WO2017009658A2

WO2017009658A2 - Method

Info

Publication number: WO2017009658A2
Application number: PCT/GB2016/052147
Authority: WO
Inventors: Bing Hu; Heng ZHUANG; Christopher Jeremy TREDWIN
Original assignee: University Of Plymouth
Priority date: 2015-07-16
Filing date: 2016-07-15
Publication date: 2017-01-19
Also published as: WO2017009658A3; GB201512410D0

Abstract

The present invention provides an ATPase inhibitor for use in the treatment or prevention of tooth cell autophagy and/or for use in the treatment or prevention of autophagy in deciduous and/or permanent teeth. Methods of treatment of the same are also provided, as are compositions comprising a local anaesthetic and an ATPase inhibitor.

Description

Method

Field of Invention

The present invention is directed to compositions comprising a local anaesthetic and an ATPase inhibitor and to uses of ATPase inhibitors for treating or preventing tooth cell autophagy.

Background to the Invention

Dental pulp found in the centre of teeth contains mesenchymal cells, which are responsible for the development of dentin and its regeneration following external damage (caused by injury or caries). Over the last decade, tooth pulp cells have been shown to be an important multipotential stem cell resource that can differentiate into various cell types including neurons. Such is the importance of these stem cells that "tooth banks" have been established in the US and Europe to store shed deciduous teeth and extracted young permanent teeth, such as impacted third molar teeth. Maintaining a healthy tooth pulp is essential for preserving a fully functional or developing tooth; this is entirely dependent on intact pulp cellular processes and functions.

Dental treatment involves similar or more frequent use of local anaesthesia than any other clinical discipline. The development of human permanent teeth from tooth bud formation to complete eruption and root formation is a long process that can take more than 10 years to complete. Children have a high risk of dental injuries and caries, and dental treatments almost invariably entail local anaesthesia to facilitate pain relief and enhance patient cooperation. The most commonly used techniques for anaesthetic administration in dentistry are local infiltrations and regional nerve blocks. Local infiltration involves the injection of an anaesthetic agent close to the operative site (usually the root apex of a tooth) and anaesthetizes only the most terminal branches of a nerve. Regional nerve blocks, on the other hand, require administration of the agent in the vicinity of a major nerve trunk where it is unprotected by the bone, thereby achieving anaesthesia in the entire area of distribution of the nerve. Local anaesthetics are known to work by binding to voltage-gated Na⁺ channels in nerves, thereby blocking sodium transportation and nerve conduction. Although the maximum doses of various local anaesthetics are well established, the side effects of these agents on dental tissues have not yet been fully investigated. The only relevant literature in this regard relates to a canine model, which reported that local anaesthetics could accumulate in natural cavities, such as the crypts of tooth buds and the mandibular canal. More importantly, a recent clinical epidemiological study showed that local anaesthesia interferes with human permanent tooth development and induces tooth agenesis (developmental failure of the permanent teeth, resulting in tooth loss) through unknown mechanisms.

The present inventors set out to address an urgent need to investigate the effects of local anaesthetics on tooth development, particularly on tooth pulp cells. Through their studies the inventors have identified the mechanisms by which local anaesthetics interfere with tooth development and induce tooth agenesis. An object of the present invention is therefore to provide compositions and methods to reduce, prevent or eliminate these harmful side effects.

Summary of the Invention

The present inventors are the first to establish that the detrimental effects of local anaesthesia on human permanent tooth development is due to a pathological increase in tooth cell autophagy. Autophagy is a catabolic process involving the degradation of unnecessary or aberrant cellular components through hydrolysis of lysosomes. It controls the turnover of organelles and proteins within cells, and of cells within organisms. During this process, targeted cytoplasmic constituents are isolated within autophagosomes, which then fuse with lysosomes to form autolysosomes where the cellular material is degraded or recycled. The inventors have also elucidated the intracellular mechanism underlying this increase in autophagy and have established that ATPase inhibitors can be used to reduce or prevent the effects of pathological autophagy in tooth cells. Accordingly, in a first aspect the present invention provides a composition comprising a local anaesthetic and an ATPase inhibitor.

In a second aspect, the present invention provides an ATPase inhibitor for use in the treatment or prevention of tooth cell autophagy. In a third aspect the present invention provides a method for treating or preventing tooth cell autophagy, the method comprising administering an ATPase inhibitor to a patient in need thereof.

Description

ATPases are a class of enzymes that catalyze the decomposition of ATP (adenosine triphosphate) into ADP (adenosine diphosphate) and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions. There are different types of ATPases, which can differ in function (ATP synthesis and/or hydrolysis), structure (F-, V- and A-ATPases contain rotary motors) and in the type of ions they transport. Suitable types of ATPase for use in the present invention include F- ATPases, V-ATPases, A- ATPases, P-ATPases and E-ATPases or combinations thereof.

In more detail, F-ATPases are commonly found in mitochondria, chloroplasts and bacterial plasma membranes where they are the prime producers of ATP, using the proton gradient generated by oxidative phosphorylation (mitochondria) or photosynthesis (chloroplasts). V-ATPases (Vacuolar-type ATPases) are primarily found in eukaryotic vacuoles, where they energise multiple transport processes and regulate pH in cells and organelles by coupling ATP hydrolysis to proton pumping. A- ATPases are found in Archaea and function like F-ATPases. P-ATPases are found in bacteria, fungi and in eukaryotic plasma membranes and organelles, and function to transport a variety of different ions across membranes. E-ATPases are cell-surface enzymes that hydrolyse a range of NTPs (nucleotide triphosphates), including extracellular ATP. In preferred embodiments of the invention the ATPase inhibitor is a V-ATPase inhibitor. Without being bound by theory, the inventors believe that over-activity of V-ATPase underlies pathological tooth cell autophagy, such as that observed following local anaesthetic treatment or acquired during the ageing process. Inhibiting the activity of ATPase, especially V-ATPase, therefore prevents or reduces the pathological effects of autophagy.

Suitable V-ATPase inhibitors for use in the compositions and methods of the present are known in the art. Such inhibitors bind to the c units of the V-ATPase V₀ complex to block the proton pumping activity of V-ATPase. Examples of V-ATPase inhibitors include plecomacrolides such as bafilomycin (including bafilomycin Al, Bl, CI and Dl) and concanamycin; macrolactones such as archazoloids; and benzolactone enamides, such as apicularen. The ATPase inhibitor may be bafilomycin Al or bafilomycin Bl, preferably the ATPase inhibitor is bafilomycin Al . In embodiments of the invention one or more V-ATPase inhibitors may be used in combination. Compositions of the invention may comprise from about 0.01 μg/ml to about 2 μg/ml, preferably from about 0.01 μg/ml to about 1 μg/ml, more preferably from about 0.01 μg/ml to about 0.5 μg/ml of ATPase inhibitor. In embodiments of the invention the compositions comprise from about 0.01 μg/ml to about 0.1 μg/ml of ATPase inhibitor.

As mentioned above, tooth cell autophagy may be induced by local anaesthetic treatment, such as dental anaesthetic treatment. The present invention therefore provides compositions comprising an ATPase inhibitor, preferably a V-ATPase inhibitor, and a local anaesthetic. In embodiments of the invention the local anaesthetic is an aminoamide or aminoester anaesthetic. Preferably, the local anaesthetic is a dental anaesthetic such as an amide-type local anaesthetic. Examples of amide-type local anaesthetics include lidocaine, articaine, bupivacaine, mepivacaine, prilocaine, or combinations thereof.

Compositions of the invention may comprise from about 1 mg/ml to about 200 mg/ml, preferably from about 10 mg/ml to about 80 mg/ml, more preferably from about 20 mg/ml to about 50 mg/ml of local anaesthetic. In embodiments of the invention the compositions comprise about 40 mg/ml of local anaesthetic.

Compositions of the invention may additionally comprise one or more pharmaceutically acceptable carriers or excipients. Suitable carriers and excipients are well known in the art and may include one or more of sodium chloride, sodium sulphite, potassium metabi sulfite, edetate disodium, sodium hydroxide, hydrochloric acid or water, or combinations thereof.

Compositions of the invention may comprise one or more additional active agents. In embodiments of the invention the additional active agent may be a vasoconstrictor, such as adrenaline or a hydrochloride salt thereof. Additionally or alternatively, compositions of the invention may comprise a preservative such as caprylhydrocuperienotoxin and/or a reducing agent such as sodium metabisulphate. Compositions may also include a water-based or adherent vehicle, such as an alumina gel-based vehicle. In embodiments of the invention the vehicle may be sterile water only.

In embodiments of the invention the composition is suitable for parenteral administration via intravenous and/or infiltration routes. The composition may be a solution for injection or infusion. In preferred embodiments of the invention the composition is a solution for injection. As mentioned above, the present invention provides an ATPase inhibitor for use in the treatment or prevention of tooth cell autophagy. The tooth cells may be tooth germ cells such as tooth pulp cells, tooth epithelial cells and/or periodontal mesenchymal cells. The tooth pulp cells may be tooth pulp mesenchymal cells and/or tooth pulp stem cell progenitors. In preferred embodiments of the invention the tooth cells are tooth pulp cells. According to the present invention ATPase inhibitors may be used to treat or prevent autophagy in permanent and/or deciduous teeth. The ATPase inhibitors can therefore help to prevent tooth agenesis and preserve cellular processes and functions (such as the ability of tooth pulp wound healing and dentine regeneration) which may be damaged by autophagy. Tooth cell autophagy as described herein may be induced, spontaneous or acquired autophagy. In all instances this refers to pathological tooth cell autophagy. In other words, levels of tooth cell autophagy are increased to pathological levels such that cell proliferation is reduced. This reduced cell proliferation can lead to tooth agenesis and/or a reduced ability of the tooth to regenerate following external damage, such as may be caused by injuries or caries. Tooth cell autophagy may be induced by exposure to an extrinsic agent, such as a local anaesthetic or other drug, disease, caries, trauma or overheating etc. Spontaneous or acquired autophagy may develop due to intrinsic processes such as part of the natural ageing process. The present invention also provides a method for treating or preventing tooth cell autophagy, the method comprising administering an ATPase inhibitor to a patient in need thereof. In preferred embodiments of the invention the patient is a mammal such as human. Preferably the patient is a human pediatric patient.

In embodiments of the invention the ATPase inhibitor may be administered in combination with a local anaesthetic, preferably a dental anaesthetic. For example, the ATPase inhibitor may be administered in combination with an aminoamide or aminoester anaesthetic. Preferably, the local anaesthetic is a dental anaesthetic, more preferably an amide-type local anaesthetic, such as one or more of lidocaine, articaine, bupivacaine, mepivacaine, prilocaine, or a combination thereof. The ATPase inhibitor may be co-administered with the local anaesthetic, in other words, the ATPase inhibitor and the local anaesthetic may be in a single formulation. Alternatively, the ATPase inhibitor and the local anaesthetic may be provided as separate formulations, administered either simultaneously or sequentially.

Preferably, the ATPase inhibitor is administered parenterally, such as by injection. When administered in combination with a local anaesthetic the ATPase may be administered as a local infiltration or regional nerve block. Brief Description of the Drawings

Figure 1. Local anaesthetics can infiltrate into tooth germ and pulp. A. Fluorescein labelled lidocaine was injected into freshly extracted 5-months-old pig mandible through foramen (asterisk) for 2 hours. The bone chamber containing the third lower permanent molar tooth germ was indicated with arrow (left panel). The tooth germ was then exposed to illustrate the close anatomical association of the tooth with mandibular foramen and canal (right panel). Note that the drug has already entered the tooth socket and stained the tooth. B. After 2 and 16 hours, the third molar germs were extracted and examined. The colour difference indicates where and how much of the anaesthetics infiltrate into the different compartments of the tooth germs. C. The concentration of the drugs was analysed by measuring green fluorescence intensity using a Leica SP5 confocal microscope and Photoshop CS6 software, and calculated in Prism 5.0 software at different sites of the tooth pulp of the third molar pulp (for nerve block) and the first molar mesial root pulp (for infiltration) (Representative images can be found in Figure ID and E). The values represent average signals from 4 independent samples. Scale bars, 5mm. D. Representative images for the fluorescein labelled lidocaine distribution inside the 3rd molar germs after receiving lidocaine nerve block injection (for illustration and quantification please see Figure 1A and C). E. Representative images of the apical part of the first molar mesial root pulp at 2 and 16 hours after infiltration injection with fluorescein labelled Ubistetin and Scandonest. The signal quantification of A and B can be found in Figure 1C. Cell nuclei were visualized using 4',6-diamidino-2-phenylindole (DAPI). Scale bars, ΙΟμπι.

Figure 2. Local anaesthetics affect tooth pulp cell proliferation but not differentiation and apoptosis. Real-time RT-PCR analysis of Ki67 (A), Bax (B), p38 (C) and Runx2 (D) mRNA expression in cells treated with increasing concentrations of the drugs tested for 16 hours, normalized to expression of the 36β4 housekeeping gene. TUNEL analysis was performed on the same samples and no difference of apoptosis index have been found (data not shown). UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. Figure 3. Local anaesthetics induce autophagy in tooth pulp cells. A. Phase contrast microscopy images of drug-treated and control cells. B. and C. Immunofluorescence analysis of LAMP- 1 and LC3 expression in control cells and cells treated with 2 mM anaesthetic drug. D. Western blot (left panel) analysis of LC3 expression in two primary dental pulp cell lines with and without drug treatment. Note that LC3 is expressed as two isoforms with molecular weights 17 kDa (LC3-II) and 19kDa (LC3- I). β-Actin was used as a loading control. Signal quantification (right panel) was performed on C-DiGit Blot scanner acquired gel images using Image Studio 4.0 software. Relative signal was calculated as LC3II (17kDa) vs LC3I (19kDa) then normalized against β-Actin signal. E. GFP-LC3 fusion protein expression in control cells and cells treated with 0.5 mM anaesthetic drug. Cells were counterstained for Phalloidin. Cell nuclei were visualized using 4',6-diamidino-2-phenylindole (DAPI). Scale bars, 20μπι. UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. Scale bars, ΙΟμπι. F. and G. Immunofluorescence analysis of LAMP-1 (F.) and LC3 (G.) expression following treatment with 0.5mM anaesthetic drug. H. and I. Immunofluorescence analysis of LC3 expression in 2-hour and 16-hour samples as indicated in Figure 1 for nerve block (H.) and infiltration injections (I). Cell nuclei were visualized with DAPI. UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. Scale bars, ΙΟμπι. Figure 4. Local anaesthetics affect tooth pulp cellular energetics. A. BrdU labeling analysis in cells treated with 0.5 or 2mM concentrations of the five anaesthetic drugs for 2, 4, 8 and 16 hours. B. Western blot analysis of LC3, p62 and mTOR protein levels in cells treated with 2mM concentration drugs. Similar results have been achieved with lidocaine (data not shown). The quantification of the individual experiment can be found in Figure 4F. C. Real-time RT-PCR analysis of p62 gene in tooth pulp cells treated with the five anaesthetics for different time periods, normalized to 36β4 housekeeping gene expression. Error bars represent standard deviation of triplicate experiments. D. and E. Key parameters of mitochondrial function including basal respiration, phosphorylation, proton leak and maximum respiration were tested by directly measuring the oxygen consumption rate (OCR) of cells treated with the five anaesthetics with 0.5mM and 2mM concentrations for 2 hours (4, 8, 16 hours results can be found in Figure 5C and D). Lido, Lidocaine; UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. Number abbreviations after individual drug abbreviation: 0.5: 0.5mM; 2: 2mM. * p<0.05; ** p<0.01. F. Quantification of the western blot experiments in Figure 4B. For LC3, LC3II/LC3I ratio and normalized with β-Actin signal. For p62 and phosphor-mTor the signal were normalized directly to β-Actin. G-L Key parameters of mitochondrial function including basal respiration, phosphorylation, proton leak and maximum respiration were tested by directly measuring the oxygen consumption rate (OCR) of cells treated with the five anaesthetics of 0.5mM and 2mM concentrations for 4 (G), 8 (H) and 16 (I) hours. 2 hours' results can be found in Figure 4D and E. Note that at 16 hours basal respiration, phosphorylation and maximum respiration were all reduced in the 2mM UBI-F, Sept, Scan and Lido treated cells. Lido, Lidocaine; UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. Number abbreviations after individual drug abbreviation: 0.5: 0.5mM; 2: 2mM. * p<0.05; ** p<0.01. Figure 5. Local anaesthetics affect tooth pulp cell proliferation via autophagy induction. A. Western blot analysis of LC3 protein levels in cells treated with 0.5mM and 2mM concentrations of the indicated anaesthetic drugs and together with ΙΟΟηΜ bafilomycin for 8 and 16 hours, β-actin was used as a loading control by stripping and re-blotting the same blot. The quantification of the individual experiment can be found in Figure 5D. B. BrdU positive cells indexing (four random fields were analysed under lOx lens in a Zeiss LSM510Meta laser-scanning microscope) of the samples indicated in (A) and Supplemental Figure 5. * p<0.05; ** p<0.01. C. Western blot analysis of LC3 protein levels in cells treated with 0.5mM and 2mM concentrations of the indicated anaesthetic drugs and together with ΙΟΟηΜ bafilomycin for 8 and 16 hours, β-actin was used as a loading control by stripping and re-blotting the same blot. The blots are from the same series of experiment showed in Figure 5A. D. Quantification of the western blot experiments in Figure 5A and C. For LC3, LC3II/LC3I ratio and normalized with β-Actin signal. Lido, Lidocaine; UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. Figure 6. p62 is the common downstream target of anaesthetic drugs on tooth pulp cells. A. Heatmap analysis of PCR array data showing changes in gene expression in tooth pulp cells after 16 hours treatment with indicated 2mM anaesthetic drugs (for 0.5mM dosage, please see Supplemental Figure 6). Colours represent fold changes in expression, as shown in the key. B. Real-time RT-PCR analysis of a panel of autophagy-related genes in primary tooth pulp cells using specific primers, normalized to 36β4 housekeeping gene expression. Error bars represent standard deviation of the triplicate experiments. C. Western blot analysis of p62 expression in drug-treated cells, β-actin was used as a loading control by stripping and re-blotting the same blot. p62 signal quantification was calculated with normalization against β-actin signal (right panel). Similar results have been achieved with lidocaine both for Western Blot and gene profiling (data not shown). UBI, Ubistesin; UBI-F, Ubistesin forte; Scan, Scandonest; Sept, Septanest. * p<0.05; ** p<0.01. C. Heatmap analysis of PCR array data showing changes in gene expression in tooth pulp cells after treatment with 0.5 mM anaesthetic drug (for 2mM dosage data see Figure 6A). Colours of the bars reflect fold changes in expression, as indicated by the key.

Examples

Materials and Methods

Drugs

The commercial anesthetic drugs used in this study were articaine based agents: Ubistesin (522721, 3M ESPE), Ubistesin forte (512987, 3M ESPE), Septanest (09091451103, Septodont), and a mepivacaine based agent: Scandonest (09091173002, Septodont) and a Lidocaine based agent: Lidocaine (Batch 4180, Dentsply). Animal model of local anesthetics application

Freshly isolated mandibles from 5-months-old Gloucester Old Spot crossed with Landrace pigs (kindly provided by a local abattoir) were used in the experiments. Local anaesthetics: Lidocaine (for nerve block), Scandonest or Ubistesin-Forte (for infiltration) was pre-labelled with 0.5% fluorescein. 1ml Lidocaine was then injected into the mandibular foramen and 0.8ml Scandonest or Ubistesin-Forte was injected respectively into buccal and lingual mesial submucous sites around the mesial apical root of mandibular first molars using 23 G needle and 1ml syringe. After 2 and 16 hours, the third permanent molar tooth germ and the first permanent molar root pulp were excised and embedded in OCT compound (Tissue-Tek; Sakura Finetek). 8μπι frozen sections were prepared. The fluorescence images of the tissues were acquired using a Leica SP5 confocal microscope and measured with Adobe Photoshop CS6 software then normalised against the signal of the labelled drugs.

Cell isolation and culture

Cell isolation and treatment protocols were approved by the Ethics Committee of the Peking University School and Hospital of Stomatology, Beijing, China. Specifically, young permanent tooth pulp cells were obtained from two patients (12 and 16 years old) whose upper first premolars were extracted for orthodontic reasons. Following extraction, the tooth chamber was first opened using a dental drill. Pulp tissue was isolated using a 23-gauge fine needle and digested with 3% collagenase I (C0130-1G; Sigma) dissolved in Hank's balanced salt solution (Gibco, Cat. No.14175-053) supplemented with 1% penicillin-streptomycin (SV30079.01, Hyclone) using a final volume of 2ml enzyme solution per gram of tissue for 1 hour at 37°C with constant agitation. The enzyme reaction was then stopped by adding an equal volume of complete cell culture medium, comprising Dulbecco modified Eagle's medium/F12 (31331-028, Gibco) supplemented with 20% fetal bovine serum (F7524, Sigma) and 1%) penicillin-streptomycin. The culture medium was changed every 2 days and cells were passaged when they reached 70-80% confluence by digestion with 0.05% Trypsin-EDTA (25300-054, Gibco). Passage 7-9 cells at 70% confluence were used for all experiments. Autophagy Inhibitor assay

A lOmM stock solution of the autophagy inhibitor bafilomycin Al (No. 11707, Sigma) was prepared in DMSO. Cells were treated with ΙΟΟηΜ bafilomycin and control cells were treated with vehicle alone. For the autophagy inhibition assay, cells were exposed to anaesthetics and bafilomycin at the same time. Plasmid preparation and transfection GFP-LC3II plasmids 11 were used for visualization of autophagosome formation. The GFP-LC3 fusion protein is expressed throughout the cytoplasm in the absence of autophagy, but translocates to the autophagosome membrane upon autophagy induction to form multiple bright green fluorescent spots. Plasmids were amplified and extracted using Plasmid Maxi Kit (12162, Qiagen), according to the manufacturer's recommendations. Cells were seeded at 2x10⁵ cells/ml into 6-well plates and cultured for 24 hours until 60-80% confluence. For each transfection, 2μg of plasmid DNA was mixed with 200μί of jetPRIME buffer (114-15, PolyPlus) and the mixture was added to an equal volume of jetPRIME solution and added to cells. Transfected cells were treated with anaesthetic drug treatment after 24 hours.

Immunostaining

Cells cultured on glass coverslips were fixed in 4% ice-cold paraformaldehyde (diluted in lOmM PBS), incubated at room temperature for 20-30 minutes, and then rinsed three times for 5 min in PBST (lOmM PBS containing 0.1% Triton-XlOO). Non-specific binding sites were first blocked by incubation in PBST containing 5% donkey serum, 0.25% cold water fish gelatin and 0.25% BSA for 60 minutes. Primary antibodies were anti-LC3A/B (D3U4C; Cell Signaling, Cat. No. 12741; 1 : 1000 dilution), and anti-lysosome-associated membrane glycoprotein 1 ([LAMP-1] D2D1 1; 9091, Cell Signaling; 1 :250 dilution). These were diluted in blocking buffer and incubated overnight at 4°C with cells. After three 5 minute washes in PBST, cells were incubated with fluorochrome-conjugated secondary antibodies diluted in blocking buffer for 1-2 hours at room temperature in the dark. Secondary antibodies were Alexa 488 donkey anti-rabbit IgG (A21206, Life Technologies) and Alexa 568 donkey anti-rabbit IgG (A10042, Life Technologies). Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI, D9542, Sigma- Aldrich, 1 : 10000) for 10 minutes. The cytoskeleton was visualized by counterstaining with DyLight 554 Phalloidin (13054, Cell Signaling, 1 :200 dilution). Immunofluorescence images were obtained using either a Leica SP5 or a Zeiss LSM510Meta laser-scanning microscope.

BrdU staining and quantification BrdU (RPN201, Amersham-GE) was diluted in complete cell culture medium at a ratio of 1 : 1,000 and added on top of tooth pulp cells for 2 hours. Cells were then fixed in 4%PFA then treated with of 2N HC1 for 30 minutes before they were stained with anti-BrdU antibodies (ab6326, Abeam, 1 :500 dilution). The antibody staining procedures were exactly as above except the blocking buffer was prepared with 2.5% BSA. After imaging, the images were processed in Image J software and BrdU positive cells and total cell number were quantified using "Analyze Particles" function.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Apoptosis was assayed using an In Situ Cell Death Detection Kit (11 684 795 910, Roche), following the manufacturer's standard protocol.

Western blotting

Proteins were extracted using Ripa buffer (89901, Pierce) supplemented with Halt Protease and a Phosphatase Inhibitor Cocktail (78440, Pierce), and quantified using a BCA Protein Assay (23225, Pierce). Protein separation and membrane transfer were performed using NuPage precast gels ( P0335BOX, Life Technologies) and transfer buffer (NP0321, Life Technologies). Antibody incubation and washes were performed using the iBind system (Life Technologies). Anti-LC3A/B (D3U4C; 12741, Cell Signaling, 1 :5,000), anti-p62 (PM045, MBL International, 1 :3,000) and anti-phospho mTor (Ser2481) (2974, Cell Signaling, 1 :2,000) primary antibodies were used. As a loading control, blots were simultaneously probed with anti-P-actin antibodies (8457P, Cell Signaling, 1 :5,000) or re-probed after treating with Restore Western Blot Stripping Buffer (21059, Pierce). Protein bands were visualized using a C-DiGit system (Li cor) with C-Digit Image Studio (Version: 1.0.19, Licor) software. Detected molecular weight: LC3I: -19 kDa; LC3II: -17 kDa; β-actin: 45 kDa; p62: 62kDa; pmTor: 289kDa.

Real-time RT-PCR and result analysis

RNA and cDNA preparation, real-time RT-PCR, and statistical analysis were performed as previously described (Hu et al 2012). Primers to the human genes used in this study were Atg3-F, 5 '-TTTGGCTATGATGAGCAACG-3 '; Atg3-R, 5'- AAGTTCTCCCCCTCCTTCTG-3'; Atg5-F, 5 '-C AGATGGAC AGTTGC AC AC A-3 '; Atg5-R, 5 '-CTGTTGGCTGTGGGATGAT A-3 '; Atg7-F, 5'-

CTGGGGACTTGTGTCCAAAC-3 '; Atg7-R, 5 '- AGAGGTTGGAGGCTC ATTC A- 3'; Atgl2-F, 5'-AATCAGTCCTTTGCTCCTTCC-3'; Atgl2-R, 5'- CACGCCTGAGACTTGCAGTA-3 '; Bax-F, 5 '-GGC ATC ATT AACTGGGGAAG-3 '; Bax-R, 5 '-TCC AGCC AGATTT AGGTTC AA-3 '; Beclin-F, 5'-

AGGTTGAGAAAGGCGAGACA-3 '; Beclin-R, 5'-

AATTGTGAGGAC ACCC AAGC-3 '; Ki67-F, 5 '-GAATTGAACCTGCGGAAGAG- 3'; Ki67-R, 5 '-TTTGCTGTTCTGCCTC AGTC-3 '; LC3-F, 5'- CGTCCTGGACAAGACCAAGT-3 '; LC3-R, 5'-TCCTCGTCTTTCTCCTGCTC-3'; p38-F, 5 '-GTC AACTGGAGC AAGAAGGA-3 '; p38-R, 5'-

ATGTGGTCACATGTGCAAAG-3 '; p62-F, 5 '-TGGACCC ATCTGTCTTC AAA -3'; p62-R, 5 '- ATGGAC AGC ATCTGGGAGAG-3 '; Runx2-F, 5'-

AAATGCTGGAGTGATGTGGT-3 '; and Runx2-R, 5'- TATGAAGCCTGGCGATTTAG-3 '.

PCR array

Changes in human signal transduction genes were measured using an RT² Profiler PCR Array (Human Signal Transduction PathwayFinder; PAHS-014ZG-4, Qiagen) on a Lightcycler 480 Instrument II 384-well block real-time PCR machine according to the manufacturer's instructions. cDNA template was used for each sample. Results were exported into Microsoft Excel and heatmap analysis was performed using Multiple Experiment Viewer (MeV) 4.9.0 open source software.

Mitochondrial energetic assay

The XF Cell Mito Stress Test (#103015-100, Seahorse Bioscience) was used in XF 96 Extracellular Flux Analyzer (Seahorse Bioscience) to measure key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR) of live cells. The materials used included: XFe96 FluxPak (Seahorse Bioscience), XF96 Cell Culture Microplates (Seahorse Bioscience), XF Calibrant (Seahorse Bioscience), XF Base Medium (Seahorse Bioscience), Sodium Pyruvate (lOOmM, Sigma S8636), L-Glutamine (G-5763, Sigma), Glucose (1.0 M, Sigma G8769). Compound(s) ETC target Effect on OCR

ATP Synthase (Complex

Oligomycin V) Decrease

Inner Mitochondrial

FCCP Membrane Increase

Complex I and III

Rotenone/ antimycin A (respectively) Decrease

Table 1 Compounds used in XF Cell Mito Stress Test

Firstly, optimal concentrations of three compounds (final concentration luM) and cell seeding density were empirically determined prior to the assay. Cells were seeded at lOx lO³ cells/well in 100 μΐ of DMEM/F12 with 20%FBS in XF96 Cell Culture Microplates using 8 replicates and incubated for 24 hours at 37 °C in 5% CO² atmosphere. Five local anaesthetics of 0.5mM and 2mM concentration were added with one control group. After 2/4/8/16 hours, the microplates were ready for check in the machine. The drug injections ports of the XF Assay Cartridge were loaded with the assay reagents in assay medium. 25 μΐ of the three compounds were added sequentially. Culture medium was exchanged with assay medium prior to measurements. Culture medium was aspirated and 80 μΐ pre-warmed assay medium added twice, aspirated and 175 μΐ pre-warmed assay medium added. The microplate was equilibrated in a C02 free incubator at 37°C for 60 minutes. During this equilibration period, the XF96 Analyzer was calibrated with a calibration plate that had been hydrated at 37°C overnight using the standard XF calibration protocol. Following calibration, the calibration plate was replaced with the XF96 cell culture microplate containing pre-treated cells with local anaesthetics and the experimental run started. Data were normalized by cell number and expressed as pmol of O² per minute per 10^A 4 cells.

Statistical analysis PRISM 5 software (Graph Pad Software) was used to analyze the experimental data. One-way ANOVA followed by Bonferroni correction was performed for real-time RT-PCR analysis, and Dunnett's test was applied for mitochondrial energetic analysis. Statistical significance was set at * p < 0.05 and ** p<0.01. Results

Local anaesthetics remain at high concentration in tooth pulp cells after nerve block injection

Due to their anatomical similarity to human beings we adopted a pig model to investigate the penetration of local anaesthetics. In addition, the 5 month' old pig we used had a mixed dentitions including deciduous teeth and young permanent teeth, as well as developing third permanent molar tooth that are similar to adolescent children. Fluorescein labelled local anaesthetics were injected either around mandibular foramen (for lidocaine) for nerve block (Figure 1 A) or under the mucosa of the mesial buccal and lingual periapical regions of the first molar (for Ubistasin and Scandonest, not shown) for infiltration, exactly simulating clinical situations. It is noticeable that local anaesthetics were able to penetrate into developing third molar in a posterior- anterior direction (Figure IB), with a concentration of 19.88 ± 14.19 mM at the posterior site and 8.72 ± 9.43 mM at the anterior site 2 hours after injection and could reach to 16.39 ± 8.36 mM and 22.23 ± 17.45 mM respectively after 16 hours inside the two proximities of the tooth (Figure 1C and ID). In contrast, at 2 hours, the infiltration injection could reach to very high concentration at the outermost cell layer of the tooth pulp with 49.54 ± 22.57 mM for Ubistesin and 21.16 ± 15.44 mM for Scandonest (Figure 1C and IE). However notably the inner layer cells had much lower concentrations of the drugs at 6.67 ± 7.21 mM for Ubistesin and 9.89 ± 10.28 mM for Scandonest (Figure 1C and IE). Contrary to nerve block methods, in infiltration injection, the drug concentration rapidly decreased after 16 hours with the outermost cell layer only held 16.65 ± 10.70 mM for Ubistesin and 9.89 ± 10.28 mM for Scandonest, whilst in the inner cell layer of the tooth pulps the drugs were entirely eliminated (Figure 1C and IE). Local anaesthetics affect tooth pulp cell proliferation in a dose dependent manner As we found that local anaesthetics remained high concentrations particularly in the nerve block method even after 16 hours. We then decided to test the dose effect of the agents on tooth pulp cells by measuring cell proliferation, differentiation and apoptosis, the key parameters that control tooth germ tissue and cell development. At increasing concentrations of the drugs tested, there was a significant dose dependent reduction of Ki67 mRNA expression (Figure 2A). In contrast, there was no change in the levels of cell differentiation and cell death upon anaesthetic challenge in mRNA expression for the tooth pulp differentiation markers Runx2 and p38, and the apoptosis marker Bax at different concentrations of the four drugs (Figure 2B-D). The TUNEL assay confirmed that apoptosis was not induced by these drugs (data not shown).

Local anaesthetics induce autophagy in tooth pulp cells

In parallel to the decreased cell proliferation, in the presence of all the drugs, increased vacuole formation was also observed in the cytoplasm of dental pulp cells (Figure 3 A and data not shown). Immunofluorescence analysis showed that the vacuoles contained LAMP-1 (Figure 3B and 3F). As LAMP-1 overexpression is often associated with an accumulation of autophagic vacuoles, we therefore measured the level of autophagy. Autophagosome numbers correlate with the levels of autophagosome-associated protein LC3-II or the number of LC3 -positive vesicles (Korolchuk et al 2011). Immunofluorescence microscopy (Figure 3C and 3G) and immunoblotting (Figure 3D) results showed that all anaesthetics tested markedly increased levels of LC3, particularly the short half-life form, LC3II (Figure 3D). To provide further evidence of autophagy induction, we analyzed GFP-positive vesicles in cells transfected with GFP-LC3 plasmids. The results showed that GFP production was highly increased in drugs-treated cells, suggesting that all of the local anaesthetics tested could induce autophagy (Figure 3E and data not shown). Consistent with our in vitro findings, in the local anaesthetic treated pig mandibles, LC3II was indeed induced in the third molar tooth pulps both at 2 hours and 16 hours after nerve block injection (Figure 3H) and in the first molar teeth received infiltration injections, LC3II induction could only be mainly identified at the outermost cell layer of the tooth pulp at 2 hours but was quickly removed after 16 hours (Figure 31). Local anaesthetics-induced cell growth arrest is not linked with mitochondrial dysfunctions

To understand if the anti-proliferative effect is time dependent, we performed BrdU labelling analysis of the cells at 0.5mM and 2mM concentrations. The results showed that local anaesthetics could already block cell proliferation at 2 hours at a rate of twofold change while at 16 hours most of the drugs reduced BrdU positive labelling by more than 5 times (Figure 4A). Western blotting analysis of LC3, p62 and phosphorylated form mTor suggested that LC3 is the earliest induced molecular by the local anaesthetics administration while the induction of p62 and p-mTor only became significant after 8 hours (Figure 4B and 4F), although p62 mRNA induction could be already seen after 2 hours (Figure 4C), suggesting autophagosome formation is among the earliest cellular reactions upon local anaesthetic drug challenge.

Local anaesthetics have been known to be able to induce cellular stress and autophagy that has been linked with mitochondrial dysfunctions. We therefore measured dynamic cellular energetics using the Seahorse XF reader in cells received local anaesthetics challenge. The results showed unexpectedly that at time points 2 hours, 4 hours and 8 hours, all the parameters tested including basal respiration, phosphorylation, proton leak and maximum respiration were induced in the agents treated cells for most of the cases (Figure 4D, E and G-I) with the exception of the 16 hour time point at 2mM but not 0.5mM concentration 4 out of 5 drugs tested reduced basal respiration, phosphorylation and maximum respiration but not proton leak (Figure 4D, E and G-I). These results indicate that mitochondrial functions were disturbed only at high concentration (2mM) after 16 hours.

Early autophagy inhibition can reverse anti-cell proliferation effects of local anaesthetics on tooth pulp cells

Autophagy has been reported to be able to reduce cell proliferation. 14 We therefore addressed this possibility by treating cells with an autophagy inhibitor, bafilomycin that specifically inhibits vacuolar-type H+ ATPase (V- ATPase) 15 together with each anaesthetic drug at 0.5mM and 2mM concentrations for 8 and 16 hours. The results showed that at 8 hour time point autophagy inhibition did antagonize the anti- proliferative effects of anaesthetics on tooth pulp cells (Figure 5 A, B, C and D), while at 16 hours, bafilomycin still had the rescuing effects in the 0.5mM agent treated group but failed to do so in the 2mM group (Figure 5A, B, C and D). p62 is the key target of local anaesthetics

To investigate which downstream molecular signalling pathways mediate the effects of local anaesthetics on tooth pulp cells, we analysed the changes in gene expression using the PCR array (PathwayFinder) assay. p62 was the only gene to consistently show dose-dependent changes in expression for all drugs tested and at different concentrations (Figure 6A and D). Upregulation of p62 mRNA and protein was validated by real-time RT-PCR and western blot analysis, respectively (Figure 6B, C). Interestingly, transcriptional analysis of a panel of key autophagy genes (including LC3) showed p62 was the only common gene changed by all the drugs tested (Figure 6B). Importantly, p62 mRNA and protein levels appeared to be linked with local anaesthetic concentrations (Figure 6C). Discussion

Very little has been known about the impact of local anaesthetics on the tooth, particularly on young tooth pulp cells. Articaine, mepivacaine and lidocaine are the most prevalent injectable local anaesthetic agents not only in dental clinics but also in other practices. Among them, articaine has been used as the first choice by most of dentists as it has been proven to be more efficient than the other drugs such as lidocaine due to better nerve block and tooth pulpal anaesthesia results. Articaine contains a thiophene ring and mepivacaine and lidocaine contain a benzene ring that can increase drug lipid solubility. Local anaesthetics are known to work by binding to voltage-gated Na⁺ channels in nerves, thereby blocking sodium transportation and nerve conduction (Strichartz 1976). To our knowledge this is the first systematic analysis of the effects of these agents on tooth pulp cell activities. The aim of our study was to find out more about common side effects of the drugs that could explain the clinically observed increased tooth agenesis ratio in patients who receive local anaesthesia treatment (Swee et al 2013) and therefore improve our clinical instruction of drug application. The side effects of local anaesthetics have been well evaluated in view of metabolism, particularly their effects on mitochondrial energetic activities, but little has been investigated in relation to side effects on tooth pulp cells. It has been reported that local anaesthetics such as bupivacaine can uncouple mitochondrial oxygen consumption and ATP synthesis and reduce ATP synthesis (Sztark et al 1997). Mepivacaine can also inhibit mitochondrial respiration (di Jeso et al 1988) and lidocaine can cause mitochondrial injury and induce apoptosis in the cells. As previously reported, local anaesthetic drugs can induce vacuolation (Morissette et al 2004). Using various approaches, we have shown that LC3-II (a marker of autophagy) levels were increased in tooth pulp cells following treatment with local anaesthetic drugs. By using an LC3-GFP reporter assay, we were also able to show that the LC3 protein was translocated into autophagosomes when primary tooth cells were treated with the anaesthetic drugs.

Mechanistically, autophagy has often been linked with mitochondrial dysfunctions and localized inside mitochondria (i.e. also named as mitophagy). However, through detailed dynamic cellular energetic analysis we have identified that upon challenge by the different drugs tested, for at least 8 hours after treatment, mitochondrial functions of tooth pulp cells were not diminished. Instead we have observed a significant increase of mitochondrial respiration, a possible surviving mechanism having been initiated in the mitochondria to counteract the toxicity effect of the drugs. It is noticeable that longer treatment at high (but not low) concentration for most of the tested drugs did reduce basal respiration, phosphorylation and maximum respiration of mitochondria, suggesting at this stage and condition mitochondrial functions may have been damaged. Interestingly proton leak has been identified in all the tested drugs in all the conditions, suggesting it is a direct consequence of local anaesthetic drug application. Indeed, another local anaesthetic drug bupivacaine has been found to have an uncoupling function through a protonophore-like mechanism. It would therefore be interesting to understand whether articaine, mepivacaine and lidocaine cause proton leak by the same mechanism. Continuous H vacuolar (V)-Atpase activity is required to maintain the pH during proton leak. Given that an acidic lysosomal condition is required for autophagosome maturation, we believe that V-Atpase's function has been over-activated upon drug challenge. Indeed, this hypothesis is supported by the fact that adding a V-Atpase specific inhibitor, bafilomycin, efficiently rescued cell proliferation.

A key role of autophagy is to remove excess aggresomes, associated p62 and dysfunctional organelles that can induce double strand DNA breaks. p62 is a receptor for cargo destined to be degraded by autophagy, including ubiquitinated protein aggregates. The p62 protein can bind both ubiquitin and LC3, thereby targeting ubiquitinated proteins to the autophagosome for clearance. p62 has also been shown to regulate cell proliferation through Twistl stabilization. Therefore intracellular p62 protein levels are critical for cell viability. Using a signalling pathway specific PCR array, we identified the p62 gene as a unique downstream target of all the drugs tested. We also found that concentrations of the anaesthetic drugs are linked with p62 protein levels, especially for later stages, while reduced cell proliferation by long time high concentration local anaesthetic treatment could not be rescued using bafilomycin. This observation suggests it is highly possible that DNA damage could be induced by excess p62 when it reaches a theshold concentration and is not cleared in time.

In conclusion, our findings that local anaesthetics can induce autophagy in tooth pulp cells have important clinical implications due to the potential impact on tooth agenesis and the development of other teeth. These findings require the attention of both clinicians and pharmaceutical companies.

Acknowledgements

The authors thank the Biotechnology and Biological Sciences Research Council of the UK (grant No. BB/L02392X/1) and the EU Marie Curie Action (grant No. 618930, OralStem FP7-PEOPLE-2013-CIG) to B.H., and the National Natural Science Foundation of China to B.H. (grant No. 30500566) and H.Z. (grant No. 30801289 and 81371138). References

Hu B, Castillo E, Harewood L, Ostano P, Reymond A, Dummer R, Raffoul W, Hoetzenecker W, Hofbauer GF, Dotto GP: Multifocal epithelial tumors and field cancerisation from loss of mesenchymal CSL signaling. Cell 2012; 149: 1207-20. Korolchuk VI, Rubinsztein DC: Regulation of autophagy by lysosomal positioning. Autophagy 2011; 7: 927-8

Morissette G, Moreau E, R CG, Marceau F: Massive cell vacuolization induced by organic amines such as procainamide. J Pharmacol Exp Ther 2004; 310: 395-406

Strichartz G: Molecular mechanisms of nerve block by local anesthetics. Anesthesiology 1976; 45 : 421 -41

Swee J, Silvestri AR, Jr., Finkelman MD, Rich AP, Alexander SA, Loo CY: Inferior alveolar nerve block and third-molar agenesis: a retrospective clinical study. J Am Dent Assoc 2013; 144: 389-95

Claims

1. An ATPase inhibitor for use in the treatment or prevention of tooth cell autophagy.

2. An ATPase inhibitor for use according to claim 1, wherein the tooth cells are tooth germ cells such as tooth pulp cells, tooth epithelial cells and/or periodontal mesenchymal cells.

3. An ATPase inhibitor for use according to claim 2, wherein the tooth pulp cells are tooth pulp mesenchymal cells and/or tooth pulp stem cell progenitors.

4. An ATPase inhibitor for use according to any of claims 1 to 3, wherein the tooth cell autophagy is induced, spontaneous or acquired autophagy.

5. An ATPase inhibitor for use according to any of claims 1 to 4, wherein the ATPase inhibitor is a V-ATPase inhibitor.

6. An ATPase inhibitor for use according to claim 5, wherein the V-ATPase inhibitor is selected from one or more of a plecomacrolide, a macrolactone, a benzolactone, or a combination thereof

7. An ATPase inhibitor for use according to claim 5 or 6, wherein the V-ATPase inhibitor is selected from one or more of bafilomycin, concanamycin, archazolid, apicularen, or a combination thereof.

8. A method for treating or preventing tooth cell autophagy, the method comprising administering an ATPase inhibitor to a patient in need thereof.

9. A method according to claim 8, wherein the tooth cells are tooth germ cells such as tooth pulp cells, tooth epithelial cells and/or periodontal mesenchymal cells.

10. A method according to claim 9, wherein the tooth pulp cells are tooth pulp mesenchymal cells and/or tooth pulp stem cell progenitors.

11. A method according to claim any of claims 8 to 10, wherein the tooth cell autophagy is induced, spontaneous or acquired autophagy.

12. A method according to any of claims 8 to 11, wherein the ATPase inhibitor is a V- ATPase inhibitor.

13. A method according to claim 12, wherein the V- ATPase inhibitor is selected from one or more of a plecomacrolide, a macrolactone, a benzolactone, or a combination thereof.

14. A method according to claim 12 or 13, wherein the V- ATPase inhibitor is selected from one or more of bafilomycin, concanamycin, archazolid, apicularen, or a combination thereof.

15. A method according to any of claims 8 to 14, wherein the ATPase inhibitor is administered in combination with a local anaesthetic.

16. A method according to claim 15, wherein the local anaesthetic is an amide- type local anaesthetic.

17. A method according to claim 16, wherein the local anaesthetic is selected from one or more of lidocaine, articaine, bupivacaine, mepivacaine, prilocaine, or a combination thereof.

18. An ATPase inhibitor for use in the treatment or prevention of autophagy in deciduous and/or permanent teeth.

19. An ATPase inhibitor for use according to claim 18, wherein the autophagy is induced, spontaneous or acquired autophagy.

20. An ATPase inhibitor for use according to claim 18 or 19, wherein the ATPase inhibitor is a V- ATPase inhibitor.

21. An ATPase inhibitor for use according to claim 20, wherein the V- ATPase inhibitor is selected from one or more of a plecomacrolide, a macrolactone, a benzolactone, or a combination thereof

22. An ATPase inhibitor for use according to claim 20 or 21, wherein the V- ATPase inhibitor is selected from one or more of bafilomycin, concanamycin, archazolid, apicularen, or a combination thereof.

23. A composition comprising a local anaesthetic and an ATPase inhibitor.

24. A composition according to claim 23, wherein the ATPase inhibitor is a vacuolar-type ATPase (V- ATPase) inhibitor.

25. A composition according to claim 24, wherein the V- ATPase inhibitor is selected from one or more of a plecomacrolide, a macrolactone, a benzolactone, or a combination thereof.

26. A composition according to claim 24 or 25, wherein the V- ATPase inhibitor is selected from one or more of bafilomycin, concanamycin, archazolid, apicularen, or a combination thereof.

27. A composition according to any of claims 23 to 26, wherein the local anaesthetic is an amide-type local anaesthetic.

28. A composition according to any of claims 23 to 27, wherein the local anaesthetic is selected from one or more of lidocaine, articaine, bupivacaine, mepivacaine, prilocaine, or a combination thereof.

29. A composition according to any of claims 23 to 28, comprising from about 10 mg/ml to about 80 mg/ml local anaesthetic.

30. A composition according to any of claims 23 to 29, further comprising one or more carriers or excipients selected from sodium chloride, sodium sulphite, potassium metabisulfite, edetate disodium, sodium hydroxide, hydrochloric acid or water.

31. A composition according to any of claims 23 to 30, further comprising adrenaline or a hydrochloride salt thereof.

32. A composition according to any of claims 23 to 31, wherein the composition is a solution for injection.