcript were significantly reduced (fig. 3B, 3C and 3F and 12C), indicating that SOX2 maintenance requires innervation. To determine post-denervation SOX2⁺Whether cells can still repopulate tissue, we performed genetic lineage tracingWherein the endogenous Sox2 promoter (Sox 2)^CreERT2；Rosa26^mTmG) The driver Cre was activated 3 days after denervation and tracked until day 14. SOX2 at 14 days after transection, as shown in FIGS. 3G and 3H⁺Acinar cell replacement of progenitor cells was significantly reduced (-50%). Similarly, in SLG in which recombination was induced before nerve transection (for tamoxifen, 1 day before transection), SOX2⁺Acinar cell replacement of progenitor cells was clearly depleted (-50%) after 14 days (fig. 12E and 12F). The decreased acinar cell replacement was probably due to decreased cell proliferation rather than cell death, as we measured a decrease in Ccnd1, but no cell death marker was observed [ activated caspase-3 in fig. 12G (CASP 3)⁺) Cells or Bax, Pmaip1(NOXA) and Bbc3(PUMA) in FIG. 12H]Or such markers remain unchanged. Cell-free death also indicated that cells previously positive for SOX2 were still present, but cholinergic innervation was critical for maintaining SOX2 expression.

To confirm that denervation preferentially affects acinar lineages in SLG, we also analyzed other epithelial cell lineages. KRT8 in denervated glands⁺The catheters were similar to the corresponding catheters in the innervated controls, as were the transcript levels of the catheter genes Krt7, Krt8, and Krt19 (fig. 3B, 3D, and 3F). Furthermore, progenitor cells KRT5 in developing SMG/SLG maintained by parasympathetic nerves (Knoxet et al, 2010)⁺Cells (Knoxet et al, 2010; Lombaert et al, 2013) were not affected by denervation (FIGS. 3F and 12; for expression of transcript Krt5, see FIG. 3B). Based on these findings, we concluded that adult SLG tissue homeostasis requires parasympathetic innervation, modulating SOX2 and SOX2⁺The cells preferentially maintain and replace the functional acini.

To determine whether re-supplementation of salivary glands with nerves can save acinus and SOX2, we examined murine salivary glands 30 days after denervation. The murine salivary glands will regain innervation over time due to plasticity of the peripheral nervous system, e.g., TUBB3 30 days after transection⁺The re-emergence of the nerves (FIG. 9A) and the re-expression of the neuronal genes Tubb3, Vip and Vacht (FIG. 3B, blue column; Yawo, 1987). Surprisingly, we found that the expression of these neuronal genes increased at day 30 (fig. 3B) -indicating innervation in response to the original injury. Notably, after regaining innervation, the levels of Sox2 and Aqp5 transcripts and Sox2 protein and Sox2⁺And AQP5⁺Both the number of cells and the size of acinar cells were restored to or above control levels (FIGS. 3B and 9A-9C).

To ensure SOX2 of SLG⁺Cells were able to respond directly to parasympathetic acetylcholine production, and we analyzed SOX2⁺Expression of acetylcholine/muscarinic receptors by cells in vivo and their ability to respond to muscarinic agonists. SOX2⁺Cells expressing CHRM1 and CHRM3 (95% and 99%; FIGS. 4A and 4B, respectively), wild-type mice were given short-term (i.p. and sacrificed at 18 hours) doses of the muscarinic agonist pilocarpine resulting in proliferating SOX2⁺Cell (SOX 2)⁺Ki67⁺A cell; fig. 4C and 4D) percent increase. However, SOX2⁺The percentage of cells did not change significantly at 18 hours post-injection (fig. 4D), indicating that muscarinic activation did not induce ectopic expression of SOX 2. Thus, these data support our hypothesis that parasympathetic nerves maintain SOX2 through acetylcholine type muscarinic signaling⁺Progenitor cells and acini and promote the recruitment of acinar cells.

Regeneration of murine salivary glands by SOX2 following radiation-induced injury

Murine salivary glands (mainly SMG) have been widely used to study the effects of Ionizing Radiation (IR) on gland function and structure, with typical analysis limited to degenerative reactions. The C57BL/6 background line was reported to exhibit acinar cell loss and decreased salivary flow rate after a single 10Gy dose of irradiation (Zeilstra et al, 2000; Coppeset et al, 2001, 2002). However, it is not clear whether the tissue has regenerative capacity and whether it remains innervated after IR. To test the effect of radiation on SOX 2-mediated salivary gland regeneration, we analyzed the innervation in mouse SLG, SOX2, after a single dose of gamma radiation on the head and neck⁺Cell and SOX2 mediated acinar cell recruitment. Study with previous salivary glands (Avila et al)2009), we found that the 10Gy dose resulted in DNA damage and cell cycle arrest and reduced cell proliferation in SLG the first day after IR, as can be seen from the significant increase in the pro-apoptotic gene Bax and cell cycle inhibitor Cdknla (p21) (fig. 13A) and the reduction in the transcriptional level of the cell proliferation marker Mki67 (fig. 13A). Then, we pass through the pair TUBB3⁺Nerves were immuno-labeled and semi-quantitative pcr (qpcr) was performed on Tubb3 and parasympathetic derived neurotransmitter Vip to measure changes in innervation and neurological function in IR SLG. As shown in FIGS. 5A and 5B, TUBB3 in IR SLG⁺Nerves were unchanged compared to the non-IR control at 1 and 3 days post irradiation. However, the transcriptional levels of Tubb3 and Vip were significantly reduced at 1 and 3 days post IR, indicating that neural function was reduced at an early stage (fig. 13B). Similarly, the transcript levels of Sox2, Mist1, and Aqp5 decreased immediately after IR, but returned to control levels on day 7 after IR (fig. 13B). On day 1 after IR, SOX2⁺The number of cells was significantly reduced (fig. 5C), while CCND1⁺SOX2⁺The number of cells increased significantly at day 3 and 7 after IR (5C). To determine whether SOX 2-mediated cellular replacement was affected after IR-induced injury, we analyzed SOX2 14 days after IR^CreERT2；Rosa26^mTmGThe extent of SOX 2-mediated supplementation in mice. Surprisingly, we found that acinar cells were protected by SOX2 in IR SLG⁺Cell replacement (GFP)⁺Cells) -this was similar to the non-IR control and produced SOX2⁺GFP⁺And SOX2 negative progeny (fig. 5D, white arrows indicate SOX2 negative progeny), indicating the presence of a transiently expanded cell that is no longer SOX2⁺But rather from SOX2⁺Cells (i.e., lineage tracing). Further, SOX2 in IR SLG⁺The number of cells was similar to the control group at day 14 (fig. 5D). Thus, although nerve signaling, SOX2, and acinar cell markers were initially lost, following radiation-induced injury, the acinar compartment could be protected from SOX2⁺The cells are replenished.

Next, we tested whether SLG could be regenerated following IR injury without Sox 2. As shown in FIG. 5E, Sox2 irradiated with a single 10Gy dose^CreERT2；Sox2^f1/flMice were unable to repopulate the tissues with functional AQP5+ acini. Indeed, in the absence of Sox2, we observed AQP5 at 14 days post-IR, compared to wild type mice⁺Acinar cells were lost and tissue structure was destroyed (fig. 5E). This result further confirms that Sox2 is critical for SLG regeneration after radiation-induced injury.

SOX2+ cells can respond to cholinergic mimetics to complement irradiated salivary glands

Since our data indicate that cholinergic cues can complement acinar lineages in SLG, we used our ex vivo lineage tracing model to determine SOX2⁺Whether the cells can recruit acini in response to muscarinic activation in healthy and irradiated SLGs. As shown in FIGS. 6A and 6B, GFP in healthy SLG tissues cultured with the acetylcholine mimic carbachol (CCh) for 48 hours⁺The cloning is increased. GFP (green fluorescent protein)⁺This increase in cloning was associated with proliferation of CCh-treated cells (Ki 67)⁺Cells) increased (fig. 13E). In our IR model, 24 hours before animals receive a single dose of IR, at Sox2^CreERT2；Rosa26^mTmGRecombination was induced in mice (fig. 6A). This time point was chosen because it was previously reported that the lag time for tamoxifen-induced recombination in the Cre strain of mice was 12-24 hours (Nakamura et al, 2006). Thus, a single SOX2⁺Cells were labeled 24 hours after injection (fig. 6B, see 0 hour panel). SLG was collected within 1 hour after radiation exposure and cultured ex vivo for 48 hours with or without CCh. As shown by the quantification results in FIGS. 6B and 6C, GFP in IR SLG was compared with that in the non-IR control group⁺More clones, indicating IR-activated SOX2⁺The cells refill the tissue. This was probably due to cholinergic signaling from the remaining nerves, as IR explants cultured with the muscarinic receptor antagonist 4-DAMP showed similar acinar cell recruitment to non-IR cultured salivary glands (fig. 6B and 6C). Importantly, treatment of IR explants with CCh resulted in GFP compared to IR treatment alone⁺Cells were increased (fig. 6B and 6C, and 13C and 13D). Based on these data, we conclude that SOX2⁺The cells can be refilled in response to muscarinic activationIR SLG。

Acetylcholine/muscarinic signaling can maintain SOX2 and acinar lineages in human SG

For humans, IR causes an irreversible decrease in markers of parasympathetic innervation (increase of sympathetic innervation) in SG (Knox et al, 2013; fig. 14A; GFRA2) and acinar (AQP3, miss 1, AMY1) but not ductal (EGFR, KRT19) lineages compared to the non-IR control group (fig. 7A; IR delivered about 2 years before surgery, n ═ 7(IR) and 11 (non-IR)). Lineage markers in human tissues (SMG was used due to availability) were confirmed by immunofluorescence (fig. 14B). Furthermore, we found that the transcriptional levels of SOX2, GFRA2, CHRM1, and CHRM3, but not tyrosine hydroxylase (TH, sympathetic marker), were also significantly or tend to be down-regulated after IR (fig. 7A and 14A), indicating that parasympathetic function and the ability of the cells to respond to acetylcholine had been exhausted. Therefore, we hypothesized that the loss of regeneration capacity of human salivary glands after IR is due to SOX2⁺Reduced levels of parasympathetic innervation of the progenitor cells results and acetylcholine/muscarinic signaling is assumed to be sufficient to maintain SOX2 expression and promote the acinar lineage. To validate this hypothesis, we established a novel human explant-murine nerve co-culture system. In this model, non-irradiated human SMG was dissected into pieces <1mm and placed next to the submandibular parasympathetic ganglia (containing mesenchyme) or mesenchyme only (control, no nerves) of a rat at 13 days of embryonic age. The tissue was then placed on a filter floating above serum-free medium for 7 days of co-culture. Nerves migrated inside and around the tissue (fig. 7B), and tissue architecture was actively maintained, as can be seen from the fact that CDH1 (E-cadherin, gene and protein expression) levels were higher in explants co-cultured with nerves compared to the case where only mesenchyme was used (fig. 7C and 7D). We analyzed the explants of the neuropsychological elicitor neurotrophic factor (NRTN) and Nerve Growth Factor (NGF) (Knox et al, 2013) in an attempt to determine the factors that induce this nerve migration. However, we found that expression was not increased in the presence of ganglia compared to the use of mesenchyme alone (fig. 7D), indicating that other nerve attractants are being synthesized, with or without nerves, epithelial cells produce these nerve attractantsEither mesenchymal (i.e. mesenchymal enough to maintain intraepithelial homeostasis) or to indicate that this phenomenon occurs early in culture (i.e. at an early stage, where nerves have just begun to coat the epithelium), any difference being resolved at this later time point (day 7). Surprisingly, the transcript levels of SOX2 (about 1.8-fold to 2-fold), acinar markers miss 1 and CD44, muscarinic receptor CHRM3 (also expressed by acinar cells; Giraldoet et al, 1988) were increased for glandular explants cultured with nerves compared to controls using mesenchyme only, but there were differences in ductal genes KRT19, KIT and EGFR (fig. 7D, data only normalized to mesenchyme, n ═ 2 individual individuals). In addition, with Ki67⁺The presence of cells was marked by an increase in epithelial cell proliferation in the presence of nerves (fig. 7C and 14C). Taken together, these data indicate that parasympathetic nerves are able to maintain SOX2 expression in human salivary glands as well as acinar and ductal markers.

To address whether acetylcholine/muscarinic signaling can sufficiently maintain acini and SOX2 in human salivary glands⁺Cell problem, we performed muscarinic stimulation of unirradiated human SMG or PG (both expressing SOX2) tissue explants in an ex vivo system. Patient-derived human tissues from four different individuals (n ═ 4) were cultured with CCh such that muscarinic stimulation was achieved within 4 hours of SOX2, muscarinic receptors CHRM1 and CHRM3[ all expressed by adult acinar cells (Giraldo et al, 1988; Mei et al, 1990)]And the expression of AQP3, AQP5(Gresz et al, 2001) and miss 1 were significantly increased (fig. 7E, n-4, single dataset see fig. 14D). While surgical denervation did not adversely affect the expression of Mist1 (FIGS. 3B and 3E), muscarinic stimulation was sufficient to increase MIST1 in human culture, suggesting that acetylcholine/muscarinic signaling, although not essential for acinar cell properties, may act as a positive regulator of the secretory process. Differences in response between samples of four patient origin may be due to biodiversity between human patients, types of glandular origin (using SMG and PG), and patient age (donor age range 30 to 78 years). However, in all cases we observed an increase in SOX2 and many acinar markers in the presence of CCh. Although we have also measured at CChIncreased KRT19 gene expression with effect, but the other unique ductal markers EGFR, KRT7, and KRT8 remained unchanged, indicating that ductal cells were generally unresponsive to muscarinic agonists (fig. 7E). Given that nerves increased ductal genes in human salivary glands in co-culture, nerves may produce other factors to cause ductal changes (compare fig. 7D and 7E). Thus, neuronal acetylcholine/muscarinic signaling is sufficient to promote acinar lineages in adult salivary glands and to maintain SOX2 expression.

Discussion of the related Art

Our study found that the progenitor cell SOX2 in adult salivary glands⁺Cells are essential for acinar recruitment, with the unexpected ability to repopulate tissue following radiation-induced injury. We further demonstrated that cholinergic nerves play a crucial role in controlling SOX 2-mediated acinar cell replacement during homeostasis, and that this neuronal effect can be replicated by adding cholinergic mimetics to the irradiated tissue. Thus, in contrast to the current theorem that murine salivary glands do not regenerate following radiation-induced injury (Zeilstra et al, 2000; Coppes et al, 2001, 2002), these data indicate that, at least in mice, salivary glands have extensive regeneration capacity following radiation-induced injury. Furthermore, we found that the acinar lineage (and SOX2) in human tissue also responded to cholinergic mimetics, targeting SOX2⁺Cells and maintenance of cholinergic nerves may contribute to the restoration of functional salivary acini after injury due to radiotherapy.

SOX2 plays an important role in the development of organisms and can regulate the homeostasis of epithelial tissues such as the stomach, trachea and intestine (Arnold et al, 2011). Similar to the decrease in homeostatic capacity of adult tracheal cells following Sox2 ablation (Que et al, 2009), we found that, under homeostatic and injury conditions, Sox2⁺AQP5 following ablation of Sox2 in acinar progenitor cells⁺Depletion of acinar cells. Interestingly, although atrophied, we did not find increased cell death in the absence of Sox2, but circulating acinar cells were significantly reduced, suggesting that after ablation, these cells exited the cell cycle and differentiated to produce dormant AQP5 deficient aciniA cell. SOX2⁺Significant loss of acini following cell ablation further confirms that these cells are the only progenitors of the acinar lineage in SLG, and this conclusion is consistent with a recent report that indicates that pancreatic acinar cells are not equally potent, but contain a subset of progenitors (Wollny et al, 2016). Our results are in contrast to a recent report published by Aure and colleagues that suggests that salivary gland acinar cells are complemented by the self-replication of mature cells rather than by progenitor cell differentiation (Aure et al, 2015), which is similar to that of pancreatic beta cells (Dor et al, 2004). The conclusion that tissue re-proliferation is due to self-replication of salivary gland acinar cells is based on the use of an inducible Mist1 promoter, which is derived from all acinar cells (including SOX2)⁺MUC19^-Cells) are expressed. Thus, the subpopulation of cells that are tracked after recombination may include progenitor cells; however, no analysis was performed to determine whether these subpopulations had stem cell characteristics.

As with all epithelial organs, peripheral nerves are required to maintain the structural homeostasis of rodent and human salivary glands (Schneyer & Hall, 1967; Mandour et al, 1977; Wang et al, 1991; Fu & Gordon, 1995; Lujan et al, 1998; Kang et al, 2010; Batt & Bain, 2013). During organogenesis parasympathetic nerves maintain a population of progenitor cells that exert effects on tissues through acetylcholine/muscarinic signaling following radiation injury (Knox et al, 2013). Although previous salivary gland denervation studies have shown a reduction in organ and acinar cell size (Schneyer & Hall, 1967; Mandour et al, 1977; Kang et al, 2010), it is not clear whether acinar cell replacement requires innervation. Our studies indicate that nerves directly modulate SOX2 to drive acinar cell replacement from lineage restricted progenitor cells. This result is consistent with the known role of SOX2 in regulating self-renewal and cell fate in many other organs (Arnold et al, 2011). However, to date, only intracellular signaling pathways, including those mediated by the WNT, FGF and EGFR families, have been shown to modulate SOX2(Hashimoto et al, 2012; Dogan et al, 2014; Rothenberg et al, 2015; Lee et al, 2016). This extrinsic nerve-based model has distinct advantages over intracellular signaling, and unlike its target organs, the neurons themselves are more resistant to radiation damage (Tofilon & Fike, 2000; Wong & Van der Kogel, 2004). Whether this mechanism also regulates the maintenance of other SOX 2-expressing epithelial organs, such as taste buds (Suzuki, 2008), prostate and seminal vesicles (Wanigasekara et al, 2004), stomach (Tatsuta et al, 1985; Zhao et al, 2014) and cornea (Ueno et al, 2012), has yet to be investigated. However, these results suggest that cholinergic nerves may play a role in the regeneration of these tissues.

Previous studies have used murine salivary glands as a model for radiation-induced degeneration (Zeilstra et al, 2000; Coppes et al, 2001, 2002). These studies are based on the hypothesis that regenerative capacity is impaired after moderate to high doses of radiation, supported by the fact that salivary flow is reduced in animals receiving radiotherapy (Redman, 2008). However, to date, in vivo analysis of cell replacement after irradiation has not been performed. Our data indicate that murine acinar cells are highly regenerative, at least for the first 30 days after radiation exposure, and are capable of refilling the acinus like an uninjured control. However, it is clear that this regenerative capacity cannot be maintained for a long period of time, since the salivary glands in mice denature/age 3-6 months after irradiation (Urek et al, 2005; Marmarmarary et al, 2016). Thus, SOX2⁺The regenerative capacity of the cells is likely to eventually fail, requiring further analysis to find the cause. Whether human salivary glands can regenerate within days/months following therapeutic radiation therapy, and whether this ability to regenerate is due to SOX2⁺The long-term failure of cells and parasympathetic deficits has yet to be determined. In fact, the time course of analyzing the changes in the patient's salivary glands needs to be mastered to understand the way these organs are affected in both the short and long term. However, our results suggest that controlling and maintaining tissue regeneration in response to radiation damage or possibly providing a means of long-term maintenance/repair of tissue is directed to these stem cells and their innervations.

Some recent studies have been aimed atThe problem of gland regeneration after radiation injury in a mouse model was solved by isolating a putative stem cell population for reimplantation (Nanduri et al, 2013, 2014) or by preserving regions in the glands thought to have stem cells (van Luijk et al, 2015). However, the nature of these endogenous stem cells and whether they contribute to the acinar cell compartment are not known at present. Furthermore, the effect of such manipulations on salivary gland innervation has not been reported. According to our study, the unexpected increase in innervation potential due to tissue perturbation may lead to SOX2⁺Progenitor cells are maintained and expanded, which regenerate acini to restore salivary gland function. Xiao et al (2014) reported that mouse salivary gland function and structure was restored by addition of Glial Derived Nerve Factor (GDNF), a nerve attracting factor, following radiation-induced injury (Knox et al, 2013). However, a recent study showed that GDNF by itself failed to directly protect SG stem cells from radiation-induced damage (Peng et al, 2017), suggesting that this result may be a result supporting niche improvement.

In conclusion, our studies have highlighted the extensive regenerative capacity of salivary glands produced by the expansion and differentiation of progenitor cell populations, even in the face of genotoxic shock. Based on these data, we suggest that by directly targeting SOX2 in tissue⁺Cells, or by isolating and expanding these cells for transplantation and activation, we have the potential to regenerate salivary gland secretory units and restore the quality of life of the patient. As shown by the previously proposed views (Knox et al, 2013), this also requires maintenance of parasympathetic function to maintain SOX2⁺And (4) a group. Given that organs such as the gut, glandular stomach, trachea and taste buds expressing SOX2 are heavily innervated by the autonomic nervous system and are damaged by therapeutic radiation used to eliminate cancer, such strategies may be applicable to repair multiple organ systems.

Materials and methods

Mouse strain

All procedures were approved by the UCSF Institutional Animal Care and Use Committee (IACUC) and followed the guidelines for care and use of NIH laboratory animals. This studyThe mouse allele used in (1) was provided by Jackson laboratories and included Sox2^eGFP(Arnold et al, 2011), Sox2^CreERT2(Smith et al, 2009), Sox2^fl/fl(Taranova et al, 2006), Rosa26^mTmG(Muzumdaret et al, 2007), Rosa26^DTA(Wu et al, 2006) and Kit^CreERT2(Klein et al, 2013).

Animal experiments

Adult female mice (6 to 8 weeks old) were used for all experiments unless otherwise stated. Mice were housed in the experimental animal resources center (LARC) in the pennaxos school district, university of california, which was certified AAALAC. If possible, a maximum of 5 mice were housed per cage, and the mice were housed in individually ventilated cages (WC) with fresh water, which were regularly cleaned and kept in a high environment. The appropriate sample size is calculated using efficacy calculations. For transgenic studies, the sample size is limited by the length of time required to reproduce a sufficient number of animals of the desired genotype and sex. Wild type animals were randomly assigned to each experimental group using Microsoft Excel software. Transgenic animals were assigned to each group based on genotype. All animals had a unique ID number, so the investigators were blinded during the analysis.

Sox2 or Sox2⁺Gene ablation of cells

Sox2 conditional ablation by daily dose of Sox2 for four consecutive days before euthanasia was performed on day 18^CreERT2；Sox2^fl/f1：R26^mTmGMice were injected with 2.5mg/20g tamoxifen and then 2.5mg/20g tamoxifen every third day. SOX2⁺Cell ablation was performed by daily exposure to Sox2 for four consecutive days before euthanasia was performed on day 7^CreERT2；Rosa26^DTA：R26^mTmGMice were injected with 2.5mg/20g tamoxifen. Rosa26^mTmGMice provide a valuable tool for lineage tracing in conjunction with cell ablation/recombination (Muzumdar et al, 2007). Briefly, the model consists of a dual fluorescent Cre reporter expressing a membrane-targeting tandem dimer (tomato) prior to Cre-mediated excision and a green-targeting membrane after excisionFluorescent protein (mG) (FIG. 10D). Thus, in these mice, cells that have recombined and therefore lack Sox2 or express DTA will express GFP. Endogenous GFP was imaged in experiments using frozen sections, while GFP antibodies were used to paraffin-embed the tissue (chicken anti-GFP; 1: 500, Aves Labs, GFP-1020).

SOX2⁺Cell lineage tracking

To Sox2^CreERT2；Rosa26^mTmGMice were injected with 2.5mg tamoxifen and euthanized after 24 hours, 14 days, or 30 days.

KIT + cell lineage tracking

Four consecutive days each day to Kit^CreERT2；Rosa26^mTmGMice were injected with 2.5mg tamoxifen and euthanized after 14 days or 6 months.

In vivo denervation experiments

C57BL/6 or Sox2 was administered 30 minutes before surgery^eGFPThe mice were administered analgesics (carprofen and buprenorphine; Patterson Veterinary and Buprenex; 0.1 and 100mg/kg (IP), respectively) and by inhalation 2% isoflurane/O₂The mixture is anesthetized. The surgical area was shaved and an incision was prepared, washed with alternating iodine and alcohol, and then a local anesthetic (lidocaine; Hospira inc., 8mg/kg) was applied. As described in previous studies (Klimaschewski et al, 1996), an incision was made in the ear and anterior portion of the tympanum and completely transected using a spring-loaded scissors. The skin was sutured using a non-absorbable suture thread (Ethicon) and the wound was further covered with surgical adhesive (Vetbond). The contralateral nerve remained intact as a control. Mice were euthanized 7 or 30 days later.

Pilocarpine assay

Adult male C57BL/6 mice (6 to 8 weeks old) were dosed with pilocarpine (Sigma-Aldrich, P0472; 0.68mg/ml in 0.9% sterile saline). Mice were anesthetized with isoflurane and injected intraperitoneally with 4.5mg/kg body weight (2001) of pilocarpine or 0.9% saline as vehicle controls. Mice were euthanized 18 hours later and glands were subjected to immunofluorescence analysis.

Gamma radiation experiment

Dissolving in 0.9%C57BL/6 mice were anesthetized with 1.25% 2, 2, 2-tribromoethanol (Alfa Aesar) in saline (Vedco Inc.). Mice were placed in Shepherd Mark-I-68A¹³⁷Cs irradiator (JL Shepherd)&Associates), use¹³⁷The Cs source irradiated the mice. Two lead blocks 1.5cm apart were used to protect the body and the foremost part of the oral cavity (snout) of the mouse, exposing only a portion of the neck and head. 1.5em opening is located in the center (distance from) of position 3¹³⁷Cs source 20em, 15.5em from the edge of the irradiator chamber). Mice were irradiated twice with 5Gy dose for 2.59 minutes (once on each side of the head, bilaterally, continuously but on the same day) at a radiation rate of 167Rads/min for a total dose of 10Gy to irradiate the salivary glands. The dose was calculated from an isodose map (dose distribution) provided by the manufacturer and the 100% exposed area was located using EBT membrane (Brady et al, 2009) to position the mice. Control mice were anesthetized in the manner corresponding to the experimental mice, but were not treated with radiation. All mice were allowed to recover completely before returning to normal rearing environment and were given ad libitum access to soft food (ClearH)₂O). Mice were euthanized after 1 hour or 1, 3, 7, 14 or 30 days.

Organ culture experiments

In vitro lineage tracing of adult salivary glands

Will be taken from Sox2^CreERT2；Rosa26^mTmGThe salivary glands of the mice (24 hours before recombinant induction) were mechanically cut into pieces <1mm, then placed in complete medium (with or without 200nM CCh (Sigma-Aldrich) or 10M 4-DAMP (Tocris)), incubated for 48h, and then fixed for immunofluorescence analysis. In some cases, the mice received 3 irradiation doses of 5Gy before the glands were cultured as above.

Isolation culture of human salivary gland tissue

Adult salivary glands were obtained from discarded, unidentified tissue with consent of patients (28-78 years old, male and female) who underwent neck resection. All subjects had received informed consent and the experiments were in accordance with the principles specified in WMA declaration of helsinki and the report of belmont of the department of health and public service. Patients had not received radiotherapy (non-IR) or fractionated radiotherapy (IR) within 2 years prior to surgery. Tissues were immediately placed in 4% PFA, RNAlater (Qiagen) or DMEM (thermo Fisher) for culture of viable cell explants. For ex vivo culture, non-IR tissues were dissected into pieces <1mm and then cultured in serum-free DMEM/F12 containing holotransferrin and ascorbic acid. For explant assays, tissues (SMG and PG) were incubated with 50-200nM CCh (200 nM CCh shown in results) for 4 hours prior to lysis against RNA. For salivary gland explant-parasympathetic mandibular ganglion (SMG) co-culture, tissues were dissected into pieces <1mm and cultured on floating filters above serum-free medium. The parasympathetic mandibular ganglion of the E13 mouse was isolated as previously described (Knoxet et al, 2010). One parasympathetic mandibular ganglion of each explant was placed next to the human salivary glands and cultured for 7 days, then fixed for immunofluorescence analysis or lysed for RNA.

Tissue treatment

After fixation, salivary glands (human and mouse) were processed for OCT or paraffin embedding. To obtain frozen sections, tissues were incubated in increasing concentrations of sucrose (25-75%) and embedded in OCT. 12-m sections were cut using a cryostat (Leica) and stored at-20 ℃. The tissues were dehydrated by incubation in increasing concentrations of ethanol followed by Histo-clear (national diagnostics) prior to embedding in paraffin (Sigma-Aldrich) for paraffin treatment. 12-m sections were cut using a microtome (Leica) and stored at room temperature.

Immunofluorescence assay

Immunofluorescence analysis of whole specimen embedded salivary glands and tissue sections has been previously described (Knox et al, 2010). Briefly, tissues were fixed with ice cold acetone/methanol (1: 1) for 1 minute or 4% PFA for 20-30 minutes, followed by permeabilization with 0.1-0.3% Triton X. Tissues were blocked overnight at 4 ℃ with 10% donkey serum (Jackson laboratory, Maine), 1% BSA (Sigma-Aldrich) and MOM IgG blocking solution (Vector Laboratories, Calif.) in 0.01% PBS-Tween 20. Salivary glands were incubated overnight at 4 ℃ with the following primary antibodies: goat anti-SOX 2 (1: 200, Neuromics, GT 15098); goat anti-SOX 10 (1: 500, Santa Cruz Biotechnology, sc-17342); mouse anti-TUBB 3(TUJ1 clone, 1: 400, R & D Systems, MAB 1195); goat anti-GFRa 2 (1: 100, R & D Systems, AF 429); rabbit anti-tyrosine hydroxylase (1: 100, Millipore, AB 152); rat anti-E-cadherin (1: 300, Life Technologies, 13-1900); rabbit anti-EGFR (1: 200, Abcam, ab 52894); rabbit anti-KRT 5 (1: 1,000, Covance, PRB-160P); rat anti-KRT 8 (1: 200, DSHB, troma I); mouse anti-KRT 7 (1: 50, Covance, MMS-148S); rat anti-CD 44 (1: 200, BioLegend, 103001); mouse anti-Ki 67 (1: 50, BDbiosciences, 550609); rabbit anti-CCND 1 (1: 200, Abcam, ab 16663); rabbit anti-caspase-3 (1: 100, Invitrogen, 34-1700): rabbit anti-AQP 3 (1: 400, Lifespan Biosciences Inc., LS-B8185); rabbit anti-AQP 5 (1: 100, Millipore, AB 3559); goat anti-MUC 19 (1: 200, Abcore, AC 21-2396); mouse anti-aSMA (1: 400, Sigma-Aldrich, C6198); chicken anti-GFP (1: 500, Aves Labs, GFP-1020); rabbit anti-CHRM 3 (1: 1,000, Research and Diagnostics, AS-3741S); and rabbit anti-MIST 1 (1: 500, donated by Stephen Konieczny, university of Prion). Antibodies were detected using Cy2-, Cy3-, or Cy 5-conjugated Fab fragment secondary antibodies (Jackson laboratories) and nuclear staining was performed using Hoechst 33342 (1: 1,000, Sigma-Aldrich). Fluorescence was analyzed using a Leica Sp5 confocal microscope and NIH ImageJ software.

Morphological analysis and cell counting

For immunofluorescence analysis (e.g., fig. 2D), marker-positively stained cells were counted using ImageJ. Acinar cell size was measured using ImageJ. All data were obtained by 3-5 fields/group, with 3 replicates per experiment. For the neurodensitometric analysis, the immunofluorescence of the neural marker TUBB3 was analyzed and the raw integrated density (shown as arbitrary units, AU) was calculated using ImageJ.

Quantitative PCR analysis

RNA was isolated from whole tissues using RNAqueous Micro kit (Ambion). Total RNA samples were subjected to DNase treatment (Ambion) using SuperScript reagent (Invitrogen, ca) prior to cDNA synthesis. SYBRgreenqPCR was performed using 5ng (mouse) or 4-10ng (human) cDNA and primers designed using Primer3 and Beacon Designer software or described in PrimerBank (pga. mgh. ha. rvard. edu/Primer bank /). Primer sequences are listed in tables 1 and 2. Melting curves and primer efficiencies were determined as described previously (Hoffman et al, 2002). Gene expression was normalized to housekeeping genes S18 and S29(Rps18 and Rsp29) or GAPDH (applicable to mice) and GAPDH (applicable to humans) and the corresponding experimental controls. The reaction was repeated three times and the experiment was performed 2-3 times.

Flow cytometry

Adult mice sublingual salivary glands (CD1) were dissected and washed in PBS containing gentamicin (Sigma-Aldrich). Cell isolation and flow cytometry were performed as described previously (Muench et al, 2002; Pringle et al, 2011). Briefly, tissues were minced in HBSS + 1% BSA (Sigma-Aldrich) with a surgical knife blade and placed in a knife containing 50mM CaCl at 37 deg.C₂(Sigma-Aldrich), 40mg/ml hyaluronidase (Sigma-Aldrich) and 23mg/ml collagenase type II (Sigma-Aldrich) in HBSS for 15-45 minutes to form single cell suspensions. The enzyme reaction was quenched by addition of BSA and the solution was filtered through a 70m strainer (BDfalcon) and centrifuged at 400g for 8 min. The resulting cell pellet was washed with sterile HBSS + 1% BSA, centrifuged and resuspended in blocking buffer (5% serum and 0.01% NaN3, BioLegend). The cell suspension was incubated with anti-CD 326 antibody (EpCAM; Miltenyi, 130-. Subsequently, intracellular staining was performed after fixation and permeabilization using an intracellular staining kit (eBioscience, 00-5523-00) and anti-SOX 2(BD Pharmingen, 562195) and Ki67(BioLegend, 652405) antibodies. Flow cytometry was performed on lsrii (bd) using appropriate single stain controls, data were collected using facsdiva (bd) and analyzed using FlowJo. Unless otherwise stated, 100,000 events were collected for each sample.

Statistical test

Normal distributions were assessed using the D' Agostino-Pearson comprehensive test. Statistical significance of the data was analyzed using Student's t-test (unpaired, two groups) or one-way ANOVA (multiple groups) and post-hoc tests using Dunnett or Tukey's test (GraphPad Prism or SPSS). For multiple tests, we used a haircut occurrence of 0.05. All graphs show mean + Standard Deviation (SD) or mean ± standard error of the mean (SEM), as shown in the legend.

Reference to the literature

Andreadis D, Epivatianos a, Poulopoulos a, Nomikos a, Papazoglou G, antoniads D, Barbatis C (2006) detect the C-KIT (CD117) molecule in benign and malignant salivary gland tumors, oral oncology 42: 57-65

Arnold K, Sarkar A, YRam MA, Polo JM, Bronson R, Sengutta S, Senndel M, Geijsen N, Hochedlinger K (2011) Sox2(+) adult stem and progenitor cells are important for mouse tissue regeneration and survival

9：317-329

Aure MH, Konieczny SF, Ovitt CE (2015) maintain salivary gland homeostasis through acinar cell self-replication developmental cells 33: 231-237

Salivary gland dysfunction induced by Avila JL, Grundmann O, Burd R, Limesand KH (2009) radiation is caused by p 53-dependent apoptosis international journal of radiation oncology, biology, physics 73: 523-529

Barker N (2014) adult intestinal stem cells: natural reviews: molecular cell biology 15: 19-33

Batt JA, Bain JR (2013) tibial transection-a standardized model of mouse denervation-induced skeletal muscle atrophy. e50657

Brady SL, Toncheva G, Dewhirst MW, Yoshizumi TT (2009) characterized 137Cs irradiators from a new perspective using modern dosimetry tools health physics 97: 195-205

Effect of Brown LR, Dreizen S, Handler S, Johnston DA (1975) radiation-induced xerostomia on human oral microflora the journal of dental research 54: 740-750

Coppes RP, Zeilstra LJ, Kampinga HH, Konings AW (2001) avoid early to late radiation injury to the parotid gland by adrenergic and muscarinic receptor agonists. 1055-1063

Radiation sensitivity of rat parotid and submandibular glands after different radiation plans for Coppes RP, Vissink a, Konings AW (2002). radiotherapy and oncology 63: 321-328

Dogan I，Kawabata S，Bergbower E，Gills JJ，Ekmekci A，Wilson W III，Rudin

CM, Dennis PA (2014) SOX2 expression is mouse

Early events in the EGFR mutant lung cancer model promote proliferation of EGFR mutant lung adenocarcinoma cell line subpopulations lung cancer 85: 1-6

Dor Y, Brown J, Martinez OI, Melton DA (2004) adult pancreatic beta cells are formed by self-replication rather than stem cell differentiation

429：41-46

Prevention of xerostomia-related caries in cancer patients irradiated by Dreizen S, Brown LR, Daly TE, Drane JB (1977.) J.dental research 56: 99-104

Dusek M, Simmons J, Buschang PH, al-Hashimi I (1996) chewing function in patients with dry mouth.geriatric dentistry 13: 3-8

Emami B, Lyman J, Brown A, Coia L, Goitein M, Munzender JE, Shank B, Solin LJ, Wesson M (1991) Normal tissue resistance to therapeutic irradiation. International journal of radiation oncology, biology, Physics 21: 109-122

Emmelin N, Holmberg J, Ohlin P (1965) receptors for catecholamines in rat submaxillary glands. british journal of pharmacology and chemotherapy 25: 134-138

Emmerson E, May AJ, Nathan S, Cruz-Pacheco N, Lizama CO, Maliskova L, zotein AC, Shen Y, Muench MO, Knox SM (2017) SOX2 regulates acinar cell development in salivary glands. e26620

Erb W(1868)Zur Pathologie und pathologischen Autonomie peripherischer

Paralysis, Dtsch Arch klin Med 4: 535

Fu SY, Gordon T (1995) delayed the contributing factors of functional recovery following nerve repair: long term denervation. journal of neuroscience 15: 3886-3895

Garrett JR, Ekstrom J, Anderson LC (1999) the neural mechanism of salivary gland secretion Bussel: karger

Giraldo E, Martos F, Gomez A, Garcia A, Vigano MA, Ladinsky H, Sanchez de La Cuesta F (1988) characterization of muscarinic receptor subtypes in human tissues. 1507-1515

Gresz V, Kwon TH, Hurley PT, Varga G, Zelles T, Nielsen S, Case RM, Steward MC (2001) identification and localization of aquaporin water channels in human salivary glands. Gastrointestinal and hepatic physiology 281: G247-G254

Sensitivity of the salivary gland to radiation by Grundmann O, Mitchell GC, Limesand KH (2009): from animal models to treatments. dental research journal 88: 894-903

Hashimoto S, Chen H, Que J, Brockway BL, Drake JA, Snyder JC, Randell SH, Stripp BR (2012) β -catenin-SOX 2 signaling regulates the fate of airway epithelial development. 932-942

Gene expression profiling for submandibular gland development in mice by Hoffman MP, Kidder BL, Steinberg ZL, Lakhani S, Ho S, Kleinman HK, Larsen M (2002): FGFR1 regulates branched morphogenesis in vitro through BMP and FGF dependent mechanisms development 129: 5767-5778

Kang JH, Kim BK, Park BI, Kim HJ, Ko HM, Yang SY, Kim MS, Jung JY, Kim WJ, Oh WM, et al (2010) parasympathetic denervation induced morphological changes in rat submandibular glands and altered gene expression profiles oral biology profile 55: 7-14

The interstitial cells of Klein S, Seidler B, Kettenberger A, Sibaev A, Rohn M, Feil R, Allescher HD, Vanderwin JM, Hofmann F, Schemann M et al (2013) Cajal integrate excitatory and inhibitory neurotransmission with intestinal slow wave activity. Communication 4: 1630

Klimaschewski L, Grohmann I, Heym C (1996) target-dependent plasticity of galanin and vasoactive intestinal peptide in the upper ganglion of rat neck after nerve injury and re-innervation.nervous system science 72: 265-272

Knox SM, Lombaert IM, Reed X, Vitale-Cross L, Gutkind JS, Hoffman MP (2010) parasympathetic innervation maintains epithelial progenitor cells during salivary gland organogenesis science 329: 1645-1647

Knox SM, Lombaert IM, Haddox CL, Abrams SR, Cotrim A, Wilson AJ, Hoffman MP (2013) parasympathetic stimulation improves epithelial organ regeneration. Communication 4: 1494

New advances in radiation therapy for head and neck cancer, Lee NY, Le QT (2008): modulated intensity radiation and hypoxia targeting oncology study 35: 236-250

Lee MJ, Kim EJ, Otsu K, Harada H, Jung HS (2016) Sox2 promoted tooth development by Wnt signaling cell tissue study 365: 77-84

Lombaert IM, Brunsing JF, Wierenga PK, Faber H, Stokman MA, Kok T, Visser WH, Kampinge HH, de Haan G, Coppes RP (2008) rescue salivary gland function after irradiated gland stem cell transplantation. e2063

Lombaert IM, Abrams SR, Li L, esparakumar VP, Sethi AJ, Witt RL, Hoffman MP (2013) in combination with KIT and FGFR2b signaling regulate epithelial progenitor cell expansion during organogenesis stem cell report 1: 604-619

van Luijk P, Pringle S, Deasy JO, Moiseenko VV, Faber H, Hovan A, Banstra M, van der Laan HP, Kierkels RG, van der Schaaf A et al (2015) preserve the salivary gland area containing stem cells for sustained salivary production following head and neck cancer radiotherapy. 305ral47

Role of autonomous innervation in rat prostate architecture maintenance in Lujan M, Paez a, Llanes L, Angulo J, Berenguer a (1998): morphometric analysis. journal of urology 160: 1919-1923

The effects of Mandour MA, Helmi AM, E1-Sheikh MM, E1-Garem F, E1-Ghazzawi E (1977) tympanostomy on human parotid salivary glands histopathology, histochemistry, and clinical studies. 338-341

Marmary Y, Adar R, Gaska S, Wygoda A, Maly A, Cohen J, Eliashar R, Mizrachi L, Orfaig-Geva C, Baum BJ et al (2016) radiation-induced loss of salivary gland function driven by cellular senescence and inhibited by IL6 regulation. cancer study 76: 1170-1180

Mei L, Roeske WR, Izutsu KT, Yamamura HI (1990) characterization of muscarinic acetylcholine receptors in human salivary gland poisoning of the lips european journal of pharmacology 176: 367-370

Muench MO, Suskind DL, Barcena A (2002) isolated, cultured, grown and identified colony forming cells with erythrocyte, myeloid, dendritic and NK cell potential from human fetal liver. 10-23

Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) whole-body dual-fluorescent Cre reporter mouse Genesis 45: 593-605

Nakamura E, Nguyen MT, Mackem S (2006) kinetic characterization of tamoxifen-modulated Cre activity in mice was determined using cartilage-specific creer (t) to determine temporal activity windows along the proximal and distal limb bones. 2603-2612

Nanduri LS, Lombaert IM, van der Zwaag M, Faber H, Brunsting JF, van Os RP, Coppes RP (2013) Salisphere-derived c-Kit cell transplantation can restore tissue homeostasis in irradiated salivary glands. 458-463

Purification and in vitro expansion of Nanduri LS, Banstra M, Faber H, Rocchi C, Zwart E, de Haan G, van Os R, Coppes RP (2014) fully functional salivary gland stem cells. 957-964

Nelson DA, Mannhardt C, Kamath V, Sui Y, Santamaria-Pang A, Can A, Bello M, Corwin A, Dinn SR, Lazare M et al (2013) quantitative unicellular analysis of cell population dynamics during submandibular salivary gland development and differentiation, open biology 2: 439-447

Ogawa M, Oshima M, Imamura a, Sekine Y, Ishida K, Yamashita K, NakajimaK, Hirayama M, Tachikawa T, Tsuji T (2013) achieve functional salivary gland regeneration by bioengineered organ embryo transplantation. Communication 4: 2498

Owens DM, Watt FM (2003) Stem cells and differentiated cells contribute to epidermal tumors Natural reviews: cancer 3: 444-451

Secretion and structural changes in the parotid salivary glands of sheep and lambs following parasympathetic denervation by Paterson J, Lloyd LC, Titchen DA (1975). 223-232

Peng X, vardendi K, maiets M, Andressoo JO, Coppes RP (2017) glial-derived neurotrophic factor plays a role in salivary gland stem cell response to radiation. 448-454

Peterson SC, Eberl M, Vagnozzi AN, Belkadi a, veniaminonova NA, Verhaegen ME, Bichakjian CK, Ward NL, Dlugosz AA, Wong SY (2015) basal cell carcinoma preferentially originates from stem cells in hair follicles and mechanosensory niches. 400-412

Pringle S, Nanduri LS, van der Zwaag M, van Os R, Coppes RP (2011) isolation of mouse salivary gland stem cells. J.VIEW.EXPERIMENTAL 8: 2484

Pringle S, Maimets M, van der Zwaag M, Stokman MA, van Gosliga D, Zwart E, Witjes MJ, de Haan G, van Os R, Coppes RP (2016) human salivary gland stem cells normalize radiation-damaged salivary gland function. 640-652

Multiple roles of Que J, Luo X, Schwartz RJ, Hogan BL (2009) Sox2 in development and adult mouse trachea.development 136: 1899-1907

Raz E, Saba L, Hagiwara M, Hygino de CruzLC Jr, Som PM, Fatterpekar GM (2013) parotid atrophy in chronic trigeminal denervation patients AJNR, american journal of neuroradiology 34: 860-863

Redman RS (2008) method for functional recovery of salivary glands damaged by radiotherapy of head and neck cancer and for review of salivary gland morphology and development related aspects biotechnology and histochemistry 83: 103-130

Inhibition of mutant EGFR in lung cancer cells by Rothenberg SM, Concannon K, Cullen S, Boulay G, Turke AB, Faber AC, Lockerman EL, Rivera MN, Engelman JA, Mahanshan S et al (2015) triggers SOX2-FOXO6 dependent survival pathway eLife 4: e06132

Effect of Schneyer CA, Hall HD (1967) denervation on parotid gland function and structural development in immature rats. journal of physiology in usa 212: 871-876

Seidel K, Marangoni P, Tang C, Houshmand B, Du W, Maas RL, Murray S, Oldham MC, Klein OD (2017) resolved stem and progenitor cells in adult mouse incisors by gene co-expression analysis. e24712

Siegel RL, Miller KD, Jemal a (2015) cancer statistics, 2015.CA clinicians journal of cancer

65：5-29

The phase-dependent pattern of Smith AN, Miller LA, Radice G, Ashery-Padan R, Lang RA (2009) Pax6-Sox2 ectopic dominance regulates lens development and ocular morphogenesis.development 136: 2977-2985

Sullivan CA, Haddad RI, Tishler RB, Mahadevan A, Krane JF (2005) radiotherapy-chemotherapy induced cell loss in human submandibular glands laryngoscope 115: 958-964

Expression of Suzuki Y (2008) Sox2 in mouse taste buds and its relationship to innervation cell tissue study 332: 393-401

Taranova OV, Magness ST, Fagan BM, Wu Y, Surzenko N, Hutton SR, PevnyLH (2006) SOX2 are dose-dependent regulators of retinal neural progenitor cell capacity. 1187-1202

Tatsuta M, Yamamura H, Iishi H, Ichii M, Noguchi S, Baba M, Taniguchi H (1985) vagotomy pairs

Promotion of N-methyl-N' -nitro-N-nitrosoguanidine-induced gastric carcinogenesis in Wistar rats cancer study 45: 194-197

Tofilon PJ, Fike JR (2000) central nervous system radiation response: radiation study 153: 357-370

Ueno H, Ferrari G, Hattori T, Saban DR, Katikireddy KR, Chauhan SK, Dana R (2012) dependence of corneal stem/progenitor cells on ocular innervation. 867-872

Early and late effects of Urek MM, Bralic M, Tomac J, Borcic J, Uhac I, Glazar I, Antonic R, Ferreri S (2005) X-radiation on the submandibular glands: medical research archive 36: 339-343

Von Vintschgau M，Honigschmied J(1877)Nervus Glossopharyngeus undSchmeckbecher.Arch Gesammte Physiol 14：443-448

Wallace H (1972) supports the regenerative nervous component of irradiated salamander limb regeneration, the embryology and experimental morphology journal 28: 419-435

Wang JM, McKenna KE, McVary KT, Lee C (1991) maintain the innervation requirements of rat prostate structural and functional integrity. 1171-1176

Waniganekara Y, Airaksinen MS, Heuckeroth RO, Milbrandt J, Keast JR (2004) signaling of neurotrophic factors through GFRalpha2 is critical for the innervation of the glands of the sacral parasympathetic ganglion neurons, but not the muscle targets. 288-300

Wollny D, ZHao S, Everlien I, Lun X, Brunken J, Brune D, Ziebell F, Tabansky I, Weichert w, Marciniak-Czochra A et al (2016) single cell analysis revealed clonal acinar cell heterogeneity in adult pancreas. 289-301

Mechanism of radiation injury to the central nervous system by Wong CS, Van der Kogel AJ (2004): effect on neuroprotection molecular intervention 4: 273-284

Wu S, Wu Y, Capecchi MR (2006) motor neurons and oligodendrocytes are produced sequentially from neural stem cells, but do not appear to share common lineage-limiting progenitor cells in vivo. 581-590

Xiao N, Lin Y, Cao H, Sirjani D, Giaccia AJ, Koong AC, Kong CS, Diehn M, Le QT (2014) neurotrophic factor GDNF improves the survival of salivary gland stem cells. 3364-3377

Xiao Y, Thoresen DT, Williams JS, Wang C, Perna J, Petrova R, Brownell I (2015) hedgehog signaling maintains stem cell renewal in sensory tactile dome epithelium. 7195-7200

Yawo H (1987) post-ganglionic axonal microsurgery post-cervical ganglion cell dendritic geometry changes in mice j neuroscience journal 7: 3703-3711

Zeilstra LJ, Vissink A, Konings AW, Coppes RP (2000) radiation-induced rat submandibular gland cell loss and its relationship to gland function. International journal of radiobiology 76: 419-429

Zhao CM, Hayakawa Y, Kodama Y, Muthupalani S, Westphalen CB, Andersen GT, Flatberg a, Johannessen H, Friedman RA, Renz BW et al (2014) denervation inhibits gastric tumorigenesis. 250ra115

Example 2

Therapeutic activation of endogenous saliva by local delivery of muscarinic agonists in injectable alginate hydrogels Liquid gland stem cell population

Another regenerative method to restore salivary gland function is stem cell therapy, i.e., transplantation of autologous stem/progenitor cells into damaged organs or reactivation of viable stem cells within tissues. However, the progenitor cells that give rise to salivary acinar cells are the major cell types that are damaged by radiation and autoimmune diseases, which was not previously known. We have recently identified SOX2 as a marker of epithelial progenitor cells in murine salivary glands (Emmerson et al, 2018). SOX2+ acinar cells are present in all major human salivary glands (submandibular glands (SMG)) and constitute 20% of the acinar compartment in the murine sublingual gland (SLG). We have found that the maintenance of SOX2+ cellular and tissue homeostasis is dependent on parasympathetic nerve production of acetylcholine, which activates muscarinic receptors to promote cell proliferation. We further demonstrate that muscarinic mimetics carbachol and pilocarpine can promote SOX2+ cell proliferation after in vivo and/or ex vivo delivery, and that muscarinic agonists can promote salivary gland regeneration after radiation-induced injury. Taken together, these studies strongly suggest that reactivating existing stem cells is a viable strategy for regenerating organs.

In this study, we tested the ability to deliver cevimeline locally and continuously to promote stem cell-mediated salivary gland re-proliferation. In particular, we have designed a combination product that involves encapsulation of muscarinic agonists in biodegradable cross-linked calcium alginate hydrogels for local, targeted and sustained delivery into the salivary glands. In combination with in vitro and in vivo tests, we have determined the chemistry of alginate-based hydrogels, which are capable of linear distribution within tissues and sustained delivery of the muscarinic receptor agonist cevimeline. In vivo testing in mice showed that the encapsulated drug was effective in promoting stem cell proliferation in the salivary glands of control and irradiated animals in a manner consistent with sustained delivery. Therefore, a new treatment method is provided for salivary gland regeneration and salivary flow recovery.

Results

Cevimeline promotes cell proliferation when administered by local injection

In view of our previous findings that systemic administration of muscarinic mimetics can promote salivary gland proliferation, we next determined whether local intraglandular injection can also promote mitotic responses. To determine whether cevimeline can act locally on the salivary glands to induce acinar cell proliferation, we injected cevimeline directly into the sublingual gland at a dose of 10mg/kg body weight (20 μ L) and measured proliferation after 18 hours. As shown in fig. 15, acinar cell proliferation was significantly increased (fig. 15), indicating that cevimeline can act directly on salivary glands to promote mitosis of cells.

Engineered biodegradable cross-linked calcium alginate hydrogel

Based on the efficacy of cevimeline local delivery in promoting cell proliferation, we next set out to develop an injectable drug delivery platform to locally release and slowly release cevimeline to drive acinar regeneration. To this end, we chose to use biomaterials based on cross-linked calcium alginate to deliver cevimeline. Alginate is a naturally occurring anionic polymer extracted from brown algae. Alginates are FDA-approved hydrogels for many biomedical applications, are biocompatible, minimally toxic, relatively low cost, and have the ability to use divalent cations (e.g., Ca) under cell-compatible conditions²⁺) The ability to undergo cross-linking. (Lee et al (2012) progress in Polymer science. 37 (1): 106-. To pairFor our application, it is important that the alginate be re-suspended at a concentration that enables injection through a clinically relevant syringe-needle system, during which shear forces break the electrostatic cross-links, allowing linear deformation of the material, but the gel spontaneously reforms in vivo.

Alginates are not inherently degradable in mammals due to the lack of the polymer chain cleaving enzyme, alginase. However, the ionically crosslinked alginate gel dissolves by releasing divalent ions, which cross-links the gel into the surrounding medium by an exchange reaction with monovalent cations (e.g., sodium ions). The alginate degradation rate can be further controlled by partial oxidation of the polymer chains. Alginate hydrogels must degrade 30 days after injection, which is critical for FDA approval as a therapeutic agent rather than an implantable article. However, to achieve sustained delivery of cevimeline, the alginate should be present for more than 7 days. To meet this degradation profile, we partially oxidized 2% or 5% of the alginate chains. In vitro degradation tests show that the two hydrogels have significantly different degradation curves, and the degradation speed of the 2% Oxidized Alginate (OA) hydrogel is slower. (FIG. 16A) the 2% OA hydrogel maintained more than 50% of the gel mass in vitro for approximately 5 days, whereas the 5% OA hydrogel failed to maintain this mass after 1 day. After 7 days of incubation in the in vitro degradation test, approximately 30% of the 2% OA hydrogel and 14% of the 5% OA hydrogel remained.

Based on this longer physical retention time, we chose to continue using the 2% OA hydrogel. We next tested how the weight percent (wt%) of the 2% OA hydrogel affected physical degradation. While the 10 wt% 2% OA hydrogel degrades at a significantly slower rate than either the 2 wt% or 5 wt% hydrogels, this concentration cannot be easily injected through the small bore needle (> 23 gauge) required for in vivo delivery to the salivary gland. (FIG. 16B)

The physical degradation of the 2% OA hydrogel (5 wt%) was then tested under more physiologically relevant conditions by injecting the 1001 hydrogel into the subcutaneous pocket on the dorsal side of wild type mice. This amount of hydrogel can form a small disc in a few minutes, which can then be removed and weighed for analysis. Compared to the in vitro system, we found that there was almost no mass loss after 7 days of in vivo injection (fig. 16C), indicating that this hydrogel is suitable for in vivo delivery of cevimeline.

Another factor critical to commercial potential is the storage stability of the dissolved alginate. To slow down the degradation we assume that the dissolved oxidized alginate material must be stored at 4 ℃ or < -20 ℃, but the specific effect of these storage conditions on the stability of the material is not clear at present. Thus, we tested the change in compressive modulus of the cross-linked calcium alginate after storage of the dissolved oxidized alginate at 4 ℃ or-20 ℃ for 30 days. When 2% OA hydrogel (5 wt%) was stored under refrigerated conditions (4 ℃), the crosslinking efficacy was significantly affected and the compressive modulus showed a decrease at20 days of storage, indicating that the oxidized alginate was degraded (fig. 16C). However, when the alginate is stored under freezing conditions (-20 ℃), the initial material properties of the alginate remain stable for 30 days after synthesis.

Local and sustained release of cevimeline from alginate hydrogel

Pharmaceutical applications of alginate have traditionally involved the controlled release of a variety of low molecular weight drugs using hydrogels. Crosslinked alginates form nanoporous materials, which we use have a theoretical pore size of about 5nm and an initial molecular weight of 197 kDa. Controlled drug release is mainly regulated by electrostatic interactions between positively charged cevimeline (pKA ═ 9.5) and negatively charged alginate hydrogels. In vitro cevimeline release studies showed that we could release the drug slowly over a2 day period. (fig. 17) despite the change in physical degradation characteristics (fig. 16A-16B), neither the oxidized amount (OA%) nor the weight percent (wt%) of the alginate hydrogel had a significant effect on the drug release kinetics, and more than 85 ± 5% of cevimeline had been released in vitro within 2 days. (FIGS. 17A-17B) to test whether varying the initial concentration of drug affects release, we loaded 3000, 60000, or 12000ng cevimeline into each pan and tested the in vitro release profile. The release rate remained relatively consistent (fig. 17C), and thus the amount of drug delivered increased predictably over a two-day period (fig. 17D).

The cevimeline can promote gland cell proliferation when encapsulated in alginate

In view of this successful drug delivery platform, we next tested the in vivo efficacy, toxicity and degradation profile of alginate + cevimeline. Because of the small size of mouse salivary glands, we chose not to inject the hydrogel directly into the gland to avoid complications from tissue damage, but rather to inject the hydrogel into the region directly adjacent to the gland that can be observed by X-ray (fig. 18A; Omnipaque encapsulation as a radiopaque contrast agent). Alginate or saline with or without 10mg/kg cevimeline was injected into the vicinity of the salivary glands and the salivary glands were analyzed at 18 hours (fig. 18C, n-3-4/group) or 3 days (fig. 18, n-3-6/group) after injection. As shown in fig. 18, we measured a significant increase in the number of proliferation of Ki67+ and EdU + acinar cells in response to 10mg/kg alginate + cevimeline on day 3 compared to the alginate alone (fig. 18C, 18D), which is consistent with the prolonged drug activity profile when used in combination with alginate. We observed a similar increase in Ki67+ acinar cell proliferation at day 3 in response to the higher 25mg/kg alginate + cevimeline, indicating that increasing the dose did not significantly improve the efficacy (figure 18). We also tested whether alginate itself is mitogenic by comparing cell proliferation in mice treated with alginate alone to glands of mice that received only Intraperitoneal (IP) injections of saline. We found that the use of alginate alone was sufficient to promote epithelial cell proliferation and that such effect was still achieved within a period of 3-7 days (fig. 23), indicating that this combination had a beneficial effect.

To determine whether alginate or alginate + cevimeline treatment would have a toxic effect on mice, we measured mouse body weight and characterized inflammatory cells in their salivary glands. We found that by day 3 post-injection, increasing the dose of alginate + cevimeline did not result in weight loss (figure 24). In addition, no toxic reaction such as diarrhea was observed compared to the control group injected with physiological saline. Our immunohistological analysis of CD3+ T cells also showed no increase compared to tissues from naive or aged (positive control) mice (fig. 19A, 19B). Our analysis of CD68+ macrophages, a key innate immune cell and the first responder to injury, showed a slight increase in these cells in response to alginate or alginate + cevimeline administration directly compared to the non-administered control group (fig. 19C, 19D). Further analysis of the macrophage population showed a significant increase in repair-promoting CD206+ macrophages in tissues receiving alginate administration compared to the control group receiving saline administration (fig. 19E), indicating that alginate may have a beneficial effect on salivary glands by activating anti-inflammatory macrophages.

Effect of cevimeline-alginate on salivary glands after radiation-induced injury

Next, we tested whether cevimeline or alginate + cevimeline could promote cell proliferation in salivary glands after gamma radiation-induced injury. Necks of 7-8 week-old C57BL6 received a single 10Gy dose of radiation therapy, after 14 days, saline or cevimeline 10mg/kg was injected systemically or intraglandularly, and after 18 hours, the amount of acinar cell proliferation was analyzed (see the graph in FIG. 20A). This level of radiation reduced citrate-induced salivary flow by 30% (fig. 20B). We found that both systemic and topical administration of cevimeline resulted in increased proliferation of acinar cells in irradiated mice (fig. 20C-20E), indicating that cevimeline is effective for damaged salivary glands.

Finally, we tested whether cevimeline encapsulated in alginate could promote cell proliferation and salivary flow. Mice were injected with alginate or alginate + cevimeline 14 days after radiation therapy and sacrificed for proliferation analysis on day 3 (fig. 21A schematic). Alginate + cevimeline resulted in higher proliferation rates than the level obtained with alginate alone (fig. 21B, 21C). We further determined whether treatment with alginate or alginate + cevimeline helped saliva flow. Surprisingly, both alginate and alginate + cevimeline were able to increase citrate-induced salivary flow and the effect lasted for 7 days (fig. 21D), indicating that alginate itself is beneficial to irradiated organs.

Discussion of the related Art

Oral muscarinic agonists stimulate the temporary production of saliva in dry mouth patients, but cause common side effects such as hyperhidrosis, flushing, urgency, gastrointestinal upset and headache. In this study, we demonstrated that local release of the muscarinic agonist cevimeline from an injectable cross-linked calcium alginate hydrogel could promote acinar cell recruitment and improve salivary gland function following radiation-induced injury without eliciting an immune response. This represents the first in vivo evidence that acinar progenitor cells in irradiated tissue can be stimulated to proliferate, and that muscarinic receptor activation is sufficient to achieve this result. We have also shown that in addition to the sustained release of small molecule cevimeline over three days, the use of cross-linked calcium alginate alone also stimulates a pro-regenerative response. Taken together, these findings provide a potential novel combination therapy useful for reversing salivary gland dysfunction following radiation-induced injury, which may be applicable to a range of dry mouth conditions.

Our treatment is based on relevant evidence over the past 100 years that suggests that peripheral nerves are critical for achieving salivary gland function, homeostasis and regeneration. Parasympathetic ablation leads to secretory gonadal atrophy, which can be reversed by revascularizing the tissue (Emmerson et al, 2018). Since parasympathetic innervation in salivary glands of patients receiving therapeutic radiation therapy for head and neck cancer is severely diminished (Emmerson et al, 2018), we challenge whether re-supply of atrophic tissue with neurogenic factors can restore tissue structure and function. The in vivo experiments shown here are a follow-up of our previously performed in vitro studies showing that increased neuronal survival and improved function in fetal salivary glands after radiation exposure or treatment of irradiated adult tissue with muscarinic agonists is sufficient to promote acinar cell recruitment by a progenitor-mediated mechanism (Knox, 2013; Emmerson, 2018). Furthermore, our current data also indicate that the improvement in salivary flow in mice and humans with irradiated salivary glands after long-term oral muscarinic agonist (Barbe 2017; Taniguchi 2019) is likely due to acinar cell recruitment rather than decreased cell death. However, whether human acinar cells can proliferate in response to muscarinic stimulation remains to be determined.

Many strategies for irradiated salivary gland regeneration have been explored. Our therapeutic application aims to mimic the endogenous modulation of tissues by the parasympathetic nervous system by providing acetylcholine homologs. This approach is consistent with recent gene therapy studies aimed at re-supplying functional nerves to organs through the expression of neurotrophic factor, a neurotrophic growth factor. Other strategies utilize growth factors known to promote branch morphogenesis during embryonic development: intraglandular administration of fibroblast growth factor 7 (FGF 7; Lombaert et al, 2009; Xiao et al, 2014), or systemic administration of insulin-like growth factor 1 (Grundmann (2010) BMC cancer 10: 417) or delivery of sonic hedgehog (Shh) (Hai et al, (2016) human gene therapy.27 (5): 390-. However, the uptake of these factors is clinically hindered by their potential to accelerate cancer cell growth. Although long-term oral administration of pilocarpine does not exhibit tumorigenicity, further studies are needed to determine whether topical administration of cevimeline can affect the onset, progression, or metastasis of cancer.

Previous studies have shown that cross-linked calcium alginate hydrogels promote wound healing in various organ systems including bone, cartilage and skin (Wang et al (2015) J.International J.Clin & Experimental Pathology 8 (6): 6636-. However, little is currently known about the fact whether this function is directly mediated through cell proliferation, survival and/or migration. Our data indicate that calcium alginate is able to promote tissue regeneration in irradiated salivary glands by inducing cell proliferation. A potential mechanism for achieving this is the increase in extracellular calcium. Calcium that is not cross-linked to the polysaccharide backbone is released directly from the alginate into the extracellular space (Doyle et al (1996), J. biomedical materials research 32 (4): 561-. Then, the Ca²⁺The depot can activate calcium sensing receptors to mediate intracellular Ca²⁺Release and/or other signal transduction pathways, promoting cell proliferation. Due to a large number of cell types (includingAcinar cells), we therefore propose Ca from alginate²⁺Ions can act as mitogens by stimulating these receptors.

Recently, it has been reported that Ca is contained in calcium alginate gel²⁺The release of (A) may also have pro-inflammatory properties (Chan et al (2013) BioMaterial letters.9 (12): 9281-9291). Calcium alginate injected subcutaneously into mice can significantly up-regulate IL-1 in peripheral tissues and enhance the inflammatory action of LPS. However, our results show that calcium alginate, with or without cevimeline, is highly biocompatible and causes little inflammation when injected in the immediate vicinity around the salivary gland.

One of the key properties that a hydrogel needs to provide is the ability to sustain drug release. Drug molecules, from smaller chemical drugs to large molecular proteins, can be released from alginate gels in a controlled manner, depending on the type of cross-linking agent and the cross-linking method. In fact, a variety of chemicals have been used to control the kinetics of drug release, such as cross-linking.

Materials and methods

Synthesis of OA

Oxidized Alginate (OA) was prepared by modifying the previously described method by reacting sodium alginate (Protanal LF 20/40, alginate MW 197,000Da, FMC Biopolymer cat # S18407) with sodium periodate (Sigma, cat # 311448). [1]Briefly, sodium alginate (10g) was placed in ultrapure deionized water (diH)₂O, 900ml) was dissolved overnight. Sodium periodate (0.22g) was dissolved in 100ml of diH₂O, then added to the alginate solution at Room Temperature (RT) and stirred in the dark. After 24 hours of reaction, ethylene glycol (521, Sigma) was added to stop the reaction. By pairing with diH₂OA was purified by O dialysis (MWCO 3500Da, Spectrum Laboratories Inc.) for 3 days, followed by treatment with activated carbon (5g/l, 50-200 mesh, Fisher, 05-690B) for 30 minutes, filtration (0.22 μm filter) and lyophilization.

To analyze the oxidation efficiency of OA, OA was dissolved in deuterium oxide (5 w/v%) and placed in an NMR tube. Using 3- (trimethylsilyl) propionic acid-d₄Sodium salt (0.05 w)V%) as internal standard on a Bruker ADVANCE III 400MHz NMR spectrometer (Bruker)¹³C-NMR spectrum. Based on the integral ratio of carbon to carbonyl carbon of aldehyde newly formed by alginate oxidation, according to¹³The H-NMR spectrum determines the actual oxidation. (FIG. 25).

Synthetic calcium crosslinked OA hydrogels

OA is stored in lyophilized form at-20 ℃ until a hydrogel is ready to be prepared. The hydrogel was prepared as follows: OA is dissolved in Dulbecco's modified Eagle medium (DMEM, Gibco cat # 11885084) and stirring is continued at room temperature for at least 30 minutes until the desired weight/volume ratio (wt%) is reached. OA by reaction with supersaturated calcium sulfate (CaSO)₄84mg/mL) of the solution at a concentration of 20L CaSO₄1 wt%/mL alginate (e.g., 40, 100 and 200L CaSO)₄Mixed with 1mL of alginate 2 wt%, 5 wt% and 10 wt%, respectively) to crosslink. OA solution and CaSO₄The slurry was loaded into two 1mL syringes, respectively. After connecting the two syringes together with female-female luer lock (Value Plastics), the two syringes were mixed (about 30 times).

Physical degradation of oxidized alginate hydrogels

In vitro physical degradation of OA hydrogels was tested by forming 1mm x 4mm disks with an oxidation rate of 2% or 5% and a weight/volume ratio of 2, 5 or 10 wt%. The discs were formed by injecting OA hydrogel between two glass plates separated by a 1.0mm spacer using a 25 gauge needle. After the gel was polymerized at room temperature for 30-45 minutes, it was fabricated into disks with a diameter of 4mm using a biopsy punch. The gel was transferred to a 0.4m transwell cell (Millicell, cat # MCHT12H48) in a 12-well plate containing 3.2m1 DMEM and the plate was shaken continuously (100RPM) at 37 ℃. At T₀(initial control), 4 hours, 1 day, 3 days, 5 days and 7 days (D) the hydrogels were collected. Three hydrogels were pooled together with an average of 3-5 independent replicates (samples) per time point. The samples were frozen at-80 ℃ and subsequently lyophilized (VirTis Wizard 2.0). Dry weight was measured with an analytical balance (Mettler Toledo AT 201).

In vivo degradation assays were performed by murine subcutaneous assays. All experiments were approved by the UCSF Institutional Animal Care and Use Committee (IACUC). Animals were anesthetized with isoflurane (2.5% induction, 1.5% maintenance), the animal skin was cleaned with 70% ethanol, and 1001OA hydrogel was injected into each subcutaneous pocket on the back side of the mice using a 25 gauge needle (up to 6 hydrogels per animal). Mice were housed in colonies and monitored for 48 hours for signs of pain and distress. Animals survived for 18 hours, 3 days, 5 days, or 7 days. At the end time point, the gel in the subcutaneous bag was microdissected, placed in a freezer at-80 ℃, lyophilized and the dry weight recorded as indicated by the in vitro degradation test.

Stability of OA

The 2% OA polymer was dissolved at 5 wt% as described above and then pipetted into a 3ml syringe and stored at 4 ℃ or-20 ℃. The stored syringe (n 5/time) was removed 1, 4, 10, 20 or 30 days after dissolution, allowed to reach room temperature and then crosslinked by mixing the OA solution with the supersaturated calcium sulphate slurry described above. Immediately after mixing, the crosslinked OA was placed between two glass plates separated by a 0.75mm spacer and allowed to stand at room temperature for 30 minutes to form a hydrogel. Calcium crosslinked OA hydrogel disks were made using a biopsy punch with a diameter of 6 mm. To evaluate the stability of the OA solutions, the modulus of the calcium crosslinked OA hydrogels was measured over time. The elastic modulus of the hydrogel was determined as follows: uniaxial, unconstrained, constant strain rate compression testing was performed on a mechanical tester (225lbs activator, TestResources) equipped with a 5N load cell at room temperature at a constant crosshead speed of 1%. The modulus of elasticity is calculated from the first non-zero linear slope of the stress-strain plot and is limited to the corresponding value when the strain reaches the first 5%.

Mass spectrometry measurement of cevimeline

The Shimadzu research LC-20ADXR system was connected to a 4500QTrap mass spectrometer with an electrospray ionization (ESI) source for LC-MS/MS analysis. Using Phenomenex(50X 3mm, 2.6 μm) column and Phenomenex SecurityGuard C18 guard column (3)mm × 2.0mm) to effect chromatographic separation. The column temperature was controlled at 30 ℃. Mobile phase a was 0.1% trifluoroacetic acid in water and mobile phase B was Acetonitrile (ACN). The amount of sample was 10. mu.L, and the flow rate was 0.5 mL/min. The gradient conditions were: (time/min,% mobile phase B): (0, 15), (1, 15), (2.5, 22.8), (2.6, 98), (5, 98), (5.1, 15) and (8, 15). The source parameters included gas curtain pressure 45psi, ion spray voltage 4000V, ion source temperature 550, and neutral collision gas. The MS/MS parameter settings in positive ion ESI mode are summarized in Table 1.

A standard working solution of cevimeline was prepared in methanol at a concentration ranging from 10.0 to 10000.0 ng/mL. The working solution was added to 100. mu.L of DMEM to generate standards or QC samples. Final concentrations of calibration standards in DMEM were 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 and 100.0, 250.0 and 500 ng/mL. QC sample concentrations were 0.9, 4, 30.0, and 400.0 ng/mL.

For the sample preparation procedure, 10. mu.L of IS working solution (1000ng/mL) was added to each 100. mu.L of DMEM. Protein precipitation was performed using 96-well protein precipitation filter plates Sigma-Aldrich (St. Louis, Mo.) and 300pl MeOH ACN (15: 85). After filtration, 200. mu.l NH₄OH 5% was added to 200. mu.l of the sample, and 10. mu.l was injected into LCMS system. The bioanalytical method was validated against current U.S. FDA guidelines, including selectivity, linearity, intra-and inter-day precision and accuracy, recovery and stability ("industrial guidelines, bioanalytical method validation", 2018FDA gov/media/70858/download).

TABLE 1 optimized ESI (+) mass spectrometry conditions for compound Multiple Reaction Monitoring (MRM).

Release of cevimeline from alginate hydrogels

In vitro release of Cevimeline (CV) from OA hydrogels was tested by forming 1mm x 4mm disks with an oxidation rate of 2% or 5% and a weight/volume ratio of 2, 5 or 10%. Alginate + cevimeline (Abcam, ab141317) hydrogel was prepared as follows: in and withPrior to freeze-dried OA mixing, the drug was resuspended in DMEM at the desired concentration, followed by crosslinking as described above to encapsulate the drug with OA hydrogel. Three 4mm alginate + cevimeline hydrogels were placed in 0.4m transwell cells in 12-well plates containing 3.2ml DMEM and incubated at 37 ℃ with slow continuous stirring (100 RPM). Sample media (3ml DMEM) was collected and supplemented at 4 hours, day 1, day 2, day 3, day 4, day 5 and day 7 (D). Three hydrogels were pooled together with an average of 3-5 independent replicates (samples) per time point. The sample medium was stored at-20 ℃ until mass spectrometry was performed. The graph shows only relevant data up to day 4, since then all cevimeline has been released. Will T₀(initial control) the gel was dissolved in DMEM to determine the baseline of cevimeline encapsulated in the hydrogel.

Animal experiments

Unless otherwise indicated, adult female mice (6-8 weeks old) from the C57/BL6 inbred strain (Harlan Laboratories) were used for all experiments. All procedures were approved by the UCSF Institutional Animal Care and Use Committee (IACUC) and followed the guidelines for care and use of NIH laboratory animals.

In vivo cevimeline delivery

Cevimeline was prepared as described before, and 0.5ml Monoject was used^TMTuberculin syringes and 28G 1/2 needles (Covidien) delivered free cevimeline by Intraperitoneal (IP) injection, or alginate and cevimeline mixtures by subcutaneous injection using 1ml TB syringes and 25G x 5/8 needles (Becton Dickinson).

For the intraglandular injection of cevimeline, the mice were administered analgesics (carprofen and buprenorphine; Paterson Veterinary and Buprenex; 0.1mg/kg and 100mg/kg, respectively, (IP)) 30 minutes prior to surgery and by inhalation 2% isoflurane/O₂The mixture is anesthetized. The surgical area was shaved and an incision was prepared, washed with alternating iodine and alcohol, and then a local anesthetic (lidocaine; Hospira inc., 8mg/kg) was applied. A cut is made in the throat along the craniocaudal axis using spring scissors. Then using tuberculin syringe mounted on sliding tipA227G x 1/2 needle (Becton Dickinson) on (Becton Dickinson) injected cevimelin into the inframandibular and sublingual salivary gland leaves. The skin was sutured using a non-absorbable suture thread (Ethicon). Mice were euthanized at 18 hours, 3 days, or 7 days, and some animals were injected 2 hours prior to sacrifice with 0.9% saline containing 0.25mg/25g of 5-ethynyl-2' -deoxyuridine (EdU, thermo fisher Scientific).

-radiation therapy

Animals were anesthetized with 100 μ l/10g bw 2.5% avermectin (2, 2, 2-tribromoethanol (Alfa Aesar), tert-amyl alcohol (2-methyl-2-butanol) (Spectrum)) in 0.9% normal saline (Vedco Inc.). The mice were placed in Shepherd Mark I irradiator (JL Shepherd)&Associates). The body was shielded from radiation using a lead block and the head and neck regions were exposed to 2 doses of 5Gy (10Gy total) of radiation to irradiate the Salivary Glands (SG). Control mice received anesthesia but no radiation treatment. All mice were allowed to recover completely before returning to normal rearing environment and were given ad libitum access to soft food (ClearH)₂O)。

Saliva Collection

Mice were anesthetized with 2% inhaled isoflurane or avertin (for radiation studies). 5mg of sodium citrate (Spectrum Chemical) dissolved in 12.5. mu.l of sterile water was dropped into the mouse mouth. After 2 minutes, a piece of filter paper was inserted into the animal's mouth and left for 5 minutes under anesthesia. The salivary secretion amount was determined by measuring the weight of the filter paper before and after collection using a precision scale (Denver Instrument SI-64, di ═ 0.1 mg).

Tissue treatment

After euthanasia was performed, SG was collected, snap frozen and OCT embedded. 10 μm cryosections were cut using a cryostat (Leica) and used for immunofluorescence studies.

Immunofluorescence Studies

Immunofluorescence analysis of tissue sections was performed as follows. Briefly, freshly frozen tissue was fixed with 4% PFA for 10 minutes at room temperature, then permeabilized with 0.5% Triton-X100 for 10 minutes at room temperature. Tissues were blocked for 2 hours at room temperature with 10% donkey serum (Jackson laboratory, Maine), 5% BSA (Sigma-Aldrich) and MOM IgG blocking solution (Vector Laboratories, Calif.) in 0.01% PBS-Tween 20. SG sections were incubated overnight at 4 ℃ with the following primary antibodies: rabbit anti-AQP 5 (1: 200, Millipore, AB3559), goat anti-SOX 2 (1: 200, Neuromics, GT15098), rabbit anti-Ki 67 (1: 200, Abcam, AB16667), rat anti-Ki 67 (1: 200, DAKO, M7249), rabbit CHRM3 (1: 1000, research diagnostics, AS-3741S), rat anti-E-cadherin (1: 400, Invitrogen, 13-1900). Antibodies were detected using Cy2-, Cy3-, or Cy 5-conjugated Fab fragment secondary antibodies (1: 300, Jackson laboratories) and nuclear staining was performed using Hoechst 33342 (1: 1000, Anaspec Inc.). EdU staining was performed using the Click-iT EdU Alexa-Fluor 488 or 647 kit (Invitrogen). Slides were fixed using fluorocount-g (southern biotech) and fluorescence was analyzed using Leica Sp5 confocal microscopy. Image processing and quantification was performed using NIH ImageJ software.

Statistics of

Statistical tests were performed using GraphPad Prism software v 8. Data are presented as mean ± SD, and subjected to either a two-tailed unpaired student test (applicable to both datasets) or a common one-way ANOVA using Tukey multiple comparison test (applicable to more than two datasets). Significance was assessed using P-value cutoff as follows: p < 0.05, P < 0.01, P < 0.001, P < 0.0001. The specific data set analysis is described in the figure.