Identifying novel radioprotective drugs via salivary gland tissue chip screening

doi:10.21203/rs.3.rs-3246405/v1

Download PDF

Article

Identifying novel radioprotective drugs via salivary gland tissue chip screening

https://doi.org/10.21203/rs.3.rs-3246405/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

During head and neck cancer treatment, off-target ionizing radiation damage to the salivary glands commonly causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients have trouble eating, speaking and are predisposed to oral infections and tooth decay. While the radioprotective antioxidant drug Amifostine is FDA approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use, motivating the discovery of alternative therapeutics. To address this issue, we previously developed a salivary gland mimetic (SGm) tissue chip platform. Here, we leverage this SGm tissue chip for high-content drug discovery. First, we developed in-chip assays to quantify glutathione and cellular senescence (β-galactosidase), which are biomarkers of radiation damage, and we validated radioprotection using WR-1065, the active form of Amifostine. Other reported radioprotective drugs including Edaravone, Tempol, N-acetylcysteine (NAC), Rapamycin, Ex-Rad, and Palifermin were also tested to validate the ability of the assays to detect cell damage and radioprotection. All of the drugs except NAC and Ex-Rad exhibited robust radioprotection. Next, a Selleck Chemicals library of 438 FDA-approved drugs was screened for radioprotection. We discovered 25 hits, with most of the drugs identified exhibiting mechanisms of action other than antioxidant activity. Hits were down-selected using EC50 values and pharmacokinetic and pharmacodynamic data from the PubChem database. This led us to test Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic) for in vivo radioprotection in mice using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited radioprotection equivalent to Amifostine. This body of work demonstrates the development and validation of assays using a SGm tissue chip platform for high-content drug screening and the successful in vitro discovery and in vivo validation of novel radioprotective drugs with non-antioxidant primary indications pointing to possible, yet unknown novel mechanisms of radioprotection.

Biological sciences/Drug discovery/Drug screening/Phenotypic screening

Biological sciences/Biotechnology/Assay systems

During head and neck cancer treatment, ionizing radiation damage to the salivary glands often causes a permanent loss of secretory function and reduced salivary flow. Due to decreased saliva production, patients have trouble eating, speaking, and swallowing^{1, 2}. Additionally, patients are at an increased risk of oral infections and tooth decay and suffer a reduced quality of life^{2, 3}. Current treatment options, including sialogogues, mouthwashes, and chewing gum, only provide temporary relief, and there is no cure². Several strategies have been proposed to alleviate this damage, including cell transplantation^4-7 and gene therapy^8-11. Despite promising results, these methods have remained experimental and are targeted toward patients already experiencing xerostomia. Hence, there is an unmet need to provide current and future head and neck cancer patients with preventative therapies to protect salivary gland function.

Intensity-modulated radiation therapy (IMRT) and the radioprotective drug Amifostine are used clinically to prevent salivary gland damage. IMRT involves using 3D imaging to target the radiation beams at the tumor and away from sensitive organs such as the salivary gland¹². While this method can be beneficial in some cases, there are mixed results in patient-reported claims of dry mouth. Furthermore, IMRT is sometimes impossible to use due to tumor location^{12, 13}. The antioxidant Amifostine is the only FDA-approved drug to prevent radiation-induced xerostomia. However, its use is often discontinued during fractionated radiation regimens due to severe side effects, including nausea, vomiting, and hypotension^{14, 15}. Additionally, its short half-life in vivo limits its efficacy, as the drug is cleared within minutes of administration¹⁶. These drawbacks highlight the critical need to discover new radioprotective drugs to prevent xerostomia.

To address this need, we developed a microbubble (MB) array-based tissue chip consisting of 3D salivary gland tissue mimetics (SGm) entrapped within a matrix metalloproteinase degradable poly(ethylene glycol) (PEG)-based hydrogel engineered extracellular matrix (eECM) that maintains secretory behavior¹⁷. The spherical architecture of the MB combined with the cellularly degradable eECM creates a distinct niche that promotes cell viability and maintenance of the secretory acinar cells based on gene and protein expression as well as secretory function based on tissue architecture, gene expression, and secretory agonist-responsive calcium signaling¹⁷. In addition, this platform was validated for use in radioprotection studies using immunohistochemical staining of individual SGm to quantitate foci of DNA damage markers γH2AX and 53BP1 after irradiation. Analysis of control chips versus chips treated with WR-1065, the active form of Amifostine, showed a reduction in DNA damage with drug treatment¹⁷.

Array-based assays that can interrogate each SGm on the chip (~280 MBs/cm²) are necessary to enable high-content drug screening. Based on literature, several assays commonly used to report radiation-induced cellular damage were tested in the tissue chip format. Assays tested included reactive oxygen species (ROS), apoptosis, secretion, and cytotoxicity at various time points post-radiation (Table S.1). Based on signal-to-noise ratio and reproducibility, the glutathione¹⁸ and cellular senescence¹⁹ assays were selected for further development for high-content screening of radioprotective drugs.

Assays were tested with 0 Gy, 15 Gy, and 15 Gy + 4 mM WR-1065 to confirm usefulness for measuring radiation damage and radioprotection of individual SGm within the MB-hydrogel tissue chip (40-50 MBs per chip). The assays were further tested with other reported radioprotective drugs, including Tempol^{20, 21}, N-acetylcysteine²², Edaravone²³, Rapamycin²⁴, Ex-Rad^{25, 26}, and Palifermin^{27, 28}. Next, a library of 438 FDA-approved drugs was screened for radioprotection using both assays, identifying 25 double hits that were further investigated for suitability as potential therapeutics informed by data within the PubChem database and experimentally determined dose-response relationships. Three of the hits, Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic), were chosen for in vivo radioprotection testing of the mouse submandibular gland using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited equivalent radioprotection to Amifostine.

Materials

Detailed information on the drugs used for assay development and validation, including, Tempol, N-acetylcysteine, Edaravone, WR-1065, Rapamycin, Ex-Rad, and Palifermin is listed in Table S.2. Drug screening was completed using a 438 Selleck Chemicals library of FDA-approved drugs (Table S.3). The screen's 25 top drug hits (Table S.4) were purchased from Selleck Chemicals for dose-response studies. The drugs were prepared and stored per manufacturer’s instructions.

Animals

Female SKH1 hairless mice, backcrossed 6 generations with C57BL/6J mice, aged 6-12 weeks were used in this study for in vitro assay development and drug discovery. Female C57BL/6J mice age 6-8 weeks were used for in vivo validation studies. Only female mice were used due to known sex differences in rodent salivary glands, with female glands more accurately emulating human salivary gland structure and function^{29, 30}. Animals were maintained on a 12 hr light/dark cycle and group-housed with food and water available ad libitum. All procedures were approved and conducted per the University Committee on Animal Resources at the University of Rochester Medical Center (UCAR #2010-024E, UCAR-2008-016E).

Microbubble (MB) array fabrication

Microbubble (MB) arrays were fabricated in poly(dimethyl) siloxane (PDMS) using gas expansion molding as previously described^{17, 31, 32}. PDMS (Dow Corning Sylgard 184) was mixed in a 10:1 base-to-curing agent ratio and poured over a silicon wafer template consisting of deep etched cylindrical pits with a 200 µm diameter, spaced 600 µm apart on a square lattice. The PDMS was cured at 100 °C for 2 hrs before peeling off the template, resulting in an array of spherical cavities with 200 µm opening and ~350 µm diameter. Circular chips with 0.7 cm diameter (48 well plates) or 0.5 cm diameter (96 well plates) were punched from the PDMS cast and glued into well plates using a 5:1 ratio of PDMS cured at 60 °C for 8 hours. The MB arrays were primed in a desktop vacuum chamber with 70% ethanol to facilitate air removal from the MBs and replacement with fluid. Ethanol was exchanged for PBS and arrays were incubated overnight before cell seeding.

Cell isolation

Mice were euthanized and the submandibular glands were removed and chopped with a razor blade for 5 min. The tissue was then incubated in Hank’s buffered salt solution (HBSS) containing 15 mM HEPES, 50 U/mL collagenase type II (Thermo Fisher 17101015), and 100 U/mL hyaluronidase (Sigma Aldrich H3506) at 37 °C for 30 min. Cells were centrifuged, resuspended in HBSS with 15 mM HEPES, and passed through 100 µm and 20 μm mesh filters to isolate cell clusters between 20-100 µm. The digestion protocol produces cell cluster sizes evenly distributed between 20 to 100 µm³³. As described below, the isolated clusters were combined with hydrogel precursor solution and seeded into the primed MB array chips.

MB-hydrogel encapsulation of salivary gland cells

Isolated submandibular gland cell clusters (20-100 µm) were encapsulated within matrix-metalloproteinase (MMP) degradable poly(ethylene glycol) (PEG) hydrogels within MB arrays (Fig. 1) as previously described¹⁷. Briefly, the cells (Fig. 1A) were resuspended in hydrogel precursor (Fig. 1B) solution containing 2 mM norbornene-functionalized 4-arm 20 kDa PEG-amine macromers, 4 mM of the dicysteine functionalized MMP degradable peptide (GKKCGPQG↓IWGQCKKG), 0.05 wt% of the photoinitiator lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP)³⁴, and 0.1 mg/mL laminin in PBS^{4, 5, 17, 35}. The cell/gel precursor solution (25 µL for 48 wells; 20 µL for 96 wells) was pipetted onto the MB chips and incubated for 30 min, pipetting every 10 min to redisperse cells that had settled onto the surface of the chip. The hydrogels were polymerized in situ using a Hand-Foot 1000 A broad spectrum UV light (UVA: 5 mW/cm²; UVB: 0.4 mW/cm²) with a UVC filter for 1.5 min and cultured with media (0.5 mL for 48 well; 150 µL for 96 well), with media changes every 2 days. Culture medium consisted of Dulbecco’s Modified Eagle Medium (DMEM):Ham’s F-12 Nutrient Mixture (1:1) supplemented with 100 U/mL Penicillin and 100 μg/mL Streptomycin, 2 mM Glutamine, 0.5x N2 supplement, 2.6 ng/mL insulin, 2 nM dexamethasone, 20 ng/mL epidermal growth factor (EGF), and 20 ng/mL basic fibroblast growth factor (bFGF).

Glutathione assay

A glutathione assay was developed for in-chip measurements by adapting the Cellular Glutathione Detection Assay Kit (Cell Signaling Technology #13859). The monochlorobimane reagent was prepared by reconstitution in DMSO per manufacturer’s directions. For 96 well plates, 10 µL of prepared reagent (1:50 ratio of monochlorobimane (MCB) to Tris assay buffer, per manufacturer’s instructions) was added to wells containing 100 µL of culture media and incubated for 30 min at 37 °C, washed with PBS, and imaged using an Olympus IX70 microscope with a DAPI filter (Excitation/Emission: 358 nm/461 nm).

Senescence assay

A cellular senescence assay was developed for in-chip measurements by adapting the Cellular Senescence Detection Kit – SPiDER-βGal (Dojindo Molecular Technologies, Inc SG04). Balifomycin A1 and SPiDER-βGal stock solutions were prepared in DMSO per manufacturer’s directions. The assay was performed by first incubating the chips with Balifomycin A1 (1:1000 dilution in media) for 1 hr at 37 °C. The solution was removed, replaced with 30 µL of media containing Balifomycin A1 (1:1000 dilution) and SPiDER-βGal (1:500 dilution), and incubated for 45 min at 37 °C. Chips were washed twice with media and imaged using a fluorescence microscope with a Texas Red filter (Excitation/Emission: 580 nm/604 nm).

Drug mechanism meta-analysis

Drug interaction data was examined using PubChem BioAssays results for the hits discovered in the Selleck Chemicals drug library screen described below ^36-38. Pathway analysis was performed using the Drug Set Enrichment Analysis (DSEA) tool to identify pathways with gene expression patterns significantly impacted by treatment with the drug hits (p<0.05) ³⁹.

Image quantification and statistical analysis

For the glutathione and senescence assays, images were quantified in ImageJ. Regions of interest (ROIs) were created by thresholding on the fluorescence signal (localized to the SGm), and each ROI's mean intensity was measured. Data were graphed and statistical analyses (ANOVA with Tukey’s post-hoc test) were performed using GraphPad Prism 9.

Drug treatment and irradiation

For radioprotection experiments, SGm were cultured in MB-hydrogel chips for 4 days, then drugs were added to the chips 30 min before radiation and washed out with media 30 min post-radiation to parallel how Amifostine is used clinically⁴⁰. A dose of 15 Gy ionizing radiation was delivered using a JL Shepherd ¹³⁷Cs irradiator. Drug treatment schemes and radiation doses were established in our previous work¹⁷. The glutathione and senescence assays were performed at 4- and 5-days post-radiation. For assay validation experiments, at least 3 chips (N = 3) were used for each drug, corresponding to > 100 MBs (n > 100); these values are listed in the figure captions for each experiment. Mean, standard deviation, and statistics were calculated based on the number of MBs (n).

The same treatment scheme was used for screening the Selleck Chemicals library, with drugs administered at 100 µM. One MB chip (N = 1) was used per drug, with ~40-50 MBs per chip (n = 40-50); statistics were calculated using the number of MBs (n) and compared to 0 Gy controls. Drugs were first screened using the glutathione assay. Drugs that exhibited statistically insignificant differences compared to the 0 Gy control (hits) with the glutathione assay were then tested with the senescence assay to discover double hits. The glutathione assay was selected as the first screen because it had higher signal-to-noise ratio, more rapid throughput (30 mins versus 2 hrs for completion of the senescent assay), lower assay kit cost, and a higher shelf-life of reagents in the kit after reconstitution.

To discriminate the effectiveness of drug candidates, for both the glutathione and senescence assays, we defined a signal range bounded by the values of the assay result for 0 Gy unirradiated and 15 Gy irradiated controls. Full radioprotection would yield a signal for 15 Gy+drug that was statistically equivalent the 0 Gy control, or a recovery of 100%.

Retroductal injection and irradiation

Retroductal injection delivery of drugs to the murine submandibular gland has been described in detail^{41, 42}. Briefly, 6-10 week old female C57Bl/6J mice were anesthetized by intraperitoneal injection of sterile saline solution of 100 mg/kg ketamine and 10 mg/kg xylazine. Maxillary incisors were secured over a metal beam, while an elastic band provided tension from behind the mandibular incisors. The mouth was widened using a custom steel retractor to apply pressure to the buccal mucosa and the tongue was retracted and cotton placed in the oral cavity. The wire inset of a 32G intracranial catheter was cut at 45° to create a bevel. The beveled wire created a shallow puncture in the left salivary papilla. A beveled catheter section containing the wire insert for support was gently inserted into the puncture site. The catheter was removed, and 1 mg/kg atropine was administered by intraperitoneal injection. After 10 min, the needle of a Hamilton syringe, loaded with vehicle or drug solution, was inserted into the catheter, and the catheter was inserted into the orifice produced in the papilla. Drug solutions were injected by hand at 10 µl/min at a volume of 1 µl/g of body weight. Following injection, the pressure was maintained on the syringe for 1 min to ensure material retention before removal of the catheter. The cotton⁴³ and retractor were removed from the oral cavity, and the elastic band and metal beam were released from the incisors. The known radioprotectant, WR-1065 (50 mg/kg, saline) was used as a control for radioprotection. Phenylbutazone, Enoxacin, and Doripenem hydrate were dosed at 50, 0.3, and 26 mg/kg (N = 4-6 per group). Drug doses were chosen based on solubility and published in vivo studies^{44, 45}. Saline was used as the vehicle for all compounds, except Phenylbutazone, which required corn oil due to poor aqueous solubility.

Mice injected with vehicle controls (saline or corn oil) or drug were treated with 0 Gy or 15 Gy within 15-30 min of injection, the submandibular glands were irradiated as described previously ^46-48. The head and neck region was positioned over the slit of a custom collimator, which allowed body shielding. Mouse submandibular glands were exposed to a single dose of 15 Gy gamma radiation delivered by a ¹³⁷Cs radiation source. This single dose in mice recapitulates human sequalae of salivary gland radiation damage^{17, 49}. Animals were allowed to recover for 48 hours to measure persistent long-lived DNA damage, after which, the submandibular glands were harvested, fixed, sectioned, and analyzed using immunohistochemistry.

Immunohistochemical analysis

Submandibular glands and SGm were isolated and fixed in 4% paraformaldehyde overnight at 4 °C. Tissues were paraffin-embedded and then cut into 5 μm sections. Slides were treated with HIER buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for antigen retrieval in a pressure cooker for 10 minutes then sections were blocked in CAS-block histochemical reagent (Thermo Fisher Scientific, 008120). Permeabilization was performed with 0.5% Triton X-100 in PBS for 5 minutes. Immunostaining was performed overnight (at 4 °C) with primary antibody for γH2AX (EMD Millipore, 05-636). Alexa-Fluor 594-conjugated donkey anti-mouse IgG was diluted 1:500 (Invitrogen, A21203) as secondary antibody and applied on sections for 1 hour at room temperature. Following a PBS rinse, 10 μg/ml DAPI (Invitrogen, Carlsbad, CA) in PBS was applied to sections for 5 minutes. Sections were washed thrice in PBS for 5 minutes and the slides were mounted using Immu-Mount mounting medium (Thermo). Microscopic images were acquired using a Leica TCS SP5 confocal microscope with a 100X oil immersion objective and Argon laser. Analysis of images was performed in ImageJ.

Salivary Gland Tissue Chip

Our previously reported salivary gland tissue chip¹⁷ was leveraged for high-content drug screening to identify novel radioprotective compounds. The chip platform consists seeding primary salivary gland cell clusters 20-100 µm (Fig. 1A), suspended in a poly(ethylene glycol) (PEG) hydrogel precursor and MMP-degradable peptide crosslinker solution together with the photoinitiator LAP (Fig. 1B), into an array of near-spherical microbubble (MB) cavities (Fig. 1C) formed in poly(dimethylsiloxane) (PDMS). Each chip, containing 40-50 MBs, is affixed within wells of a 96-well plate. In situ polymerization of the hydrogel was achieved using long-wave, low intensity UV light. Over time, the cell clusters aggregate and proliferate to form SGm (Fig. 1D).

High-throughput methods to assess drug radioprotection

In prior studies¹⁷, immunohistochemical (IHC) staining was used to quantify the number of γH2AX and 53BP1 puncta within nuclei, which are sensitive markers for double-stranded DNA breaks⁵⁰ and direct measures of radiation damage. IHC staining is, however, a laborious time and resource-consuming process requiring retrieval of the SGm from the MB array chip, tissue sectioning, staining, and imaging. While IHC enables sensitive analyses of radiation damage, it completely abrogates the goal of in situ high-content screening for which the tissue chip was developed. As mentioned above, several assays commonly used to assess radiation-induced cellular damage were tested in the tissue chip format at various time points post-radiation (Table S.1). Based on signal-to-noise ratio and reproducibility, the glutathione ¹⁸ and senescence¹⁹ assays were selected for further development. The glutathione assay was used to measure glutathione levels at various time points post-radiation to determine the optimal time for detecting differences between 0 Gy and 15 Gy. Based on the data, the greatest signal separation was measured at 4 days post-irradiation (Fig. S1). This time point is similar to previous reports on decreases in glutathione post-radiation¹⁸ and was used for all experiments moving forward.

Next, WR-1065 was tested to identify effective concentration(s) that prevented changes in glutathione levels post-radiation. A SGm tissue chip was cultured for 4 days to allow spheres to form¹⁷ . The chip was then treated with WR-1065 30 min before and during radiation, followed by drug wash out with media 30 min post-radiation (Fig. 2A). This dosing scheme is consistent with the use of Amifostine clinically¹⁴ and with our previous work¹⁷. The glutathione assay was performed 4 days post-radiation (Fig. 2A). Example images show high levels of glutathione at 0 Gy (Fig. 2B) that is decreased by 15 Gy radiation (Fig. 2C) and maintained with 4 mM WR-1065 (Fig. 2D). Quantification shows that 0.1 mM and 0.4 mM WR-1065 were ineffective at preventing radiation damage. In contrast, 1 mM and 4 mM WR-1065 provided significant protection (Fig. 2E). The 4 mM dose corresponds with our previous work on DNA damage markers γH2AX and 53BP1¹⁷ and values from literature⁵¹ and establishes the range of effective concentrations for WR-1065 treatment in vitro. Moreover, it is clinically relevant as 15-30 minutes before radiation, patients are administered Amifostine intravenously at 200 mg/m² ^{15, 40}. Assuming an average adult body surface area of 17,000 cm² and a blood volume of 1.35 gallons (5.1 liters)^{52, 53}, Amifostine is administered at 300 µM.

Since WR-1065 is an antioxidant and mediates radioprotective effects through free radical scavenging and induction of superoxide dismutase expression⁵⁴, we tested other antioxidants implicated as radioprotective (Tempol, Edaravone, N-acetylcysteine). Tempol exhibited excellent radioprotection at both 1 and 4 mM (Fig. 2F) which is consistent with prior studies^{20, 49, 55}. Edaravone showed complete radioprotection at 1 mM but not at 4 mM (Fig. 2G). Edaravone maintained ~43% of glutathione levels at 0.1 mM (Fig. S2A), suggesting that the optimal concentration range for Edaravone might be lower than WR-1065, which is consistent with literature indicating radio-protective dose ranges are 0.1-1 mM^{23, 56, 57}. For N-acetylcysteine (NAC), no improvement in glutathione levels was observed at 1 or 4 mM compared to untreated SGm (Fig. 2H); however,glutathione levels were rescued by 56% when treated with 10 mM NAC (Fig. S2B), also consistent with literature²².

Drugs reported with non-antioxidant radioprotective mechanisms were also tested using the glutathione assay. Rapamycin is an mTOR inhibitor reported to restore salivary flow rate post-irradiation in swine²⁴. Ex-Rad reduces p53-dependent and independent apoptosis²⁵. Palifermin (keratinocyte growth factor) has been reported to stimulate salivary gland stem/progenitor cell expansion post-radiation²⁷. Using drug concentrations based on literature^{26, 28}, our data shows some protection against glutathione level changes resulting from radiation with 50 µM rapamycin (58%, Fig. 2I) and 100 ng/mL Palifermin (47%, Fig. 2J), but no protection from 50 µM Ex-Rad (Fig. 2K).

For senescence, a protocol similar to the glutathione assay was followed to determine the optimal time point post-radiation for detecting a change in senescence between 0 Gy and 15 Gy, as measured by senescence-associated β-galactosidase activity. A time point of 5 days post-radiation was optimal (Fig. S3), similar to a previous study¹⁹. WR-1065 radioprotection was tested by adding drug to the chips 30 min before radiation followed by wash out 30 min post-radiation (Fig. 3A). An expected increase in senescence was detected for SGm exposed to 15 Gy compared to 0 Gy (Figs. 3B,C) , which was restored to levels equivalent to 0 Gy with the addition of WR-1065 (Fig. 3D). Quantification shows that both 1 mM and 4 mM WR-1065 treatment resulted in complete radioprotection (Fig. 3E), consistent with the glutathione assay results.

Next, the level of senescence was measured using the other reported radioprotective drugs. Tempol (Fig. 3F) and Edaravone (Fig. 3G) reduced senescence at both 1 mM and 4 mM by 75% and 91% for Tempol, 94% and 113% for Edaravone, at 1 and 4 mM, respectively, versus untreated, irradiated controls. Edaravone also reduced senescence by 96% at 0.1 mM versus untreated, irradiated controls (Fig. S4A). For NAC, 83% and 57% reduction in senescence was observed with 1 mM and 4 mM (Fig. 3H) and 82% at 10 mM (Fig. S4B) versus untreated, irradiated controls. For drugs with non-antioxidant mechanisms, rapamycin showed complete protection (Fig. 3I), whereas Palifermin provided 64% protection (Fig. 3J) and Ex-Rad conferred only 45% radioprotection (Fig. 3K).

A summary of results from the glutathione and senescence assays shows similar trends for radioprotection (Table S.5). The few differences may result from different mechanisms of action and/or assay targets. Logically, the glutathione assay may be more sensitive to antioxidant function (NAC, Tempol, Edaravone), while the senescence assay may be more appropriate for drugs such as rapamycin, which has anti-senescence properties⁵⁸. These results highlight the trade-offs in developing screening assays and point to the benefit of screening with two assays. Although the assays developed are indirect measures of radiation-induced DNA damage, they nonetheless were validated to detect radiation induced cell-damage and drug radioprotection. More importantly, these assays can be used for in situ high-content drug screening with multiple replicates (40-50) per test and enhanced throughput compared to IHC staining for γH2AX.

Drug library screening identified several promising radioprotective compounds

The glutathione and senescence assays were used to screen a library of FDA-approved drugs (Selleck Chemicals) at 100 µM. Drugs were first screened using the glutathione assay according to the timeline in Fig. 2A. Any compound resulting in statistically equivalent glutathione levels compared to the 0 Gy control was considered a hit (Fig. 4, orange circles). Hits with the glutathione assay were then tested uisng the senescence assay and considered a “double hit” if senescence levels were statistically equivalent to levels at 0 Gy (Fig. 4, blue circles). A list of the 438 drugs screened and relevant statistics are shown in Table S.3. Overall, 438 drugs from the library were tested with a hit rate of 5.7%, for a total of 25 double hits (Fig. 4B) listed in Table 1. While this hit rate is higher than many other drug screening reports (0.1 – 0.3%)^{59, 60}, this may be due to the high statistical rigor afforded by the tissue chip format. Additionally, phenotypic screens generally have higher hit rates than target-based screens and they maybe a more successful strategy for discovery of first-in-class medicines (> 1%)^61-63.

Identification of potential radioprotective drug mechanisms

Of the 25 potential radioprotective compounds, 20 have known interactions with proteins involved in calcium signaling identified within the BioAssay database in PubChem (Fig. 5A). These compounds may impact secretory signaling in the salivary gland, which can be radioprotective^{64, 65}. While degranulation may not be key to radioprotection, secretory stimulation may play a role in proliferation and survival of the secretory cells⁶⁴. Similarly, using the Drug Set Enrichment Analysis (DSEA) tool to identify pathways, the Reactome analysis related to secretion appear to be upregulated by many of the identified drugs, supporting this potential mechanism (Fig. 5B,C, and Fig. S5). Interestingly, only 9 of the 25 compounds have known antioxidant properties, and 12 are anti-inflammatory. This is critical data indicating that alternate mechanisms of radioprotection may be achievable and represented in the identified hits. A reduction in pathway activity related to cell adhesion, cell-cell and cell-matrix interactions also represents a potential area of exploration. Multiple studies have identified changes in integrin expression, cell adhesion and matrix interactions upon radiation exposure, which may be linked to cell response^{1, 66-69}. Manipulation of these mechanisms in the salivary gland may convey radioprotection.

Hit down-selection using drug promiscuity data and EC₅₀values

A systematic approach was used to down selection the 25 double hits for in vivo testing. Since drugs within the library are FDA-approved, considerable information on their pharmacology in mice and humans is readily available through resources such as PubChem. Within PubChem, the BioAssay database was created by the National Institute of Health (NIH) as an open repository containing results of small molecule screening data³⁸. We used the BioAssay data to analyze drug promiscuity, which refers to the ability of a drug to bind multiple molecular targets with distinct pharmacological outcomes, often causing unwanted side effects¹⁰⁸. The drugs exhibiting bioactivity in a large number of assays were deprioritized. Data for each double hit was obtained from the database and promiscuity was calculated as the percent of assays reported as “active” (Table 1). Drugs with high activity (>10%) were excluded from further testing. Additionally, Etidronate, Melatonin, and Albendazole were excluded due to poor bioavailability^109-111, and Eplerenone was excluded due to solubility concerns¹¹².

The remaining 13 drugs were tested for glutathione levels post-irradiation in dose-limiting experiments (1 -100 µM) to identify effective concentrations. Many drugs were only effective at 100 µM and were excluded due to a lack of potency. Forthe remaining seven drugs (Phenylbutazone, Meropenem, Diethylstilbestrol, Prazosin, Enoxacin, Glipizide, and Doripenem) dose responses and heat maps summarizing the dose-response results for the glutathione and senescence assays are presented in Fig. 6 and Fig. 7, respectively. Dose-response curves for the glutathione assay show that Phenylbutazone (Fig. 6B) and Meropenem (Fig. 6C) exhibited radioprotection equivalent to 0 Gy over a concentration range 0.1 – 100 µM and Diethylstilbestrol (Fig. 6D) was radioprotective at 10-100 µM. Prazosin (Fig. 6E), Enoxacin (Fig. 6F), Glipizide (Fig. 6G), and Doripenem (Fig. 6H) showed protection only at higher concentrations.

The radioprotection trends based on the senescence assay (Fig. 7) differed somewhat, with Phenylbutazone (Fig. 7B) and Meropenem (Fig. 7C) showing only partial protection and Diethylstilbestrol (Fig. 7D) exhibiting radioprotection equivalent to 0 Gy between 50-100 µM concentrations whereas Prazosin (Fig. 7E) showed complete protection between 0.1-100 µM. Glipizide (Fig. 7G) and Doripenem (Fig. 7H) also showed variable protection.

EC₅₀ values extrapolated from dose response curves are shown in Table 2. Phenylbutazone showed the most promising results, with low EC₅₀ values for both the glutathione (0.08 µM) and senescence (0.05 µM) assays. Phenylbutazone is a non-steroidal anti-inflammatory drug (NSAID) that inhibits cyclooxygenases (COX-1 and COX-2), enzymes that produce prostaglandins¹¹³. Prostaglandins, specifically PGE₂ signaling, have been shown to increase in irradiated salivary glands, and mitigation of salivary gland damage was achieved through treatment with the anti-inflammatory drug Indomethacin^{1, 114}. Indomethacin also showed radioprotection in our drug screen but was ineffective at concentrations lower than 100 µM. Indomethacin only blocks COX-1, underscoring the greater efficacy of Phenylbutazone. Phenylbutazone was originally developed for chronic pain for conditions such as arthritis but has since been restricted to treating ankylosing spondylitis due to induction of rare but severe blood disorders, including anemia and leukopenia¹¹³. However, doses ranged from 300-1000 mg, generating a plasma concentration of 30-50 µg/mL¹¹³. In contrast, the EC₅₀ of 0.08 µM for protecting against radiation-induced glutathione changes established in this study equates to a 26 ng/mL concentration. Thus, the risk of severe adverse effects may be greatly diminished for doses necessary for radioprotection. Additionally, Phenylbutazone has excellent bioavailability (up to 90%) ¹¹³^,¹¹⁵ and long half-life (50-105 hrs)¹¹³, which may enable dose de-escalation, further decreasing risks.

Enoxacin is an antibacterial agent used for treating urinary tract infections¹¹⁶ that was previously identified as radioprotective¹¹⁷. Using a high-throughput screening method with the viability of lymphocytes as the primary readout, two classes of antibiotics (tetracyclines and fluoroquinolones) were identified as robust radioprotectors, including Enoxacin¹¹⁷. This observation corroborates our drug screening results, in which several antibiotics were identified as double hits, including Enoxacin, Meropenem, Doripenem hydrate, Rifampin, and Rifabutin. The Enoxacin EC₅₀ of 2.4 µM for the glutathione assay is similar to the 13 µM EC₅₀ reported for viability of lymphocyte cells¹¹⁷. Notably, five other fluoroquinolones reported as radioprotectors (Levofloxacin, Gatifloxacin, Ofloxacin, Moxifloxacin, and Norfloxacin)¹¹⁷ were not hits in our drug screen (Table S3). These disparities may be related to differences in cell type (salivary gland versus lymphocyte) or readouts (glutathione/senescence versus viability).

Based upon drug down selection data and measured EC₅₀ values, Phenylbutazone, Enoxacin, and Doripenem hydrate were selected for in vivo validation in mice with γH2AX foci per nucleus IHC staining as an outcome measure consistent with prior work showing correlation with the development of xerostomia^{17, 41, 42, 46}. Vehicle-treated SGm exposed to 15 Gy radiation exhibited a 3.3-fold increase in the number of γH2AX foci per nucleus, indicating a significant increase in double-stranded DNA breaks due to radiation exposure (Figs. 8A-D, I). Treatment with WR-1065 resulted in a 0.5-fold reduction in γH2AX foci per nucleus compared to 15 Gy controls (Figs. 8D, E, I). No significant differences existed between 0 Gy controls and WR-1065 treated SGm exposed to 15 Gy (Figs. 8C, E, I). These results are similar to prior studies utilizing WR-1065 in vitro and in vivo via retrograde ductal injection^{17, 42} . Treatment with the test compounds, Phenylbutazone, and Enoxacin resulted in 0.4- and 0.5-fold reduction in γH2AX foci per nucleus compared to 15 Gy controls, respectively (Figs. 8E-G, I). Results observed after Phenylbutazone and Enoxacin treatment were not significantly different from 0 Gy controls (Figs. 8C, F, G, I). Treatment with Doripenem hydrate did not reduce γH2AX foci per nucleus relative to 15 Gy controls and showed a 2.7-fold increase compared to 0 Gy controls (Figs. 8C, D, H, I).

To overcome long-standing challenges associated with off-target radiation damage resulting in life-long dry mouth and poor quality of life, we sought to leverage our salivary gland tissue chip technology¹⁷ to identify novel radioprotective drugs. First, we investigated several assays that detect cell damage following radiation exposure (Table S1). We identified reduced glutathione and β-galactosidase (cell senescence) as biomarkers of radiation damage and developed assay protocols for high-content screening. We validated the assays with known radioprotective drugs, including WR-1065, the active metabolite of Amifostine, the only FDA-approved preventative therapy for radiation-induced xerostomia^{14, 15, 51}. We next tested other drugs reported to be radioprotective including Tempol^{20, 21, 49}, Edaravone^{23, 56, 57}, N-acetylcysteine²², Palifermin^{27, 28}, Ex-Rad^{25, 26}, and Rapamycin²⁴. Our results found that Tempol and Edaravone showed complete radioprotection similar to WR-1065 by both assay whereas the other drugs showed partial radioprotection. Partial protection likely reflects differences in tissue types or the assay protocols used in the other studies but our results confirm that all the drugs tested exhibit radioprotection at some concentration, by one or both assays (Table S5). A library of FDA-approved drugs was screened, enabling the de novo identification of 25 drugs with radioprotective activity (Table 1). Of the 25 hits, 20 drugs have known interactions with proteins involved in calcium signaling (Fig. 5A), which is critical for saliva secretion¹¹⁸. Only 9 of the 25 compounds have known antioxidant properties, and 12 are anti-inflammatory. This suggests alternate mechanisms of radioprotection. A DSEA pathways analysis suggests these drugs upregulate secretion and activate pathways related to cell adhesion, cell-cell and cell-matrix interactions suggesting possible new mechanistic targets for designing novel radioprotective drugs. Using the PubChem database and experimentally-determined EC₅₀ values (Figs. 6, 7 and Table 2), the list of 25 double hits was narrowed down to 3 candidates (Phenylbutazone, Enoxacin, and Doripenem) for testing using an in vivo radiation damage model. Results demonstrate that Phenylbutazone and Enoxacin have equivalent radioprotection to WR-1065 (Fig. 8).

In conclusion, this body of work validates the SGm tissue chip as a high-content drug discovery tool. We validated glutathione and β-galactosidase (cell senescence) as biomarkers of ionizing radiation damage. Novel drugs were discovered that confer radioprotection by mechanisms other than antioxidant activity. Ongoing studies seek to investigate chemical analogs and mechanisms of action to identify lead drug candidates that can be developed for clinical translation.

Acknowledgements

The authors gratefully acknowledge the support of the National Institute of Dental and Craniofacial Research (NIDCR), and the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health, under award numbers UG3DE027695 and UH3 DE002212 to DSWB, CEO, and LAD, NIEHS Environmental Health Sciences Center T32 ES007026 to JAM, and F31 DE029658 to LRP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors would like to thank Matthew Ingalls for helpful discussions.

Jasmer, K.J., Gilman, K.E., Muñoz Forti, K., Weisman, G.A. & Limesand, K.H. Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions. J Clin Med 9 (2020).
Escobar, A.A.-S., J.P. Xerostomia: An Update of Causes and Treatments. In Salivary Glands - New Approaches in Diagnostics and Treatment. In Salivary Glands - New Approaches in Diagnostics and Treatment; IntechOpen, 15–37 (2019).
Mori, M.T., H.; Sato, M.; Sumitomo, S Antimicrobial Peptides in Saliva and Salivary Glands: Their Roles in the Oral Defense System. Oral Med Pathol 11, 1-17 (2006).
Shubin, A.D., Felong, T.J., Graunke, D., Ovitt, C.E. & Benoit, D.S. Development of poly(ethylene glycol) hydrogels for salivary gland tissue engineering applications. Tissue Eng Part A 21, 1733-1751 (2015).
Shubin, A.D. et al. Encapsulation of primary salivary gland cells in enzymatically degradable poly(ethylene glycol) hydrogels promotes acinar cell characteristics. Acta Biomater 50, 437-449 (2017).
Pringle, S. et al. Human Salivary Gland Stem Cells Functionally Restore Radiation Damaged Salivary Glands. Stem Cells 34, 640-652 (2016).
Nam, K. et al. Laminin-1 Peptides Conjugated to Fibrin Hydrogels Promote Salivary Gland Regeneration in Irradiated Mouse Submandibular Glands. Front Bioeng Biotechnol 9, 729180 (2021).
Baum, B.J. et al. Development of a gene transfer-based treatment for radiation-induced salivary hypofunction. Oral Oncol 46, 4-8 (2010).
Alevizos, I. et al. Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction. Gene Ther 24, 176-186 (2017).
Baum, B.J. et al. Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction. Proc Natl Acad Sci U S A 109, 19403-19407 (2012).
Lombaert, I.M.A. et al. CERE-120 Prevents Irradiation-Induced Hypofunction and Restores Immune Homeostasis in Porcine Salivary Glands. Mol Ther Methods Clin Dev 18, 839-855 (2020).
Wang, X. & Eisbruch, A. IMRT for head and neck cancer: reducing xerostomia and dysphagia. J Radiat Res 57 Suppl 1, i69-i75 (2016).
Eisbruch, A. Reducing xerostomia by IMRT: what may, and may not, be achieved. J Clin Oncol 25, 4863-4864 (2007).
Rades, D. et al. Serious adverse effects of amifostine during radiotherapy in head and neck cancer patients. Radiother Oncol 70, 261-264 (2004).
Eisbruch, A. Amifostine in the treatment of head and neck cancer: intravenous administration, subcutaneous administration, or none of the above. J Clin Oncol 29, 119-121 (2011).
Shaw, L.M. et al. Human pharmacokinetics of WR-2721. Int J Radiat Oncol Biol Phys 12, 1501-1504 (1986).
Song, Y. et al. Development of a functional salivary gland tissue chip with potential for high-content drug screening. Commun Biol 4, 361 (2021).
Meeks, L., De Oliveira Pessoa, D., Martinez, J.A., Limesand, K.H. & Padi, M. Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction. Physiol Genomics 53, 85-98 (2021).
Peng, X. et al. Cellular senescence contributes to radiation-induced hyposalivation by affecting the stem/progenitor cell niche. Cell Death Dis 11, 854 (2020).
Vitolo, J.M. et al. The stable nitroxide tempol facilitates salivary gland protection during head and neck irradiation in a mouse model. Clin Cancer Res 10, 1807-1812 (2004).
Hu, L. et al. Effect of Tempol on the prevention of irradiation-induced mucositis in miniature pigs. Oral Dis 23, 801-808 (2017).
Kim, H.J. et al. Protective Effects of N-Acetylcysteine against Radiation-Induced Oral Mucositis In Vitro and In Vivo. Cancer Res Treat 52, 1019-1030 (2020).
Homma, T., Kobayashi, S., Sato, H. & Fujii, J. Edaravone, a free radical scavenger, protects against ferroptotic cell death in vitro. Exp Cell Res 384, 111592 (2019).
Zhu, Z. et al. Prevention of irradiation-induced salivary hypofunction by rapamycin in swine parotid glands. Oncotarget 7, 20271-20281 (2016).
Ghosh, S.P. et al. Radiation protection by a new chemical entity, Ex-Rad: efficacy and mechanisms. Radiat Res 171, 173-179 (2009).
Kang, A.D. et al. ON01210.Na (Ex-RAD(R)) mitigates radiation damage through activation of the AKT pathway. PLoS One 8, e58355 (2013).
Lombaert, I.M. et al. Keratinocyte growth factor prevents radiation damage to salivary glands by expansion of the stem/progenitor pool. Stem Cells 26, 2595-2601 (2008).
Choi, J.S., Shin, H.S., An, H.Y., Kim, Y.M. & Lim, J.Y. Radioprotective effects of Keratinocyte Growth Factor-1 against irradiation-induced salivary gland hypofunction. Oncotarget 8, 13496-13508 (2017).
Pinkstaff, C.A. Salivary gland sexual dimorphism: a brief review. Eur J Morphol 36 Suppl, 31-34 (1998).
Maruyama, C.L., Monroe, M.M., Hunt, J.P., Buchmann, L. & Baker, O.J. Comparing human and mouse salivary glands: A practice guide for salivary researchers. Oral Dis 25, 403-415 (2019).
Giang, U.B. et al. Quantitative analysis of spherical microbubble cavity array formation in thermally cured polydimethylsiloxane for use in cell sorting applications. Biomed Microdevices 16, 55-67 (2014).
Giang, U.B., Lee, D., King, M.R. & DeLouise, L.A. Microfabrication of cavities in polydimethylsiloxane using DRIE silicon molds. Lab Chip 7, 1660-1662 (2007).
Song, Y. et al. Encapsulation of Primary Salivary Gland Acinar Cell Clusters and Intercalated Ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-Degradable Hydrogels to Maintain Tissue Structure and Function. Adv Healthc Mater 11, e2101948 (2022).
Fairbanks, B.D., Schwartz, M.P., Bowman, C.N. & Anseth, K.S. Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials 30, 6702-6707 (2009).
Shubin, A.D. et al. Stress or injury induces cellular plasticity in salivary gland acinar cells. Cell Tissue Res 380, 487-497 (2020).
Kim, S. et al. PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Res 49, D1388-d1395 (2021).
Kim, S. et al. PubChem Substance and Compound databases. Nucleic Acids Res 44, D1202-1213 (2016).
Wang, Y. et al. An overview of the PubChem BioAssay resource. Nucleic Acids Res 38, D255-266 (2010).
Napolitano, F., Sirci, F., Carrella, D. & di Bernardo, D. Drug-set enrichment analysis: a novel tool to investigate drug mode of action. Bioinformatics 32, 235-241 (2016).
Bardet, E. et al. Preliminary data of the GORTEC 2000-02 phase III trial comparing intravenous and subcutaneous administration of amifostine for head and neck tumors treated by external radiotherapy. Semin Oncol 29, 57-60 (2002).
Varghese, J.J. et al. Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation. J Vis Exp (2018).
Varghese, J.J. et al. Localized Delivery of Amifostine Enhances Salivary Gland Radioprotection. J Dent Res 97, 1252-1259 (2018).
Kim, A., Banevicius, M.A. & Nicolau, D.P. In vivo pharmacodynamic profiling of doripenem against Pseudomonas aeruginosa by simulating human exposures. Antimicrob Agents Chemother 52, 2497-2502 (2008).
Kari, F., Bucher, J., Haseman, J., Eustis, S. & Huff, J. Long-term exposure to the anti-inflammatory agent phenylbutazone induces kidney tumors in rats and liver tumors in mice. Jpn J Cancer Res 86, 252-263 (1995).
Rocha, A.L. et al. Enoxacin induces oxidative metabolism and mitigates obesity by regulating adipose tissue miRNA expression. Sci Adv 6 (2020).
Varghese, J.J. et al. Retroductal Nanoparticle Injection to the Murine Submandibular Gland. J Vis Exp (2018).
Arany, S., Benoit, D.S., Dewhurst, S. & Ovitt, C.E. Nanoparticle-mediated gene silencing confers radioprotection to salivary glands in vivo. Mol Ther 21, 1182-1194 (2013).
Varghese, J.J. et al. Localized Delivery of Amifostine Enhances Salivary Gland Radioprotection. J Dent Res 97, 1252-1259 (2018).
Cotrim, A.P. et al. Differential radiation protection of salivary glands versus tumor by Tempol with accompanying tissue assessment of Tempol by magnetic resonance imaging. Clin Cancer Res 13, 4928-4933 (2007).
Djuzenova, C.S. et al. A prospective study on histone gamma-H2AX and 53BP1 foci expression in rectal carcinoma patients: correlation with radiation therapy-induced outcome. BMC Cancer 15, 856 (2015).
Hofer, M. et al. Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells. J Med Chem 59, 3003-3017 (2016).
Mosteller, R.D. Simplified calculation of body-surface area. N Engl J Med 317, 1098 (1987).
Sharma, R. & Sharma, S. in StatPearls (Treasure Island (FL) ineligible companies. Disclosure: Sandeep Sharma declares no relevant financial relationships with ineligible companies.; 2023).
Noaparast, Z. & Hosseinimehr, S.J. Radioprotective agents for the prevention of side effects induced by radioiodine-131 therapy. Future Oncol 9, 1145-1159 (2013).
Citrin, D. et al. Radioprotectors and mitigators of radiation-induced normal tissue injury. Oncologist 15, 360-371 (2010).
Chen, L., Liu, Y., Dong, L. & Chu, X. Edaravone protects human peripheral blood lymphocytes from gamma-irradiation-induced apoptosis and DNA damage. Cell Stress Chaperones 20, 289-295 (2015).
Ishii, J. et al. The free-radical scavenger edaravone restores the differentiation of human neural precursor cells after radiation-induced oxidative stress. Neurosci Lett 423, 225-230 (2007).
Iglesias-Bartolome, R. et al. mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis. Cell Stem Cell 11, 401-414 (2012).
Blanchard, C. et al. Neomycin Sulfate Improves the Antimicrobial Activity of Mupirocin-Based Antibacterial Ointments. Antimicrob Agents Chemother 60, 862-872 (2016).
Colquhoun, J.M. et al. Identification of Small Molecule Inhibitors of Staphylococcus aureus RnpA. Antibiotics (Basel) 8 (2019).
Moffat, J.G., Vincent, F., Lee, J.A., Eder, J. & Prunotto, M. Opportunities and challenges in phenotypic drug discovery: an industry perspective. Nat Rev Drug Discov 16, 531-543 (2017).
Rottmann, M. et al. Spiroindolones, a potent compound class for the treatment of malaria. Science 329, 1175-1180 (2010).
Swinney, D.C. Phenotypic vs. target-based drug discovery for first-in-class medicines. Clin Pharmacol Ther 93, 299-301 (2013).
Coppes, R.P., Zeilstra, L.J., Vissink, A. & Konings, A.W. Sialogogue-related radioprotection of salivary gland function: the degranulation concept revisited. Radiat Res 148, 240-247 (1997).
Coppes, R.P., Zeilstra, L.J., Kampinga, H.H. & Konings, A.W. Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists. Br J Cancer 85, 1055-1063 (2001).
Lafrenie, R.M. & Yamada, K.M. Integrins and matrix molecules in salivary gland cell adhesion, signaling, and gene expression. Ann N Y Acad Sci 842, 42-48 (1998).
Rose, R.W., Grant, D.S., O'Hara, M.D. & Williamson, S.K. The role of laminin-1 in the modulation of radiation damage in endothelial cells and differentiation. Radiat Res 152, 14-28 (1999).
Cordes, N., Blaese, M.A., Meineke, V. & Van Beuningen, D. Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro. Int J Radiat Biol 78, 347-357 (2002).
Baluna, R.G., Eng, T.Y. & Thomas, C.R. Adhesion molecules in radiotherapy. Radiat Res 166, 819-831 (2006).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 2082, A.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Albendazole. (
Lee, E.H., Baek, S.Y., Park, J.Y. & Kim, Y.W. Rifampicin activates AMPK and alleviates oxidative stress in the liver as mediated with Nrf2 signaling. Chem Biol Interact 315, 108889 (2020).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 135398735, R.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Rifampicin. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 73303, D.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Doripenem. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 443872, E.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Eplerenone. (
Xiao, J., Shimada, M., Liu, W., Hu, D. & Matsumori, A. Anti-inflammatory effects of eplerenone on viral myocarditis. Eur J Heart Fail 11, 349-353 (2009).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 2754, C.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Cilostazol.
Ota, H. et al. Cilostazol Inhibits Oxidative Stress–Induced Premature Senescence Via Upregulation of Sirt1 in Human Endothelial Cells. Arteriosclerosis, Thrombosis, and Vascular Biology 28, 1634-1639 (2008).
Jung, W.K. et al. Cilostazol is anti-inflammatory in BV2 microglial cells by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases. Br J Pharmacol 159, 1274-1285 (2010).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 3305, E.A.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Etidronic-Acid. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 3950, L.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Lomustine.
Katsouri, L. et al. Prazosin, an α(1)-adrenoceptor antagonist, prevents memory deterioration in the APP23 transgenic mouse model of Alzheimer's disease. Neurobiol Aging 34, 1105-1115 (2013).
Malekinejad, Z. et al. Prazosin Treatment Protects Brain and Heart by Diminishing Oxidative Stress and Apoptotic Pathways After Renal Ischemia Reperfusion. Drug Res (Stuttg) 72, 336-342 (2022).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 4893, P.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Prazosin. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 448537, D.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Diethylstilbestrol. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 31307, T.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Triamcinolone. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 4485, N.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Nifedipine. (
Yao, J., Long, H., Zhao, J., Zhong, G. & Li, J. Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway. Life Sciences 253, 117292 (2020).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 5280453, C.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Calcitriol. (
Tukaj, S., Grüner, D., Tukaj, C., Zillikens, D. & Kasperkiewicz, M. Calcitriol exerts anti-inflammatory effects in keratinocytes treated with autoantibodies from a patient with bullous pemphigoid. J Eur Acad Dermatol Venereol 30, 288-292 (2016).
Kim, Y.H. et al. in Antioxidants, Vol. 11 (2022).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 34312, O.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Oxcarbazepine. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 441130, M.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Meropenem. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 135398739, D.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Didanosine. (
Dağli, A.F. et al. Antiinflammatory and antioxidant effects of gemcitabine in collagen-induced arthritis model. Turk J Med Sci 47, 1037-1044 (2017).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 60750, G.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Gemcitabine. (
K, P.S., K, A.A., Medammal, Z., Thayyil, M.S. & Babu, T.D. Theoretical insights into the radical scavenging activity of glipizide: DFT and molecular docking studies. Free Radic Res 56, 53-62 (2022).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 3478, G.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Glipizide. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 60871, A.d.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Adefovir-dipivoxil. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 60877, E.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Emtricitabine. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 3715, I.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Indomethacin. (
Cusan, C. et al. Anti-inflammatory and anti-oxidant activity of a new class of phenyl-pyrazolone derivatives. Curr Drug Discov Technol 3, 67-73 (2006).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 4781, P.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Phenylbutazone. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 969516, C.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Curcumin. (
Esposito, E. & Cuzzocrea, S. Antiinflammatory activity of melatonin in central nervous system. Curr Neuropharmacol 8, 228-242 (2010).
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 896, M.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Melatonin. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 3229, E.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Enoxacin. (
National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 2141, A.R.J., 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Amifostine. (
Gupta, M.N., Alam, A. & Hasnain, S.E. Protein promiscuity in drug discovery, drug-repurposing and antibiotic resistance. Biochimie 175, 50-57 (2020).
DeMuro, R.L., Nafziger, A.N., Blask, D.E., Menhinick, A.M. & Bertino, J.S., Jr. The absolute bioavailability of oral melatonin. J Clin Pharmacol 40, 781-784 (2000).
Dunn, C.J., Fitton, A. & Sorkin, E.M. Etidronic acid. A review of its pharmacological properties and therapeutic efficacy in resorptive bone disease. Drugs Aging 5, 446-474 (1994).
Ochoa, D. et al. High-Fat Breakfast Increases Bioavailability of Albendazole Compared to Low-Fat Breakfast: Single-Dose Study in Healthy Subjects. Front Pharmacol 12, 664465 (2021).
Khan, M.A. et al. Eplerenone nanocrystals engineered by controlled crystallization for enhanced oral bioavailability. Drug Deliv 28, 2510-2524 (2021).
Lees, P. & Toutain, P.L. Pharmacokinetics, pharmacodynamics, metabolism, toxicology and residues of phenylbutazone in humans and horses. Vet J 196, 294-303 (2013).
Gilman, K.E., Camden, J.M., Woods, L.T., Weisman, G.A. & Limesand, K.H. Indomethacin Treatment Post-irradiation Improves Mouse Parotid Salivary Gland Function via Modulation of Prostaglandin E(2) Signaling. Front Bioeng Biotechnol 9, 697671 (2021).
Smith, P.B., Caldwell, J., Smith, R.L., Horner, M.W. & Moss, M.S. The bioavailability of phenylbutazone in the horse. Xenobiotica 17, 435-443 (1987).
Henwood, J.M. & Monk, J.P. Enoxacin. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 36, 32-66 (1988).
Kim, K. et al. High-Throughput Screening Identifies Two Classes of Antibiotics as Radioprotectors: Tetracyclines and Fluoroquinolones. Clinical Cancer Research 15, 7238-7245 (2009).
Ambudkar, I.S. Calcium signalling in salivary gland physiology and dysfunction. J Physiol 594, 2813-2824 (2016).

Table 1: Drug activity based on data from the BioAssay database. Drugs highlighted in dark gray were excluded from further analysis due to bioactivity in >10 % of assays. Drugs in light gray were excluded based on poor bioavailability and batch variability (eplerenone)

Table 2: Calculated EC₅₀ values for the top radioprotective drugs. Values were estimated using a nonlinear fit in Prism. “Undetermined” indicates that the software could not accurately fit a curve to the data.

	Estimated EC₅₀ (µM)
Compound	Glutathione	Senescence
Phenylbutazone	0.08	0.05
Meropenem	0.1	undetermined
Diethylstilbestrol	0.7	37.7
Prazosin HCl	1	0.04
Enoxacin	2.4	0.5
Glipizide	4.0	undetermined
Doripenem hydrate	8.6	16.4

There is NO Competing Interest.

SupplementalMaterials.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Identifying novel radioprotective drugs via salivary gland tissue chip screening

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

3. Results and Discussion

4. Conclusions

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1