Tranilast, an anti-allergic drug used in the treatment of bronchial asthma, was identified as an inhibitor of the transcription factor Forkhead box O-1 (FoxO-1) by high throughput chemical library screening in the present study. Based on FoxO-1’s role in apoptotic cell death and differentiation, we examined the effect of tranilast on palmitic acid (PA)-induced cell damage in INS-1 cells. Tranilast substantially inhibited lipoapoptosis and restored glucose-stimulated insulin secretion under high PA exposure. Moreover, PA-mediated downregulation of PDX-1, MafA, and insulin expression was attenuated by tranilast. PA-induced oxidative and ER stress were also reduced in the presence of tranilast. These protective effects were accompanied by increased phosphorylation and decreased nuclear translocation of FoxO-1. Conversely, the effects of tranilast were diminished when treated in transfected cells with FoxO-1 phosphorylation mutant (S256A), suggesting that the tranilast-mediated effects are associated with inactivation of FoxO-1. Examination of the in vivo effects of tranilast using wild type and diabetic db/db mice showed improved glucose tolerance along with FoxO-1 inactivation in the pancreas of the tranilast-treated groups. Thus, we report here that tranilast has protective effects against PA-induced lipotoxic stress in INS-1 cells, at least partly, via FoxO-1 inactivation, which results in improved glucose tolerance in vivo. Four isoforms of FoxO  have been identified in mammalian cells, among which FoxO-1 is the most abundant and best studied isoform2. Previous studies have shown that overexpression of FoxO-1 in insulin-sensitive tissues is associated with type 2 diabetes mellitus3. Of note, FoxO-1 regulates the transcription of two important enzymes involved in hepatic gluconeogenesis (glucose-6 phosphatase and phosphoenolpyruvate carboxykinase)5. Thus, the regulation of FoxO-1 activity as well as its expression significantly contributes to hepatic glucose production.Forkhead box O (FoxO) proteins act as nuclear transcription factors and are involved in negative cross-talk with the insulin signaling pathway7. Elevation in free fatty acid levels, especially that of saturated fatty acids such as palmitic acid (PA), results in impairment of insulin production and secretion, and β-cell apoptosis10, which in turn leads to lipotoxicity, serving as a key player in the pathophysiology of type 2 diabetes. Among the various mediators of lipotoxicity, FoxO-1 is known to play a crucial role in β-cell function, inhibiting β-cell replication and neogenesis11. Accordingly, FoxO-1 mRNA levels were found to be increased in the islets of patients with type 2 diabetes in clinical studies12.β-cell dysfunction is a prominent feature of type 2 diabetes that results in defects in glucose-stimulated insulin secretion13, a transcription factor involved in β-cell function and survival, leading to the suppression of mRNA expression of insulin, glucagon-like peptide 1, and glucokinase14. In addition, several genes involved in apoptosis, and oxidative and ER stress are known to be downstream transcriptional targets of FoxO-1. FoxO-1 activity is regulated through various post-translational modifications, including phosphorylation, acetylation, and ubiquitination. Importantly, FoxO-1 phosphorylation by protein kinase B (PKB/Akt) results in its exclusion from the nucleus, and thus FoxO-1 remains in an inactive state15. On the other hand, decreased FoxO-1 phosphorylation allows FoxO-1 to enter the nucleus and play an active role in the transcription of several genes.FoxO-1 is known to bind to the promoter of pancreatic duodenal homeobox 1 (PDX-1)Based on the negative role of FoxO-1 in insulin action, FoxO-1 inhibitors may be applicable as a new therapeutic approach to treat metabolic disorders, including type 2 diabetes. However, FoxO-1 as a potential therapeutic target remains underutilized due to the non-availability of specific and potent inhibitors. To identify new FoxO-1 inhibitors, we screened a chemical library of the Korean Chemical Bank for FoxO-1 inhibitory activity, and identified tranilast as a FoxO-1 inhibitor.16. Initially, tranilast was developed as an anti-allergic agent and is currently used for the treatment of inflammatory diseases such as bronchial asthma, atypical dermatitis, and allergic conjunctivitis17. Its main mechanism of action is attributed to its inhibitory action on the release of the chemical mediator, histamine18. In the present study, the pharmacological profile of tranilast as a FoxO-1 inhibitor was investigated for its repositioning as a potential anti-diabetic agent with special focus on β-cell function. Since lipotoxicity induced by saturated fatty acids appears to be an important cause of impairment of β-cell function19, PA-induced lipotoxicity model was employed in the present study, and the effect of tranilast on rat INS-1 pancreatic β-cells following PA exposure was examined. The in vivo efficacy of tranilast was evaluated using two mouse strains, C57BL/6J and db/db, based on the facts that C57BL/6J strain is the popular stain for metabolic research with diabetes prone characteristics, and db/db strain is a well-known type 2 diabetes animal model with β-cell dysfunction.Tranilast  is an analog of a tryptophan metabolite50 of 30 μM  to identify new FoxO-1 inhibitors, and initially identified 260 active hits. 163 hits were excluded for further study due to their cytotoxicity, and 97 hits were confirmed. Among those, tranilast was selected for the biochemical and pharmacological characterization as a FoxO-1 inhibitor  for 24 h, which was restored in the presence of tranilast in a concentration-dependent manner. Complete restoration of cell viability was achieved following treatment with 100 μM tranilast , which was reduced following tranilast treatment (13.1% at 100 μM) Fig. A. In addp < 0.005).As β-cells are sensitive to both oxidative and ER stress, we examined the effect of tranilast on PA-induced oxidative and ER stress. First, the effect of tranilast on PA-induced ROS production was determined using a DCF-DA probe. As shown in Fig. Oxidative stress results in mitochondrial dysfunction reflected as changes in the mitochondrial transmembrane potential. Here, JC-1, a dye for monitoring mitochondrial membrane potential, was used to examine the effect of tranilast on mitochondrial function. PA induced the breakdown of mitochondrial membrane potential by 19.3 ± 1.75%, which was restored almost to normal levels by treatment with 100 μM tranilast  was markedly increased by PA, whereas the level was restored by tranilast Fig. A. The efp < 0.05), which was restored by tranilast   Fig. C lower. To elucidate the underlying mechanism of action of tranilast with a special focus on FoxO-1, the levels of phosphorylated FoxO-1 (pFoxO-1) were assessed following PA and tranilast treatments. Compared to the control group, PA treatment significantly reduced the level of pFoxO-1 in whole cell lysates Fig. A, and thNext, we determined the effects of tranilast after the cells were transfected with FoxO-1 phosphorylation mutant  to further confirm that tranilast actions are attributed to the FoxO-1 phosphorylation. As expected, the nuclear FoxO-1 induced by PA was translocated to the cytoplasm by tranilast treatment in wild type FoxO-1 transfected cells, whereas tranilast-induced FoxO-1 translocation from the nucleus to the cytoplasm was disappeared in FoxO-1 S256A mutant-transfected cells  was orally administered to C57BL/6J mice three times over 2 days, and a glucose tolerance test was performed. As expected, no difference was observed in fasting plasma glucose levels between vehicle and tranilast-treated groups, but tranilast improved glucose clearance compared to the vehicle group  for 4 weeks. The levels of non-fasting blood glucose decreased in the tranilast-treated group Fig. A, with iIn this study, we investigated the protective effect of tranilast on PA-induced β-cell dysfunction, and its mechanism of action as a FoxO-1 inhibitor using INS-1 cells. We found that tranilast ameliorated PA-induced β-cell lipotoxicity via FoxO-1 inhibition in vitro. In vivo administration of tranilast improved glucose tolerance with increased pFoxO-1 and PDX-1 levels in the pancreas of db/db mice. These data suggest the possible repositioning of tranilast as an anti-diabetic agent.7. Various genetic and environmental factors are implicated in β-cell dysfunction, and most notably chronic exposure to saturated fatty acids induces β-cell dysfunction and apoptosis, leading to the development of type 2 diabetes10. Thus, protection of β-cells from saturated fatty acid-induced damage is critically important for preventing impairment of glucose homeostasis. The detailed molecular mechanisms involved in PA-induced β-cell dysfunction and apoptosis have not been fully elucidated, although several pathways such as those mediating mitochondrial dysfunction, ER stress, and oxidative stress have been implicated22. Consistently, we found that PA (500 μM) induced mitochondrial dysfunction and ER and oxidative stress leading to INS-1 β-cell dysfunction and apoptosis in our study.Normal pancreatic β-cell function and mass are critical for maintaining glucose homeostasis, and any defect significantly contributes to the development of diabetes mellitus24. FoxO-1 is a negative regulator of the transcription factor, PDX-1 that is crucial for β-cell growth and function25. FoxO-1 inactivation leads to increased PDX-1 expression and β-cell proliferation, and FoxO-1 suppression reduces the expression of apoptotic markers11. In contrast, FoxO-1 overexpression promotes β-cell apoptosis by reducing CD24 expression26. In addition to its role in β-cell apoptosis, FoxO-1 activity also affects GSIS and modulates the transcription of FoxO-1 target genes including PDX-1, MafA, and insulin in β-cells. In agreement with the role of FoxO-1 in β-cells, tranilast prevented PA-induced apoptosis via increased PDX-1 transcription in our study, possibly acting as a FoxO-1 inhibitor.Among the many factors involved in β-cell maintenance, FoxO-1 plays a key regulatory role in β-cell mass and function27. Interestingly, FoxO-1 activity is influenced by oxidative stress through various post-translational modifications, such as phosphorylation, acetylation, and ubiquitination28. For example, FoxO-1 phosphorylation in β-cells is reduced by oxidative stress, allowing its translocation from the cytoplasm to the nucleus29. In contrast, inhibition of PDX-1 expression in response to lipotoxicity due to oxidative stress, results in its increased translocation from the nucleus to the cytoplasm30. Accordingly, our results showed that tranilast treatment attenuated PA-induced oxidative stress, in association with increased shuttling of FoxO-1 from the nucleus to the cytoplasm.Accumulating evidence suggests that oxidative stress also contributes to structural and functional damage of β-cells31, tranilast relieved mitochondrial depolarization and upregulated the Bcl-2/Bax ratio, thereby restoring PA-induced mitochondrial dysfunction. In addition, ER stress in response to lipotoxicity was shown to contribute to β-cell apoptosis11, which was mitigated by tranilast treatment, thus adding to the protective mechanisms against PA-induced β-cell damage. Consistent with our results, FoxO-1 inhibition downregulated the expression of ER stress markers and promoted β-cell survival under free fatty acid stress11. Pancreatic β-cells are highly susceptible to oxidative stress due to their poor antioxidant defense mechanisms32. Our current data revealed that PA-induced ROS production was also inhibited following treatment with tranilast.In the present study, we uncovered a novel mechanism of action of tranilast and demonstrated its protective effects against PA-induced β-cell lipotoxicity. Tranilast inhibited PA-induced apoptosis in a concentration-dependent manner. Consistent with the findings that mitochondrial depolarization induced by PA leads to β-cell apoptosis29. Real-time qPCR revealed that the mRNA levels of these FoxO-1 target genes were regulated by PA and corrected by tranilast treatment. Interestingly, increased phosphorylation of FoxO-1 was observed in the presence of tranilast that was accompanied by reduction of nuclear levels of FoxO-1. Moreover, the protective effects of tranilast from PA-induced lipotoxicity were abrogated when the cells were transfected with FoxO-1 S256A phosphorylation mutant, in association with the failure of FoxO-1translocation from the nucleus to the cytoplasm. These findings further support that tranilast inhibits FoxO-1 at least partly via increased its phosphorylation.The role of FoxO-1 inactivation in tranilast activity was confirmed through many experiments. In agreement with the inhibitory effect of tranilast on FoxO-1 activity observed using the FoxO-1 reporter assay, the expression of downstream targets of FoxO-1 was also altered by tranilast. For example, the pro-apoptotic Bcl-2 family member Bax, PDX-1, MafA, and insulin are known FoxO-1 target genes35. Furthermore, this signaling pathway is involved in the regulation of FoxO-1 transcriptional activity8. In contrast, activation of the JNK signaling pathway in response to oxidative stress leads to inhibition of Akt and subsequent dephosphorylation of FoxO-1, which results in nuclear accumulation of FoxO-136. Interestingly, we found that increased pFoxO-1 following tranilast treatment was accompanied by decreased Akt phosphorylation, but increased JNK phosphorylation (results not shown). Thus, besides its inhibitory activity on FoxO-1, tranilast may function via additional mechanisms of action, such as by regulation of PI3K/Akt/JNK phosphorylation. The detailed mechanism of tranilast-mediated induction of FoxO-1 phosphorylation needs to be elucidated further.PI3K/Akt signaling plays a central role in β-cell growth and functionIn vivo studies were carried out to examine the efficacy of tranilast using C57BL/6J and db/db mice. Improved glucose tolerance was observed following tranilast administration in both strains, with increased expression of PDX-1 and insulin, possibly via FoxO-1 inactivation in pancreatic β-cells. Other mechanisms of action of tranilast  may also contribute to the improved glucose tolerance in vivo. On the other hand, relatively mild in vivo effects of tranilast were observed, which may be attributed to several factors including the pharmacokinetic properties of tranilast and the mouse conditions . Although we focused on the FoxO-1 inhibitory effect of tranilast in pancreatic β-cells in the present study, other insulin-sensitive tissues such as the liver may also be affected by tranilast-mediated FoxO-1 inhibition. In summary, we found that tranilast protected pancreatic β-cells from PA-induced lipotoxicity and improved glucose tolerance at least partly through FoxO-1 inactivation. Notably, tranilast shows limited adverse effects and is well tolerated in patients. Based on the current study, tranilast may be of additional therapeutic value in the treatment of metabolic disorders such as type 2 diabetes mellitus.2 and 95% air. For the purpose of the present study, batches of cells were plated in 24-well plates at a density of 2 × 105 cells/well and cultured for 24 h in RPMI medium. Then, the cells were incubated with various concentrations  of tranilast for 1 h, followed by PA (500 μM) treatment for the indicated times. Bovine serum albumin (BSA)-bound PA in ethanol was prepared as described previously37.Rat INS-1 pancreatic β-cells obtained from the Korean Cell Line Bank  were grown in a cell culture flask in 12 mL of RPMI medium containing 10% FBS, 1% penicillin, and 1% streptomycin at 37 °C in an atmosphere of 5% CO6 cells) were transfected with FoxO-1 vector , and IRE vector (pGL3-4xIRE-Luc), and then seeded into 96 well plates at a density of 2 × 104 cells/well. After 24 h incubation with compounds (50 μM in DMSO), luciferase activity was measured using the Dual Luciferase Reporter Assay Kit , and normalized to Renilla luciferase activity for each compound.To discover novel FoxO-1 inhibitors, FoxO-1 luciferase reporter assay was established. HepG2 cells obtained from the Korean Cell Line Bank  in the absence or presence of various concentrations  of tranilast (1 h pretreatment) for 24 h. Then, the cells were incubated with MTT solution for 4 h at 37 °C under 5% CO2. DMSO (200 μL) was added to extract the MTT formazan, and the absorbance of each well was read at 570 nm using an automatic microplate reader .INS-1 cells were plated at a density of 1 × 10g for 10 min was incubated with 80 μL of assay buffer and 10 μL of the substrate  at 37 °C for 2 h. The optical density of the reaction mixture was determined using spectrophotometer at 405 nm.Caspase activities were determined using a colorimetric assay kit  according to the manufacturer’s protocol. After treatment with different concentrations of tranilast, the cells were harvested and lysed in lysis buffer for 5 min in an ice bath. Equal amount of protein (10 μg) obtained by centrifugation of lysates at 10,000×Treated cells were washed twice with phosphate buffered saline (PBS), fixed for 20 min in 4% paraformaldehyde, and washed three times in PBS. The cells were then blocked in 4% normal goat serum in PBS containing 0.3% Triton X-100 for 60 min. After rinsing twice with PBS, the cells were counterstained with DAPI in mounting medium . Cover slips were mounted on microscope slides, and images were obtained using an Olympus LG-PS2 microscope (Tokyo).N-2-ethanesulfonic acid/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4), and stained with 5 μL of FITC-conjugated annexin V antibody and 5 μL of PI (50 μg/mL). The mixture was incubated for 15 min at room temperature in the dark and analyzed by flow cytometry (Becton Dickinson Co.).Following the indicated treatments, cell apoptosis was measured using Annexin V and propidium iodide (PI) double staining. The cells were washed with PBS, suspended in 100 μL of binding buffer (10 mM N -2-hydroxyethylpiperazine- The apoptotic induction was determined using a cell death detection ELISA kit (Sigma-Aldrich) following the indicated treatment, which measures the specific mono- and oligonucleosomes in the cytoplasmatic fraction of cell lysates. The assay is based on a quantitative sandwich-enzyme-immunoassay-principle using monoclonal anti-histone and anti-DNA peroxidase antibodies. The relative apoptosis rate was photometrically determined by measuring the peroxidase activity of the immunocomplex at 405 nm, according to the manufacturer’s protocol.4 and/or xanthones III or V  at 37 °C in a shaking water bath for 2 h. Then, 100 μL of 75 μM DCF was added to the samples and incubated for 30 min in the dark. Finally, the samples were centrifuged at 6,000 × g for 15 min. ROS were conventionally detected in the supernatants by fluorescent spectrometry at 488 nm (excitation wavelength) and 532 nm (emission wavelength) using a Perkin-Elmer spectrometer. Results are expressed as percentage of ROS formation in treated cells versus control cells.ROS were detected by 2'-7'-dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence using a colorimetric assay kit  according to the manufacturer’s protocol. Briefly, aliquots (500 μL) of the cell lysates were incubated in the absence (control) or presence of 5 μM FeSOMitochondrial transmembrane potential was measured using a JC‐1 kit  according to the manufacturer's protocol and analyzed using a laser scanning Varioskan Flash (Thermo Fisher). Green and red fluorescence were analyzed at 590 and 530 nm, respectively, and the ratio of green/red was considered an indicator of mitochondrial transmembrane potential.2, 10 mM KCl, 0.2 mM phenylmethylsulfonylfluoride, 0.5 mM dithiothreitol, and 10 μg/mL aprotinin), incubated on ice for 15 min, lysed by adding 0.1% Nonidet P-40 and vortexed vigorously for 10 s. After centrifugation of lysates at 12,000 × g for 1 min at 4 °C, the resulting nuclei pellets were resuspended in high salt buffer , and used as nuclear fractions.To obtain nuclear extracts, the treated cells were washed once with PBS, and pelleted by centrifugation. The cell pellets were resuspended in hypotonic buffer . After incubation for 30 min at 4 °C, the lysates were centrifuged to remove cell debris, and quantified using the Bio-Rad protein assay reagent according to the manufacturer's instructions. Equal amount of cellular protein (30 μg) from treated and untreated cell extracts were run onto the sodium dodecyl sulfate–polyacrylamide gel. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes. After overnight blocking at 4 °C with 4% skim milk, the membranes were incubated with appropriate primary antibody (Santa Cruz Biotechnology) overnight. After several washes with Tween 20/Tris-buffered saline, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody  for 1 h at room temperature, washed three times with T-TBS and then developed using enhanced chemiluminescence (Amersham Life Science).GCAATGGACAACAACAGTAAA-3’; PC-FKHR(FoxO-1)_S256A, 5’-GTTGTCCATTGCTGCAGCTCTTCTCCTAGG-3’. After mutagenesis, the sequence was verified by sequencing. For immunofluorescence detection of GFP-FoxO-1, CHO cells were plated onto slide glass and were transfected with a total of 1.0 μg of wild-type or mutated pcDNA GFP FKHR(FoxO-1)-S256A using FuGENE . Then the cells were counterstained with DAPI to visualize the nuclei. After mounting, the sections were imaged with a Zeiss LSM 700 laser-scanning confocal microscope (Carl Zeiss) and analyzed with ZEN 2010 software (Carl Zeiss).To prepare FoxO-1 S256A mutant (TCC → GCA) vector, site-specific mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene) using following primers: PN-FKHR(FoxO-1)_S256A, 5’-AGAGCTGCATotal RNA was isolated from the cells using easy-Blue™ Kit (Intron Biotechnology). For each sample, 100 ng of RNA was reverse-transcribed (RT) using MuLV reverse transcriptase, 1 mM dNTP, and 0.5 µg/µL oligos (dT12-18). PCR amplification was performed using SYBR green. The PCR primers used were as follows: sense strand rPDX-1, 5′-AAATCC ACC AAA GCA CAC GC-3′, anti-sense strand rPDX-1, 5′-AAGTTGAGCACT ACT GCC AGC-3′; sense strand rMafA, 5′-AGGAGGAGGTCATCCGACTG-3′, anti-sense strand rMafA, 5′-CTTCTCGCTCTCCAGAATGTG-3′; sense strand rInsulin, 5′- GAC CCG CCAGTGCCACCA-3′, anti-sense strand rInsulin, 5′-TCCACAAGCCACGCATCTG-3′; sense strand rFoxO-1, 5′- AATTTGCTAAGAGCCGAGGA-3′; anti-sense strand rFoxO-1, 5′ -AAGTCATCATTGCTGTGGGA-3′; sense strand rDP, 5′-TCTGGAAGACACCCTCTGCT-3′, anti-sense strand rDP, 5′-CACAGAGTCCCACCATGTTG-3′; sense strand rGAPDH, 5′-GCCACTCAGAAGACTCTGGA-3′, anti-sense strand rGAPDH, 5′-GTTCAGCTCTGGGATGACCT-3′; sense strand rGRP78, 5′-CTG GAC TGA ATG TCA TGA GGA TCA-3′, anti-sense strand rGRP78, 5′-CTC TTA TCC AGG CCA TAT GCA ATA G-3′; sense rATF3, 5′-AAC TGG CTT CCT GTG CAC TT-3′, anti-sense strand rATF3, 5′-TGA GGC CAG CTA GGT CAT CT-3′; sense strand rCHOP, 5′-CCA CCA CAC CTG AAA GCA GAA-3′, anti-sense strand rCHOP, 5′-GGT GCC CCC AAT TTC ATC T-3′; sense strand mPDX-1, 5′-AAA TCC ACC AAA GCA CAC GC-3′, anti-sense strand mPDX-1, 5′-AAG TTG AGC ACT ACT GCC AGC-3′; sense strand mInsulin, 5′-AAA TCC ACC AAA GCA CAC GC-3′, anti-sense strand mInsulin, 5′-AAG TTG AGC ACT ACT GCC AGC-3′; sense strand mFoxO-1, 5′-AAA TCC ACC AAA GCA CAC GC-3′, anti-sense strand, mFoxO-1, 5′-AAG TTG AGC ACT ACT GCC AGC-3′; sense strand mGAPDH, 5′- CTC AAC TAC ATG GTC TAC ATG TTC CA -3′, anti-sense strand mGAPDH, 5′- CCA TTC TCG GCC TTG ACT GT ′. The results are expressed as a ratio of β-actin expression.2 atmosphere.Male C57BL/6J mice (8 weeks old) were injected with collagenase in the common bile duct under anesthesia, and the pancreas was isolated, minced, and placed in ice-cold Hanks solution. After incubation of the mixture at 37 °C for 15 min, the islets were hand-picked under a stereomicroscope, and cultured in RPMI medium supplemented with 10% heat-inactivated FBS, 25 mg/mL amphotericin B, 10,000 U/mL penicillin, and 10,000 mg/mL streptomycin at 37 °C in a humidified 5% CO4 cells/well) and incubated with tranilast  in the presence or absence of 500 μM PA for 24 h, and the GSIS assay was performed as described previously39.INS-1 cells or islets were plated in 24-well plates  were purchased from Orient Bio Inc. . C57BL/6J mice were acclimated for one week under constant conditions . For the experiments, the mice were divided into three groups (n = 8 each group): (1) control group (treated with vehicle solution containing 0.5% carboxymethyl cellulose (CMC)); (2) tranilast (50 mg/kg)-treated group; (3) tranilast (100 mg/kg)-treated group. Each treatment was done by oral injection at three time points .Db/db mice  were acclimated to the constant conditions  for one week prior to the experiments and maintained for 4 weeks or more. For the experiments, the mice were randomly assigned into four groups (n = 8 each group), and treated with (1) vehicle solution containing 0.5% CMC (control group), (2) tranilast (50 mg/kg in 0.5% CMC), (3) tranilast (100 mg/kg in 0.5% CMC), (4) tranilast (200 mg/kg in 0.5% CMC) by oral injection once daily for 4 weeks or more. Body weight, food intake, and blood glucose were monitored weekly at the same time every week (between 10:00 and 11:00 AM). The amount difference between the preweighed original amount of food and the weight of the food left in each cage (n = 4 per cage) were considered as food intake. Blood was obtained from the tail vein of the animals in a fed state, and glucose concentrations were determined using Gluco Dr. Plus . Animals were minimally anesthetized with 1–1.5% isoflurane using small animal anesthesia system, and tissues were rapidly isolated and weighed. Pancreatic tissues were fixed in 4% paraformaldehyde and stored at 25 °C until further analyses. Serum insulin levels were determined using ELISA kits . All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health  and were approved by the Animal Care and Use Committee of Gachon University, Seongnam, Korea . All methods involving animals are reported in accordance with ARRIVE  guidelines.For glucose tolerance test, the mice were orally given glucose (2 g/kg), and blood was collected at 0, 30, 60, and 120 min after glucose administration from the tail vein into capillary tubes (Chase Scientific Glass). Plasma glucose levels were measured using Gluco Dr. Plus (Allmedicus). The area under the curve (AUC) for glucose was calculated using OriginPro 6.1 .Pancreatic tissues fixed in 4% paraformaldehyde were embedded in paraffin, cut into 7 μm thick sections, and incubated with monoclonal antibodies against pFoxO-1, FoxO-1, PDX-1, and insulin (Santa Cruz Biotechnology) overnight at 4 °C. Then the sections were treated with biotin-conjugated goat anti-rabbit secondary antibody , followed by the treatment with streptavidin-conjugated with horseradish peroxidase . DAB peroxidase substrate (Vector Laboratories) was used for visualization, and the specimens were counterstained with hematoxylin .t-test for paired experiments or two-way analysis of variance. Results are expressed as the mean ± standard deviation (SD) of three separate experiments, and p < 0.05 was considered statistically significant.Data were analyzed using Student’s Supplementary Information.


Prostate cancer is a very common disease in men. Nowadays several life-prolonging therapies are available, also efficient in the metastatic setting. However, it is important to choose the best approach for each patient, and in this context molecular biomarkers are fundamental. Baseline high circulating tumor DNA (ctDNA) fraction in plasma and androgen receptor (AR) copy number (CN) gain correlates with worse outcomes. This study investigates correlation between PSA response endpoints, plasma DNA analysis and progression free/overall survival, underling the importance of a multimodal approach to early predict outcome.p = 0.017) and AR gain (p = 0.0003). Combining ctDNA fraction and AR CN, we found a different median PSA progression-free survival (PFS) among four groups: (1) low ctDNA/AR normal, (2) high ctDNA/AR normal, (3) low ctDNA/AR gain, and (4) high ctDNA/AR gain . In a multivariable analysis, high ctDNA, AR gain, PSA DT, PSA DT velocity remained independent predictors of PSA PFS. Conclusions: Elevated ctDNA levels and AR gain are negatively and independently correlated with PSA kinetics in mCRPC men treated with abiraterone or enzalutamide.Background: Baseline high circulating tumor DNA (ctDNA) fraction in plasma and androgen receptor (AR) copy number (CN) gain identify mCRPC patients with worse outcomes. This study aimed to assess if ctDNA associates with PSA kinetics. Methods: In this prospective biomarker study, we evaluate ctDNA fraction and AR CN from plasma samples. We divided patients into high and low ctDNA level and in AR gain and AR normal. Results: 220 baseline samples were collected from mCRPC treated with abiraterone (n = 140) or enzalutamide (n = 80). A lower rate of PSA decline ≥ 50% was observed in patients with high ctDNA ( AR locus in late stage tumors. This spurred inquiries by numerous groups to re-address the possible contribution of residual androgens remaining after castration as well as the AR itself to the progression of CRPC and its continued growth. Collectively, these efforts revealed that genetic alterations at the AR locus act to restore AR signaling in the setting of low androgens  and 205 patients (93.2%) had a performance status (PS) of 0–1. Gleason score was ≥ 8 in the 59.6% of cases. There was no treatment discontinuation due to adverse events and all patients demonstrated good tolerance to the drugs. Most patients (86.8%) had a low volume disease with 1 or 2 sites of metastases. All patients were previously treated with ADT for hormone-sensitive prostate cancer, and 156 (70.9%) received docetaxel for CRPC before ARSI. One-hundred ninety (86.4%) men had bone metastasis and 31 (14.1%) visceral metastasis . There wAR gain was detected in 73 (33.2%) patients.In all pre-treatment plasma samples, median ctDNA fraction assessed by targeted NGS was 0.180 (IQR 0.10–0.39), and p = 0.017). A meaningful shorter PSA doubling time (DT) from nadir was observed in men with high vs. low ctDNA , as well as a significant higher PSA DT velocity from nadir . .  . . The higp < 0.0001).The time to PSA progression was shorter in patients with with high ctDNA vs. low ctDNA  and a shorter PSA DT from nadir  (p = 0.045). Furthermore, the time to PSA progression was shorter in patients with AR gain vs. AR normal .Similarly, patients with AR gain experienced a lower rate of PSA decline ≥ 50% compared to patients with AR normal (= 0.001) . Both chThe 3% of patients experienced an initial PSA flare but there is no correlation with clinical outcome.p < 0.0001 and 4.2 vs. 9.5 months, p < 0.0001, respectively)  .p < 0.0001). In addition, there were similar results among these four cohorts for radiographic-PFS and OS  .Combining ctDNA fraction and AR copy number (CN), we identified four distinct groups based on median PSA-PFS: 1) low ctDNA/AR normal, 2) high ctDNA/AR normal, 3) low ctDNA/AR gain, and 4) high ctDNA/AR gain  . In a mu 0.0001) , high ctOne key point of treating CRPC patients is to prolong survival while maintaining the quality of life and preventing needless toxic effects of an ineffective treatment. To reach this aim, the early evaluation of therapeutic efficacy is a crucial goal in the patient management strategy. The current gold standard for assessing tumor response and treatment efficacy is the radiographic imaging and PSA level assessment.Various retrospective studies have demonstrated that PSA kinetics could be a prognostic biomarker during the history of prostate cancer ,41,42.In patients affected by mCRPC and treated with chemotherapy, both PSA velocity and PSA DT provide independent prognostic information . A stronIn the last decades, several efforts are been made to identify new biomarkers with a predictive and/or prognostic role, and the most relevant pathway explored in prostatic cancer depends on AR. An increase in AR copy number, known as AR gain, was detected in up to 70% of CRPC ,45,46, cp < 0.0001). In addition, the present study, including the simultaneous assessment of PSA assessments and ctDNA, offers a significant opportunity to investigate prostate tumor dynamics thanks to a sequential monitoring of PSA during therapy as a surrogate biomarker of the emergence of resistance to treatment. Similar results of a strong correlation between declines in PSA and ctDNA levels have been also observed in a recent study of 140 CRPC patients [The high expression of AR could bypass the inhibition induced by ARSI and finally result in resistance to therapy. PSA may be considered as an excellent biomarker of AR activity ,49. Indepatients .Our work showed that changes in PSA dynamics in combination with plasma DNA analysis were independent predictors of outcome and so more reliable early predictive biomarkers in mCRPC, especially in certain conditions.First, it may be a future valid aid for the subgroup of individuals for whom PSA value does not represent the real condition of the disease; particularly, in aggressive variants of prostate cancer with low or non-rising PSA levels. Second, findings obtained did not show the superiority of one biomarker over the other but rather the combination of ctDNA with PSA kinetics may improve outcome prediction, for example, adding some information about response to treatment in a subgroup of prostate cancer patients without target lesions, such as those with only bone metastatic disease, in which CT scan and bone scan could lack of sensibility and specificity in detection of response or progression. Third, PSA flare is a phenomenon that could deceive clinicians due to an initial and transient rising of PSA levels but with no correlation with poor prognosis . In factLastly, there is growing attention about the possible role of immunotherapy in prostate cancer ,52. It iIn a previous study, we generated a prognostic score based on ctDNA fraction and functional imaging to better predict treatment outcome . SimilarThis paper has many limitations: a single-center study, heterogeneous population including chemotherapy-naïve and post-docetaxel patients as well as patients treated with enzalutamide and abiraterone, a limited number of multiple sequential PSA values. Furthermore, there are not longitudinal data concerning ctDNA. However, even these limitations, the present study represents an essential first step towards achieving the introduction of serial ctDNA assessment with standard tests utilized in clinical practice, including PSA dynamics.These findings underline the importance of a multimodal approach to outcome prediction by integrating standard imaging and blood tests, such as PSA kinetics parameters, and molecular information to better understand the response to treatment and the efficacy of novel drugs not only in mCRPC but also in early stages of disease. Specifically, the study of liquid biopsy could also serve as a biomarker of disease response to allow for earlier switch or intensification of therapy in patients harboring both plasma AR gain and high ctDNA before starting ARSI.


N = 37, Mage = 80). We employed ANCOVAs, controlling for age, sex, and morbidity, to assess differences in post-treatment outcomes between AR and No-AR conditions. AR recipients (vs. No-AR) reported lower post-treatment helplessness and more perceived control over their health. Our study offers evidence for AR to increase control-related outcomes and lays the groundwork for further research into supporting older adult populations with compromised health.Older adults make up the largest portion of the population of physically inactive individuals. Health challenges, and psychological barriers , contribute to reduced activity engagement and low perceived control. This pilot study tested an attributional retraining (AR) intervention designed to increase control-related outcomes in a physical activity context for older adults with compromised health. Using a randomized treatment design, we examined treatment effects on a sample of older adults attending a day hospital ( Physical inactivity is a significant public and global health concern for all ages and especially for older adults  often experience challenges that can prevent them from activity engagement . Because individuals with compromised health are often at risk of reduced activity, they provided an optimal sample for our consideration of physical inactivity. Third, our study goes beyond past research by examining the role of AR on control-related psychological outcomes . Improvements in these psychological outcomes should logically follow when people view their inactivity as a result of controllable causes, thus fostering their motivation to become more physically active.Our pilot study extends this line of research in several ways. First, the premise of our AR intervention is to have older adults consider Given the important implications for reducing inactivity in older adults and the efficacy of altering harmful attributions for important outcomes, we sought to provide an AR intervention for older adults at-risk of limited activity. Our main study goal was to test the short-term efficacy of AR on control-related outcomes. Our directional hypotheses were tested using a randomized experimental treatment design for older adults attending a day hospital facing health challenges. AR (vs. No-AR) was hypothesized to decrease helplessness and increase perceived control over health, fitness, and life in general. One-sided tests were used since according to theory and past AR studies, treatment recipients should benefit psychologically from AR  viewed the AR video and subsequently filled out a Time 2 (T2) post-treatment survey. Participants in the No-AR condition (n = 19) simply filled out the survey. At each step, participants received $10 for participating.The study procedure spanned seven months with three sequential steps. Prior to Step 1, health records were collected from the hospital  and participants were randomly assigned to either the AR or No-AR condition. Forty-four participants were eligible for the study; however, seven chose not to participate. Step 1 entailed both groups completing a Time 1 (T1) survey comprising demographic information and control-related variables. During Step 2 (1-week later), participants in the treatment condition  and to avoid thinking about causes they do not have control over . The goal of the video was to encourage thinking in controllable ways about inactivity. The video provides a social exchange between two older adults. The first character (Jim) embraces internal and uncontrollable causes (attributions) for why he is inactive. The second character (Don) helps “retrain” Jim's causal thinking by encouraging him to use more internal and controllable causes . At the end of the video, Jim effectively models Don's adaptive way of thinking and begins seeing positive changes in his motivation and activity levels. In conclusion, Jim explains why focusing on controllable causes for inactivity can be beneficial: these causes (attributions) can be modified in the future by individuals.male, 2 = female) and medical conditions were obtained from hospital records with permission obtained from the participants and the health centre. Medical conditions were measured using a sum score from participants' reports of 39 possible medical conditions  experienced within the last year . A score was calculated by summing the conditions; thus, higher scores denote higher ratings of multimorbidity. Participants were asked to report their age at Step 1 in the Time 1 survey. See Covariate selection was guided by past research and our preliminary analyses as outlined below. Prior research has identified age, sex and morbidity as sharing important relationships with physical activity . Domain specific  and global  measures of perceived control were assessed on a 10-point scale. Participants were asked “how much influence do you feel you have  over your physical health/physical fitness/life in general.” These one-item measures that have been used in other perceived control studies in health domains have high face validity  examined zero-order correlations among all study variables. Our main analyses employed ANCOVAs to test differences between the AR and No-AR conditions for each dependent variable employing directional hypotheses. Analyses controlled for age, sex, and morbidity. Lastly, a supplemental analysis employing a Repeated Measures ANCOVA was conducted to examine change in the outcome variables from Time 1 to Time 2 for the AR and No-AR groups .Preliminary analyses (a) tested the effectiveness of our randomization procedure using independent sample t-tests established the effectiveness of our random assignment to conditions. In other words, no pre-treatment differences between conditions (AR vs. No-AR) were observed for helplessness, t(34) = 1.33, perceived control over health, t(34) = −0.42, perceived control over fitness, t(33) = −1.12, and perceived control over life in general, t(34) = −0.71 . Second, zero-order correlations revealed several relationships (see r = 0.33) and helplessness was negatively related to perceived control over health and fitness . There were no sex differences found. The results reveal expected correlations between T1-T2 perceived control variables (rs range = 0.61-0.78).First, the preliminary analyses revealed independent sample hips see . MorbidiF = 4.45, p = 0.022, Mdiff = −0.67, SE = 0.32, 95% CI = −1.217, −0.131; Our hypotheses that assessed post-treatment (T2) differences between AR and No-AR participants were partially supported. An ANCOVA revealed a significant difference between the conditions on helplessness, F = 7.07, p = 0.006, F = 2.85, p = 0.051, post-hoc comparisons indicate AR recipients (vs. No-AR) had greater perceived control over health at T2  = 4.32, p = 0.031]; and no difference for perceived control over health, fitness, or life in general. For the No-AR group, there was no significant change in helplessness from Time 1 to Time 2, but there was a significant decline in perceived control over health .A supplemental Repeated Measures ANCOVA was conducted to test for change in the outcome variables over time for the two conditions, controlling for age, sex and morbidity. For the AR group, there was a significant decline in helplessness from Time 1 to Time 2 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Flufenamic acid (FFA) is a highlypolymorphic drug molecule withnine crystal structures reported in the Cambridge Structural Database.This study explores the use of synchrotron X-ray powder diffractioncombined with differential scanning calorimetry to study crystallizationand polymorphic phase transitions upon heating FFA–polymeramorphous solid dispersions (ASDs). Ethyl cellulose  andhydroxypropylmethylcellulose (HPMC) grades with different viscositiesand substitution patterns were used to prepare dispersions with FFAat 5:1, 2:1, 1:1, and 1:5 w/w drug/polymer ratios by quench cooling.We employed a 6 cp HPMC 2910 material and two HPMC 2208 samples at4000 and 100 000 cp. Hyphenated X-ray diffraction (XRD)–differentialscanning calorimetry (DSC) studies show that the 6 and 100 000cp HPMCs and 4 cp EC polymers can stabilize FFA form IV by inhibitingthe transition to form I during heating. It appears that the polymersstabilize FFA in both amorphous and metastable forms via a combinationof intermolecular interactions and viscosity effects. Increasing thepolymer content of the ASD also inhibits polymorphic transitions,with drug/polymer ratios of 1:5 w/w resulting in FFA remaining amorphousduring heating. The comparison of FFA ASDs prepared with differentsamples of HPMCs and ECs suggests that the chemical substitution ofthe polymer  plays a more significant rolein directing polymorphic transitions than the viscosity. A previouslyunreported polymorph of FFA was also noted during heating but itsstructure could not be determined. The major problem with this is that the amorphousform is thermodynamically unstable and will spontaneously crystallizeto more stable crystalline counterparts upon storage. Crystallizationis a complicated process during which a number of polymorphs may coexistwithin the same formulation.7 It belongsto class II of the Biopharmaceutical Classification System (BCS),with low solubility and high permeability.8 This results in low bioavailability in vivo and hence to suboptimaltherapeutic outcomes. To overcome this limitation, amorphization hasbeen explored to improve the solubility of FFA.9Flufenamic acid amino]benzoicacid),also known as fenamate, is a nonsteroidal anti-inflammatory drug (NSAID)used for treating rheumatoid arthritis, osteoarthritis, and variousmusculoskeletal pain conditions.Table S1.10 The first crystal structureof FFA (form III) was elucidated in 1973, and nearly 10 years later,form I was reported.12 Form II and forms IV–VIIIwere reported more recently in 2011.12 Theprecise order of stabilities of the FFA polymorphs and their transitionsis not well understood despite extensive research efforts. In alleight characterized FFA systems, a strong intramolecular O···H–Nhydrogen bond holds the phenyl ring carboxylic group and bridgingamino group coplanar. While packing, FFA molecules form dimers andthe dominant intermolecular forces in the structures are O···H–Ohydrogen bonds between adjacent molecules.12The polymorphism of FFA has been the subject of extensiveresearchby both the crystallographic and pharmaceutical communities for morethan 40 years. According to the Cambridge Structural Database (CSD),nine polymorphs of FFA have been discovered , including eight structurally characterized forms 1. The cr13 However, structural information cannot be obtained fromDSC profiles; to acquire this, X-ray diffraction (XRD) is required.Because of the fast heating rates used in DSC, XRD and DSC measurementsare generally performed separately. Hyphenating the techniques toobtain DSC and XRD data simultaneously on the same sample has beenshown to add significant additional insight. Recently, the combinationof high-energy synchrotron XRD with differential scanning calorimetry(XRD–DSC) has been applied to study phase transitions in arange of systems including carbamazepine,14 mefenamic acid,15 paracetamol/lactoseblends,16 and spray-dried ASDs of olanzapine.13Differential scanning calorimetry (DSC) is a powerful toolthathas been widely used to investigate the phase transitions of APIs,both alone and in combination with excipients. For instance, DSC hasbeen shown to be very powerful in exploring crystallization from drug/polymeramorphous solid dispersions (ASDs) upon heating.18 Here, HPMC was used to investigatehow polymer viscosity and chemical structure affect the polymorphictransitions of FFA. EC is a hydrophobic cellulose ether used as acoating material, a tablet binder, and a matrix former, with viscosityfrom 4 to 300 cp.19 FFA/EC dispersionswere prepared and compared with FFA/HPMC systems to explore how thehydrophobicity of the carrier matrix affects the polymorphic transitionsupon crystallization.In this paper, ASDs of FFA–polymerwere prepared, and theheat-induced crystallization of FFA from the dispersion was exploredby XRD–DSC. The polymorphic phase transitions observed wereinvestigated in detail. Ethyl cellulose  and grades of hydroxypropylmethylcellulose(HPMC) with different viscosities were used as the polymer additives. HPMCs are a family of solublemethylcellulose ethers. They are hydrophilic, biodegradable, and biocompatiblepolymers having a wide range of applications in drug delivery. HPMCpolymers are available in various viscosity grades ranging from 3to 100 000 cp.14H10F3NO2; Mw = 281.23g/mol) was purchased from Acros. HPMC 2208 with a viscosity of 4000cp (K4M) and 100 000 cp (K100M) was purchased from ColorconLtd., and HPMC 2910  from ShinEtsu.EC (4 cp) was obtained from Sigma-Aldrich.Flufenamicacid  at the Diamond Light Source.20 A modified Q20 DSC (TA Instruments) was mounted and alignedon the sample stage in the experimental hutch to allow the monochromatedX-ray beam  pass throughholes in the DSC cell. A Thales Pixium RF4343 detector was located1.9 m behind the sample. The DSC was calibrated with an indium standardand the Pixium detector with cerium dioxide, prior to experimentsbeginning. Initial experiments were performed with FFA alone. FormI was heated from 0 to 150 °C and then equilibrated to 0 °C.The same sample was then heated to 70 °C at 10 °C/min andheld at 70 °C for 5 min.The experimentalsetup for XRD–DSC is very similar to that described in ourprevious work.To prepare ASDs, appropriate amountsof FFA, HPMC, and EC were weighed in 5 mL glass vials to give a finalmass of ca. 30 mg and mass ratios of 5:1, 2:1, 1:1, and 1:5 . The samples were then mixed for ca. 3 min using a vortexmixer. Approximately 20 mg of each sample was heated in the DSC from0 to 150 °C at 10 °C/min before equilibrating back to 0°C to produce ASDs. Finally, the products were heated to 160°C at 10 °C/min. The FFA/HPMC (4000 cp) 1:1 ASDs were alsoramped from 0 to 160 °C at 2 °C/min in a separate experiment.23 TOPAS-Academic V524 was employed to analyze selected patterns withthe Rietveld method26 implemented within the softwareto obtain realistic values for the unit cell parameters at elevatedtemperatures.27 Structural data for theFFA polymorphs were obtained from the CSD. Batch Rietveld refinementswere then performed to determine phase fractions as a function oftemperature.The Diamond Generic Data Acquisition (GDA) software was employedto collect diffraction patterns for 5 s, with a 1 s pause betweeneach scan, which equated to a pattern for every 1 °C of heating.The two-dimensional (2D) Pixium data sets were masked and convertedinto one-dimensional (1D) diffraction patterns by azimuthal integrationusing the DAWN Science Workbench.–1 with 64 scans at a resolution of 4 cm–1. FFA,HMPC, EC, and the samples prepared for the DSC–XRD study wereall explored. The drug/polymer mixtures were heated in the DSC from0 to 150 °C at 10 °C/min before equilibrating back to 0°C. Finally, the products were heated to the temperature aftereach recrystallization event at 10 °C/min. The samples were thenremoved from the DSC and characterized by FTIR.FTIRspectroscopy was conducted on a Perkin Elmer Spectrum 100 instrument.All spectra were recorded between 650 and 4000 cmStandard laboratory XRD experimentswere undertaken on a MiniFlex 600 diffractometer (Rigaku) suppliedwith Cu Kα radiation . FFA was loaded in low-volume glass holders and scannedfrom 5 to 50° in 0.02° steps at 2°/min. The experimentaldata were plotted with OriginPro 2017 and compared with calculatedpatterns for the various polymorphic forms obtained from the CSD.VT-XRDmeasurements were performed using a Stoe Stadi-P diffractometer equippedwith a Cu anode (Kα1), a Ge monochromator, a Dectris Mythen1K detector, and an Oxford Instruments CryojetHT (90–500 K).An in-house setup was employed to discourage the formation of iceon the goniometer head at low temperatures. FFA raw material was firstground for approx. 10 min and then loaded in a 0.5 mm glass capillary.In one experiment, the FFA-loaded capillary was heated to 145 °Cand then quenched to 0 °C before being heated from 30 to 80 °Cin 10 °C steps. In the second run, ice water was used to quenchthe sample, but all other parameters were kept the same. XRD patternswere obtained on reheating from 30 to 80 °C. The sample was scannedfrom 2 to 50° 2θ in steps of 0.5° at 10 s per step.A complete scan lasted approx. 23 min, and each 10 °C temperaturechange took approx. 12 min. Once reaching the desired temperature,the sample was kept at the set temperature for 5 min before startingthe next scan.Figures S1 and S2) before melting. The sample first recrystallizesat 54 °C and then converts to form IV at 74 °C before transforminginto form I at 97 °C. This then melts at 133 °C.FFAform I was heated from 0 to 150 °C,equilibrated to 0 °C, and then heated up to 160 °C. Duringreheating, pure FFA experiences three different transitions and finallyrecrystallizes into form I . This new FFA pattern is subsequently termed pattern X (form X).In attempts to obtain form X with better crystallinity, FFA form Iwas heated from 0 to 150 °C and then equilibrated to 0 °Cto produce amorphous FFA. The reheating process was monitored by XRD–DSC,with the sample heated to 70 °C at 10 °C/min and then heldat 70 °C for 5 min. Thecrystallinity of the material present at 70 °C upon heating theFFA glass is poor, but the pattern cannot be satisfactorily matchedwith any polymorphic form of FFA S3. This Figure S3 and confirmsthat FFA recrystallizedinto form X before the isothermal hold period. The major reflectionsof FFA form X are summarized in Table S2. Computationally predicted crystal structures were explored in attemptsto solve the structure of form X. However, none of them provides asatisfactory fit with pattern X. The closest gives an Rwp (weight profile R-factor) of 18 .The pattern obtained at 68 °Cis identical to that in Figure S6).Cooling using a CryojetHT revealed that the amorphous form had crystallizedinto form IV at 50 and 60 °C; at 40 °C, the pattern appearsto correspond to a mixture of forms IV and X, but the crystallinityof the pattern is too poor to determine the crystal structure . A further attempt to prepare phase-pureform X involved melting FFA in an oven at 145 °C  andimmediately transferring to a diffractometer , but this resulted in a mixture of forms III andIV FFA. Despite myriad attempts to solve its structure, form X couldonly be clearly identified when heating an FFA glass in a DSC. Thissuggests that it is very unstable.VT-XRDwas employed in attempts to obtain high-resolution datafor form X. For a sample cooled by ice water, the amorphous form directlyrecrystallized into form IV (Combined XRD–DSCdata for an FFA/6 cp HPMC 2910 ASD (5:1 w/w) can be seen in The XRD–DSC data illustrate the occurrenceof an amorphous-to-crystallinetransition, the conversion of one polymorph to another, and a crystalline-to-liquidtransition. There are no reflections (bar those from the Al pan) inthe contour plot until 56 °C when an exothermic event is observedin the DSC trace. A change in the position of the Bragg reflectionsappears at around 80 °C, with another exothermic peak showingon the DSC thermogram. Finally, at 126 °C, the peak of the endothermicevent, a total loss of Bragg reflections in the contour plot is seen.Figure S9). The pattern recordedat 116 °C shows the FFA to be present as form IV , the polymorphic transitionsof the FFA/HPMC 6 cp ASD (5:1 w/w) are somewhat different, as theASD recrystallizes into form X before converting to form IV. Thissuggests that HPMC inhibits the conversion from form IV to I and stabilizesform IV during heating.Compared with pure FFA, which experienced three differenttransitionsand finally recrystallizes into form I under the same heating conditions. Very weak Bragg reflectionsat 1.75, 2, and 2.25° could be observed between 0 and 20 °C,which suggests that the FFA/HPMC (2:1 w/w) mixture was not fully amorphousafter quench cooling. However, these reflections are too small toallow the identification of the polymorphic form present. More obviousBragg reflections can be seen to appear in the contour plot at about40 °C. These disappear at approx. 120 °C, correspondingto the recrystallization and melting of FFA . Rietveld refinements against the recorded pattern at86 °C show that FFA recrystallized into form IV . The endothermic peakat 120 °C thus corresponds to the melting of form IV. This isa further depression (cf. the 5:1 w/w ASD) of the literature meltingpoint for FFA IV,28 consistent with thepresence of additional polymer in the 2:1 system. Batch refinementswere then undertaken against all of the patterns, with the Rwp value ranging from 0.6351 to 5.1667. It canbe seen  that FFA form IV beginsto grow in at ca. 60 °C, reaches a maximum at around 110 °C,and then declines until no crystalline material remains at 120 °C.When the FFA/HPMC ratio decreased to2:1 w/w, XRD–DSC showsthat the diffracted intensity in the contour plot is very weak, whichmay arise from the low crystallinity of the recrystallized material, a small and broadendotherm peaking at approx. 120 °C is observed in the DSC traces,while no exothermic events are visible. For the 1:1 w/w sample, thereare some reflections between 80 and 110 °C but they are veryweak; no reflections at all can be seen with the 1:5 analogue. Thepatterns obtained at 100 °C with both samples are shown in Figure S14 and reveal that both are almost entirelyamorphous. These systems contain more polymer than the previouslydiscussed 5:1 and 2:1 w/w materials, and at such drug/polymer ratios,it appears that the steric hindrance and viscosity of the polymerprovide significant resistance to the movement of drug molecules,severely restricting (1:1 w/w) or preventing (1:5 w/w) the recrystallizationof FFA. The low intensity of the peaks present in the XRD patternsmakes it hard to identify the polymorph of FFA present, and the broadnessof the DSC endotherm also prevents a definitive assignment of whichspecies is melting. However, the reflections seen for the 1:1 systemover the temperature range of ca. 75–115 °C appear consistentwith FFA form IV.For dispersions prepared with FFA/6 cp HPMC at 1:1 and 1:5 w/wratios (28), at which point a total loss of Bragg reflections is observed.The patterns recorded at 120 and 133 °C were analyzed using theRietveld method . The pattern recorded at 120 °C reveals FFA to exist as formIV, while at 133 °C, FFA is present as form I.Combined XRD–DSCdata for the reheating of an FFA/4000 cp HPMC 2208 dispersion witha 5:1 w/w ratio are presented in Figure S16).Both samples are in the amorphous form at the beginning of the experiment,and Bragg reflections begin to emerge at about 60 °C. These correspondto FFA form IV, coinciding with the onset of an exothermic peak inthe DSC traces. Following this, there is an endothermic event peakingat 126 °C in each thermogram, after which no Bragg reflectionscan be seen. Finally, in each trace, there is another small endothermicpeak with an onset temperature at 134 °C. The diffraction patternsof the two samples do not quite mirror the DSC results. Accordingto the refinements , form IV is the only polymorph that exists in the samples, butthere are two endothermic peaks seen in each DSC trace, indicatingthat there is more than one polymorph in each sample. The onset temperatureof the second endothermic peak is 134 °C, the melting temperatureof form I. We thus hypothesize that a small amount of form IV convertsto form I at this temperature, sufficient to be identified by DSCbut not enough to be detectable by XRD.Dispersionsof FFA/4000 cp HPMC at both 2:1 and 1:1 w/w ratios have a similarphase transition behavior . However, because of the highviscosity and steric hindrance provided by the HPMC, only a tiny amountof amorphous FFA is recrystallized , barely at the detection limitation of XRD. Therefore, at thisdrug/polymer ratio, the viscosity of the polymer matrix provides ahigh level of resistance to the movement of drug molecules, preventingthe recrystallization of FFA.For an FFA/HPMC 4000cp ASD at a 1:5 w/w ratio, no obvious reflectionscan be observed on the counter plot. The two peaks in the DSC tracemay relate to the crystallization and then melting of FFA, respectively. From 113 to 123 °C, the DSC data show a smallexotherm–endotherm event, together with a change in the positionsof Bragg reflections (form IV + I). Another change in the patternof Bragg reflections is observed at ca. 123 °C (form I), followedby a total loss of diffracted intensity at about 133 °C. Theseevents are coincident with the peak of the melting endotherms of formsIV and I, respectively.The data in the previous section illustrate that thereare some discrepancies between the diffraction patterns of the 1:1and 2:1 w/w FFA/4000 cp HPMC ASDs and the DSC observations. To tryand understand this better, the experiment was repeated at a heatingrate of 2 °C/min with the 1:1 w/w FFA/4000 cp HPMC dispersion5a. The dThe integrated total diffraction intensity for each pattern asa function of temperature is plotted in 12 These observations suggest thatthe polymorphic transition from form IV to I occurred before the meltingof form IV was complete. The temperature at which the solid convertsfrom form IV to I is presumably reached by the instrument before themelting of form IV is complete. The heating rate clearly has a strongeffect on the behavior of form IV. At 2 °C/min, there is sufficienttime for the drug molecules to rearrange to form I between 113 and120 °C, with no apparent melting. However, when the temperaturereaches 120 °C (the melting onset of form IV), the polymorphictransition from form IV to I is not complete and the residual formIV in the sample begins to melt.The plot of phase fractions as a function of temperature (–1) occurs at almost the same rate as the evolutionof form I (4.0504 °C–1) . The similarity between these two numbers indicatesthat the conversion is a continuous process and supports the conversionbelow 120 °C being a solid–solid transition. The samesituation is observed from 120 to 122 °C, during which the rateof decrease of form IV (−2.1087 °C–1) is also similar to the growth of form I (2.1023 °C–1) . This indicates that bothbefore and after the start of form IV melting (120 °C), the formIV-to-form I transition proceeds via a solid-to-solid pathway, withno recrystallization from the molten FFA. The first endothermic peakin the DSC thermogram is thus the melting peak of the residual formIV .Melting is a thermodynamicevent, which occurs much faster thanrecrystallization (a kinetic event). On the other hand, during a solid–solidpolymorphic transition, the amount of each polymorphic form presentchanges at the same speed. The rate of change in the amount of eachform of FFA present was analyzed as follows: a linear fit of the integrateddata was carried out at the straightest sections of the phase fractioncurves around the intersection (116.6–119.8 and 120–122°C). It appears that the decline of form IV . It is thus clear that making a composite with4000 cp HPMC can drive FFA to recrystallize into the metastable formIV. This may be because drug molecules are dispersed in HPMC aftercooling, and the viscosity of HPMC and H-bonds formed between FFAand HPMC provides resistance to the movement of drug molecules; thus,more time and energy are required for crystallization. Therefore,instead of recrystallizing directly into form I, the high-temperaturestable form, the FFA molecules organize themselves to form the metastableform IV, which requires less energy and time. This has previouslybeen noted with olanzapine and paracetamol ASDs in polymer carriers,for instance.29 With an increasing temperature(energy providing) and slow heating rate (sufficient time for transformation),molecules tend to rearrange to a more stable structure.When pure amorphousFFA is heated at 2 °C/min, it only showsthe crystallization and then the melting of form I . Data collected during reheating a 1:5 w/w FFA/HPMC100 000 cp ASD illustrate that the sample remains almost completelyamorphous during the heating process. A weak melting peak is observedat 126 °C, which likely corresponds to the melting of form IV,28 but there is too little crystalline materialpresent to be detected by XRD .DSC–XRDdata on 5:1, 2:1, and 1:1 w/w formulations prepared with FFA–100 000cp HPMC show that the samples recrystallize into form IV and thenmelt at 127 °C . FFA/ECdispersions made at 2:1 and 1:1 w/w polymers show similar phase transitionbehavior . The first exothermic peak on the DSC trace appears at 60 °C,but no reflections are observed on the contour plot. This may be becauseduring the first exothermic event, only a very small amount of samplecrystallizes, below the detection limit for XRD. Bragg reflectionsof form IV begin to emerge at about 80 and 70 °C in the 2:1 and1:1 w/w systems, respectively, coinciding with the onset of the secondexothermic peak in the DSC traces. Following this, there is an endothermicevent peaking at 126 °C in each thermogram, after which no Braggreflections can be seen. Given the findings above, the latter peakis expected to be the melting of form IV.A 5:1 w/w FFA/EC ASD showssimilar results to the 6 cp HPMC ASD at 5:1 w/w, with the sample firstrecrystallizing to form X and then converting to form IV ,there are almost no reflections shown in the contour plot and no endothermicevents exist in the DSC trace, suggesting that the sample remainsamorphous during heating. There is a distinct reflection present at0.8° from 0 to 160 °C; the origin of this is not clear,but it is thought to arise from some impurities in the sample.For a dispersion ofFFA/EC at a 1:5 w/w ratio , the polymercontentof the blend does not lead to a huge difference and the samples showthe same phase transition properties. Compared with pure FFA, whichexperienced three different transitions and finally recrystallizesinto form I under the same heating conditions, FFA with HPMC 100 000cp recrystallizes to form IV, while FFA with HPMC 4000 cp recrystallizesto form IV and then converts to form I. The polymorphic transitionsfor FFA/HPMC 6 cp ASD are somewhat different; when the drug/polymerratio is 5:1 w/w, it recrystallizes into an unknown phase (“patternX”) before converting to form IV. The transition with the 2:1w/w 6 cp HPMC ASD is the same as that of 100 000 cp ASD, whileFFA remains almost completely amorphous when the drug/polymer ratiois 1:1 w/w. Additionally, for any HPMC grade at a 1:5 w/w drug/polymerratio, the large amount of polymer present provides significant sterichindrance to molecular motion, and the FFA remains largely amorphousthroughout the heating cycle.Considering the effects of differenttypes of HPMC, at low HPMCcontents (drug/polymer > 1:5 w/w), the system with HPMC 4000 cpfirstrecrystallizes to form IV, which then converts into form I througha mechanism involving localized melting. However, the analogous materialmade with HPMC 100 000 cp only recrystallized into form IV.This result suggests that the higher viscosity of HPMC can stabilizemetastable form IV during heating, presumably because it leads togreater steric hindrance to molecular mobility. The HPMC 6 cp composite(2:1 w/w) displays the same phase transition properties as that madewith HPMC 100 000 cp, however . The viscosity of HPMC 6 cp is ca. 15 000 timeslower than that of HPMC 100 000 cp, but there is also a differencein the amount of methoxy and hydroxypropyl groups present, with HPMC6 cp having rather more of these. Both types of HPMC show the sameinhibition effects on the polymorphic transitions of FFA, which mightsuggest that both viscosity and the substitution have an effect onthe transformation. HPMC 6 cp has more methoxy and hydroxypropyl groups,making it more branched; thus, it gives similar steric hindrance toHPMC 100 000 cp, even though the latter is much higher in viscosity.The polymorphic transitions of the FFA/EC 4 cp ASDs are similarto those with FFA/HPMC 6 cp ASDs, except for the 1:1 w/w FFA: polymerratio where with HPMC the FFA remains amorphous during heating, whilethe EC ASD recrystallizes into form IV. A summary of the polymorphictransition with the different ASDs is shown in The aim of this workwas to study heat-induced crystallizationand polymorphic phase transitions in FFA–polymer ASDs. We haveshown that FFA forms X, IV, I, and amorphous material are all obtainedupon heating the ASDs. This study shows that FFA form IV can be stabilizedby HPMC 6 and 100 000 cp and EC 4 cp, which inhibit the transitionfrom form IV to I during heating. Increasing the polymer content ofthe ASD also inhibits polymorphic transitions, with drug/polymer ratiosof 1:5 w/w, resulting in FFA remaining largely amorphous during heating.Comparing FFA–HPMC 6 and 100 000 cp dispersions suggestedthat the substitution type has a more distinct impact on the polymorphicstability than the viscosity of HPMC. These two HPMCs have differentsubstitution types, with the 6 cp material having notably more methoxyand hydroxypropyl side groups and thus being more branched. ASDs withthese two types of HPMCs experience largely the same phase transitions.In contrast, ASDs made with the 4000 cp HPMC (which has the same substitutionpattern as 100 000 cp) show a reduced stabilization of metastablepolymorphs. These findings suggest that the increased presence ofmethoxy and hydroxypropyl groups aids HPMC with low viscosity to havethe same stabilizing effects as much more viscous systems. Comparingthe HPMC data with findings in the FFA/EC system, it is again clearthat the chemical substitution of the polymer plays a more significantrole in directing polymorphic transitions than the viscosity.


Augmented reality (AR) is a rapidly developing technology that has substantial potential as a novel approach for addiction treatment, including tobacco use. AR can facilitate the delivery of cue exposure therapy (CET) such that individuals can experience the treatment in their natural environments as viewed via a smartphone screen, addressing the limited generalizbility of extinction learning. Previously, our team developed a basic AR app for smoking cessation and demonstrated the necessary mechanisms for CET. Specifically, we showed that the AR smoking cues, compared to neutral cues, elicited substantial cue reactivity (i.e. increased urge) and that repeated exposure to the AR smoking cues reduced urge (i.e. extinction) in a laboratory setting. Here we report the next step in the systematic development of the AR app, in which we assessed the usability and acceptability of the app among daily smokers in their natural environments.N = 23, 78.3% female, Mean Age = 43.4, Mean Cigarettes/Day = 14.9), not actively quitting, were instructed to use the AR app in locations and situations where they smoke  at least 5 times per day over one week. The study is registered in clinicaltrials.gov (NCT04101422).Daily smokers (Results indicated high usability and acceptability. Most of the participants (73.9%) used the AR app on at least 5 days. Participants found the AR cues realistic and well-integrated in their natural environments. The AR app was perceived as easy to use (Mean = 4.1/5) and learn (mean of 2 days to learn). Overall satisfaction with the app was also high. Secondary analyses found that 56.5% reported reduced smoking, with an average 26% reduction in cigarettes per day at follow-up.This study found that the augmented reality (AR) smartphone application that utlized cue exposure treatment for smoking cessation was perceived as easy to use and learn in the natural, day-to-day environment of daily smokers. Findings set the stage for a larger clinical trial testing the AR app as an adjuvant therapy for treating tobacco dependence, with potential applicability to other addictive behaviors.These findings set the stage for a randomized controlled trial testing the AR app as an adjuvant therapy for treating tobacco dependence, with potential applicability to other substances. Cigarette smoking remains the leading cause of cancer and preventable cause of disability and mortality in the United States . DespiteAR also has several unique practical advantages, as compared to VR, including relatively low cost in developing digital objects, high degree of realism given the simplicity of the AR objects, and real-time exposure by inserting AR objects into the user’s natural environment   that the smoking cues elicited cue-reactivity, particularly self-reported urge to smoke and (2) that repeated exposure to the cues produced evidence of extinction. Both of these conditions were met in a controlled laboratory-based setting among daily smokers . SpecifiTo advance the AR app toward evaluation in a full-scale randomized controlled trial (RCT), the current study assessed its usability and acceptability among daily smokers not currently making a quit attempt in their typical natural environments. Although the study was not powered to test clinical outcomes, we describe proxies of behavior change  and changes in cigarettes per day.N = 20 to capture 98.4% of potential usability problems )Within the AR app, participants completed a single-item assessment of daily cigarette smoking (i.e. ‘How many cigarettes did you smoke yesterday?’). Engagement with the app  was also captured.Usability and acceptability were measured using the following self-report questionnaires at follow-up based on the literature on mHealth usability ,26: (1) To describe change in motivation and self-efficacy at posttest, the Contemplation Ladder, ARME, and Self-Efficacy Scale were administered. To describe changes in the strength of urge to smoke, a single-item measure of smoking urge over the past week was collected . Although participants were not offered smoking cessation treatment and not actively attempting to quit or reduce smoking, past week smoking behavior (average CPD on the days they smoked) was assessed. Participants were also asked where , when , and with whom  they used the AR app. Additionally, the number of participants who contacted the study team for additional help using the app was tracked.Descriptive analyses  were conducted on the study variables including demographics, self-report measures on usability and acceptability, app use, motivation, self-efficacy, and change in CPD. Although the current sample was not powered for inferential statistics, paired t-tests and McNemar’s test were conducted to explore changes in the proxy variables between baseline and follow-up.M = 79.5, SD = 17.1). There was a statistically nonsignificant increase in the perceived ease of using the app over time (t(22)=-1.37, p=.184) and 87% reported that the app was overall easy to use at the follow-up survey. The average number of days to become comfortable with the app was 2 with the majority reporting only 1 day , and only 17% (n = 4) asked for additional help from the study team. The 4 participants who asked for additional help provided the following reasons: trouble downloading because they had the wrong app name; incorrect ID number; trouble placing the indicator; and asking where the practice tutorial was located. High satisfaction was reported such that 65% reported that they would recommend the app to a friend or family member to help them quit smoking and 70% expressed overall satisfaction with the app. Lastly, 48% reported that the AR app would be appealing to them if they were currently attempting to quit smoking.On the follow-up survey, participants reported an overall positive experience with the AR app. They reported that the AR cues were highly realistic, were well-integrated in the environment, and appeared as if the cues were right in front of them. The AR app was perceived as easy to use and learn as indexed by the System Usability Scale (p<.01). Participants also reported smoking slightly fewer cigarettes at follow-up as compared to baseline (t(22)=-2.90, p<.01). In particular, 13 participants (57%) reported having reduced the number of cigarettes smoked in the past week at follow-up, with an average reduction of 26% among them .Previous laboratory-based research established that the AR app met the necessary mechanistic criteria underlying CET—cue reactivity and extinction. The current study extended that research by examining the usability and acceptability of the AR app among daily smokers in their natural environments.Usability was supported by high ratings across several measures. Consistent with our earlier findings in laboratory settings ,21, AR cAcceptability of the app was deemed moderate to high. Overall, participants reported high satisfaction with the app, although only half of participants reported that the app would be appealing if they were currently attempting to quit. However, this is not surprising given that the current study focused on the usability of the extinction trials, so the basic app did not include other user engagement features that will be added later, nor did we provide other standard components of smoking cessation interventions. Whereas app functionality regarding CET was our top priority at this developmental phase, other features such as gamification or progress feedback were not yet implemented, nor was it yet integrated into a smoking cessation program. Thus, it is possible that our acceptability ratings were impacted by the somewhat bare-bones nature of this initial version of the app.Exploratory analyses revealed some small but significant changes in smoking proxies and behavior over the week of app use. No changes were found in the cessation motivation or self-efficacy indices over this short time-frame. However, both strong urge to smoke and CPD decreased from baseline to follow-up. These are the clinical indices that theoretically should be most affected by CET, so it is encouraging that we detected a change signal even within this feasibility trial.It is important to acknowledge the limitations of this study, particularly in comparison to a full-powered clinical trial. The sample size was underpowered for inferential statistics across participants, participants were not enrolling in a smoking cessation program per se, the CET intervention lasted only a week, and this version of the AR app included only AR presentation without features to enhance engagement  or additional smoking cessation assistance. Moreover, this single-armed trial did not include control or comparison arms, which limits causal conclusions about the exploratory longitudinal findings. Despite these limitations of a usability/acceptability trial, the study yielded encouraging findings with respect to both the usability and acceptability of an AR app that presents smoking-related stimuli in participants’ natural settings. One remaining concern is the availability of AR-compatible smartphones, given that a relatively high proportion of potential participants were excluded because their devices were not AR-compatible. It is likely, though, that these individuals were using older devices, since most contemporary smartphones now have this capability. Thus, this concern should attenuate with the diffusion of newer technology.Given the novelty of clinical AR apps, several research questions remain. First, this app was envisioned to be integrated into a comprehensive smoking cessation intervention. Therefore, research is needed to determine how best to pair the app with an existing treatment. Relatedly, the incremental efficacy of the AR app for smoking cessation should be tested in comparison to standard smoking cessation interventions . There are also various theoretical, functional and implementation questions that have been elaborated elsewhere . Other sFinally, the current results, together with our previous laboratory findings, have demonstrated that a smoking-related AR app elicits cue reactivity ,21, prodThe current study was the first, to our knowledge, to test and demonstrate both usability and acceptability of an AR app for smoking cessation in the real world. Together with our previous laboratory studies, this research provides evidence supporting the clinical potential of AR-based CET for smoking cessation and relapse prevention. The current findings set the stage for an RCT testing the AR app as an adjuvant therapy for treating tobacco dependence, with potential applicability to other substances.Min-Jeong Yang: Writing – original draft, Formal analysis, Interpretation of the data, Visualization, Revising it critically for intellectual content, Final approval of the version to be publishedKaren O. Brandon: Project administration, Data curation, Formal analysis, Interpretation of the data, Visualization, Writing – review & editing, Revising it critically for intellectual content, Final approval of the version to be publishedSteven K. Sutton: Data curation, Formal analysis, Interpretation of the data, Visualization, Writing – review & editing, Revising it critically for intellectual content, Final approval of the version to be publishedMarloes Kleinjan: Study Design, Interpretation of the data, Writing – review & editing, Revising it critically for intellectual content, Final approval of the version to be publishedLeslie E. Sawyer: Study Design, Interpretation of the data, Writing – review & editing, Revising it critically for intellectual content, Final approval of the version to be publishedThomas H. Brandon: Conceptualization, Study Design, Funding acquisition, Investigation, Methodology, Interpretation of the data, Writing – review & editing, Revising it critically for intellectual content, Final approval of the version to be publishedChristine Vinci: Conceptualization, Study Design, Funding acquisition, Investigation, Methodology, Interpretation of the data, Writing – review & editing, Revising it critically for intellectual content, Final approval of the version to be publishedAll authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work.


Several gastrin-releasing peptide receptor (GRPR) antagonists with improved in vivo behavior have been recently developed and tested in the clinic. However, despite the generally mild side effects of peptide receptor radionuclide therapy (PRRT), toxicity has been observed due to high doses delivered to nontarget tissues, especially in the kidneys and pancreas. Previous experiences with radiolabeled peptides opened a unique opportunity to explore GRPR pretargeting using clickable bombesin antagonists. Toward this goal, we used clickable DOTA-like radiocomplexes which have been previously evaluated by our group. We functionalized a potent GRPR antagonist with a clickable TCO moiety using two different linkers. These precursors were then studied to select the compound with the highest GRPR binding affinity and the best pharmacokinetics to finally explore the advantages of the devised pretargeting approach. Our results provided an important proof of concept toward the development of bioorthogonal approaches to GRPR-expressing cancers, which are worth investigating further to improve the in vivo results. Moreover, the use of clickable GRPR antagonists and DOTA/DOTAGA derivatives allows for fine-tuning of their pharmacokinetics and metabolic stability, leading to a versatile synthesis of new libraries of (radio)conjugates useful for the development of theranostic tools toward GRPR-expressing tumors. G protein-coupled receptors (GPCR) are a heterogeneous family of transmembrane receptors clinically relevant as imaging or therapeutic targets in a huge variety of diseases such as respiratory conditions, mental health disorders and cancer . The bomThe subtype GRPR is particularly relevant for the development of anticancer therapies and its overexpression has been reported in several cancer types such as small lung cell, head/neck squamous cell, colon, glioblastoma, breast and prostate cancer . GRPR isThe main limitation to the clinical use of radiolabeled peptides is the metabolic stability and, in recent years, several groups have been intensely working toward BBN analogs with improved in vivo behavior ,7. Howev111In]In-AMBA [Several GRPR antagonists resulting from modifications to the C-terminal sequence of bombesin have been developed and have been recently extensively reviewed . Some of]In-AMBA ,19. Furt]In-AMBA .Although the side effects of peptide receptor radionuclide therapy (PRRT) are generally mild, renal toxicity has been observed, especially with highly energetic β-emitters . In factPretargeting strategies have been extensively used in radioimmunotherapy (RIT) to improve the slow pharmacokinetics of monoclonal antibodies and optimize the delivery of their radioactive payloads ,26,27. TSince the GRPR-antagonist complex is not internalized after the binding, GRPR antagonists offer a unique opportunity to explore the pretargeting strategy using clickable bombesin antagonists for prostate cancer theranostics. Inspired by the encouraging results reported for antibodies and their fragments, the main goal of the work here described was to show that the pretargeting approach might improve the in vivo performance of GRPR-targeting peptides, namely by reducing the associated nephrotoxicity and nontargeted effects in other critical organs such as the pancreas. Toward this goal, we used the small and hydrophilic tetrazine-containing radiocomplexes previously evaluated by our group . These c2 InCl3 (370 MBq/mL in HCl) was obtained from Mallinckrodt (Curium) Medical B.V. .All chemicals were purchased from Sigma-Aldrich and used as received unless otherwise specified. Rink Amide resin, HATU, HBTU and all Fmoc-protected amino acids were purchased from Novabiochem by Merck KGaA . DOTA- NHS and DOTAGA anhydride were purchased from Chematech . InCl111In have been previously reported. The chemical synthesis of the peptide AR and the preparation of the derivative AR-PEG4-TCO have also been previously described + calculated 1253.7, found 1254.1. AR-Pip-TCO: The compound AR-Pip-NH2  was reacted with the TCO-NHS derivative  in the presence of 250 µL of DIPEA and 3 mL of dry DMF. After 3 h, the reaction was stopped, the solvent was removed under reduced pressure and the compound was purified by HPLC (Rt = 16.8 min), and lyophilized and characterized by ESI-MS to afford the final peptide in 34% yield. ESI-MS: C71H104N16O14 [M + H]+ calculated 1405.8, found 1405.8.AR-Pip-NH-Fmoc: The resin containing the AR peptide  was left swelling for 10 min with DCM (2 mL) before the reaction. After removal of the DCM, a solution containing Fmoc-4-amino-1-carboxymethyl-piperidine , HBTU  and DIPEA (200 µL) in 4 mL of DMF was added. The reaction was left stirring overnight at room temperature. The formation of the desired compound was confirmed by ESI-MS analysis of a small aliquot of the peptide cleaved from the resin. ESI-MS: C77H102N16O14 [M + H]+ calculated 1474.8, found 1476.0. AR-Pip-NH2O (TFA 0.1%) B: ACN (TFA 0.1%). Gradient program: 5 min A-95%, 20 min going from A-95% to B-100%, 1 min going back to A-95% and 4 min A-95%. Mass spectra were acquired in an electrospray ionization/quadrupole ion trap (ESI/QITMS) Bruker HCT mass spectrometer. Samples were injected in mixtures of H2O:ACN at a flow rate of 150 µL h−1.HPLC analysis of the described compounds (cold and radioactive) was performed using the same chromatographic equipment, a Perkin-Elmer LC 200 analytical HPLC coupled to an LC 290 UV/Vis detector and a Berthold LB-507 A radiometric detector. The column used was a Supelco Discovery BIO WidePore C18 . Mobile Phases A: H111In]In-DOTA-Tz and [111In]In-DOTAGA-Tz were reacted with the peptides AR-PEG4-TCO and AR-Pip-TCO to afford the corresponding four radioconjugates with activities comprised between 5–20 MBq. The formation of the radioconjugates by click reaction was monitored by HPLC analysis (γ-detection) and proceeded to completion with the disappearance of the peaks corresponding to the free radiocomplexes. The retention times of the final radiolabeled peptides are reported in The clickable radiocomplexes In-DOTA-Tz-TCO-PEG4-AR for 1, 2 and 4 h, with or without the GRPR agonist Tyr4-Bombesin (0.25 µg/0.5 mL/well).To assess the specific GRPR-mediated cellular uptake and internalization of radioconjugates (receptor blocking studies), a similar study was performed in which cells were incubated with the [125I]I-Tyr4-BBN in the presence of increasing concentrations (10−16 to 10−5 M) of Tyr4-BBN or the four natIn-conjugates for 90 min at 4 °C. The competition was interrupted by discarding supernatants and washing the cells twice with ice-cold PBS with 0.2% of BSA. Cells were then lysed with 1 M NaOH treatment and lysates were collected and counted for their radioactivity content in an automated γ–counter . IC50 values were calculated by nonlinear regression according to one-site or two-site models using GraphPad Prism 5 software  and are the average of three independent experiments.The in vitro cell-binding assays were performed with the prostate carcinoma cell line PC3, as previously reported . BrieflyThe animal studies were performed according to national and EU legislation for good practices on animal care, protection and welfare.111In-radioconjugates to be retained in tumors were evaluated in Balb/c-male nude mice with PC3 xenografts as previously reported In-DOTA-Tz after an interval of 4 h. At selected time points, the animals were sacrificed and the biodistribution was assessed using the same experimental procedure.To assess the pretargeted approach, the same animal model was previously injected in the tail vein with 1 nmol of the TCO-containing GRPR antagonist AR-PEG2 (that we will refer to as AR) was synthesized by solid-phase peptide synthesis (SPPS) using an automated synthesizer. After cleavage from the resin, the peptide was purified using HPLC analysis and identified by ESI-MS.The potent GRPR antagonist with sequence D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH4-TCO was obtained by reacting the AR peptide with TCO-PEG4-NHS in 57% yields. The final product was identified by ESI-MS analysis, purified using HPLC and lyophilized. The derivative AR-Pip-TCO was obtained by reacting first the AR peptide with the Fmoc-protected piperidine linker. Then, the protecting group was removed and the free amino group was coupled to the TCO-NHS derivative. The desired final product was obtained in a final 13% yield  and was identified by ESI-MS analysis, purified using HPLC and lyophilized.The clickable antagonist AR-PEG111In]In-DOTA-Tz-TCO-PEG4-AR, [111In]In-DOTAGA-Tz-TCO-PEG4-AR, [111In]In DOTA-Tz-TCO-Pip-AR and [111In]In-DOTAGA-Tz-TCO-Pip-AR were synthesized to perform preliminary in vitro studies and identify the most promising compound to test our GRPR pretargeting strategy.The [111In-radioconjugates shown in 4-TCO and AR-Pip-TCO and the radiocomplexes [111In]In-DOTA-Tz and [111In]In-DOTAGA-Tz.The The click reaction was performed in an aqueous solution at 37 °C. After 1 h, the HPLC analysis (γ-detection) of the mixture showed the disappearance of the peak corresponding to the clickable DOTA-like radiocomplex, indicating the completion of the reaction, as shown in natIn-conjugates with identical retention times  and human serum (HS) for 24 h, followed by HPLC analysis. All radioconjugates exhibited excellent stabilities in PBS and CCM (above 95%), with no degradation in up to 24 h of incubation see . In HS, The lipophilicity of the radioconjugates was evaluated by the determination of the partition coefficient log Po/w, using the shake-flask method; the results are shown in −1 moiety did not appear to influence the hydrophilicity of the final compound.The values of log Po/w of the radioconjugates indicate that all compounds are quite hydrophilic. The derivatives bearing the pegylated spacer, in the first two entries of 111In]In-DOTA-Tz-TCO-Pip-AR showed a more negative log Po/w value of −1.28, suggesting that the introduction of the protonable piperidinyl linker might contribute to a higher extent to the overall hydrophilicity of this DOTA-based compound. On the other hand, the derivative [111In]In-DOTAGA-Tz-TCO-Pip-AR is the least hydrophilic, with a log Po/w value of −0.9 that might be explained by a compensation between the two opposite charges from the complex and the linker at physiological pH.The radioconjugate [111In]In-DOTA-Tz-TCO-PEG4-AR, [111In]In-DOTAGA-Tz-TCO-PEG4-AR, [111In]In-DOTA-Tz-TCO-Pip-AR and [111In]In-DOTAGA-Tz-TCO-Pip-AR were studied to determine their uptake and internalization in a prostate cancer in vitro model using the well-established GRPR-positive cell line, PC3.The radioconjugates [111In]In-DOTA-Tz-TCO-PEG4-AR had the highest cellular uptake at around 35% of the total activity applied per million cells after 4 h of incubation, eventually reflecting its highest affinity for the GRPR. Furthermore, this derivative was the only one showing a slow but steady increase in uptake over time, doubling the uptake value  at the end of the assay. Most importantly, the derivative [111In]In-DOTA-Tz-TCO-PEG4-AR was mainly bound to the cell membrane, which is consistent with antagonist behavior.Despite bearing the same carrier peptide, the four derivatives showed biological behaviors influenced by both the linker and the chelator moiety inserted into the structure. The derivatives carrying the longer and flexible pegylated linker showed higher cellular uptake values and the derivatives bearing the DOTAGA chelator demonstrated lower cellular uptake than the DOTA congeners, as shown in 111In]In-DOTA-Tz-TCO-Pip-AR and [111In]In-DOTAGA-Tz-TCO-Pip-AR, bearing the protonable piperidinyl linker, showed moderate uptake values with a maximum uptake of around 10% of the total activity applied per million cells. The trend of the uptake and internalization was similar for both conjugates, without significative enhancements over time, as shown in The radioconjugates [111In]In-DOTA-Tz-TCO-PEG4-AR was confirmed by repeating the assay in the presence of an excess of the GRPR agonist bombesin (Tyr4-Bombesin). The addition of the agonist successfully saturated the GRPR binding sites and prevented the uptake of the radioconjugate, as shown in The specificity of the uptake of the radioconjugate In-DOTA-Tz-TCO-PEG4-AR that displayed the highest cellular uptake and GRPR binding affinity in the cellular studies, and we compared the biodistribution profile with the corresponding DOTA-based radiopeptide bearing the piperidinyl linker.We next extended the preclinical evaluation of the GRPR antagonists to a prostate cancer in vivo model, using male Balb/C nude mice bearing PC3 xenografts. For such purpose, we selected the derivative [111In]In-DOTA-Tz-TCO-PEG4-AR demonstrated good and fast uptake values in the tumor and in the tissues expressing the GRPR receptor such as the pancreas , as shown in [111In]In-DOTA-Tz-TCO-Pip-AR bearing the piperidinyl linker .Consequently, we injected the TCO-containing GRPR antagonist AR-PEG111In]In-DOTA-Tz complex alone: (i) at 15 min post-injection, 3.7 ± 0.1 versus 2.46 ± 0.6%I.D./g and 1.2 ± 0.4 versus 0.42 ± 0.02%I.D./g, respectively; and (ii) at 1 h post-injection, 1.19 ± 0.2 versus 0.5 ± 0.2%I.D./g and 0.4 ± 0.11 versus 0.09 ± 0.06%I.D./g, respectively. Despite the modest increase, the results are significant, as confirmed by the paired t-test value . Furthermore, in the pretargeting experiment, the tumor and pancreas uptake at 1 h post-injection were reduced upon GRPR blockade with a BBN agonist, from 1.19 ± 0.2 to 0.64 ± 0.1%I.D./g and from 0.4 ± 0.11 to 0.25 ± 0.05%I.D./g, respectively.The results obtained in the pretargeting experiment indicated a higher uptake in the tumor and pancreas when compared with the results obtained upon injection of the clickable In-DOTA-Tz and [111In]In-DOTAGA-Tz already described in previous work of our group. The synthesis of the radioconjugates by click reaction was performed in water at 37 ˚C and was complete after 1 h of incubation, as corroborated by the disappearance of the peak corresponding to the radiocomplexes in the HPLC. The final four radioconjugates demonstrated excellent in vitro radiochemical stabilities.The study hereby presented allowed the preclinical evaluation of four radioconjugates obtained upon the click reaction between a TCO-functionalized GRPR antagonist and a Tz-containing 111In]In-DOTA-Tz-TCO-PEG4-AR. Biodistribution studies with this derivative have further confirmed the promising cellular results with a good and persistent uptake in the tumor and the GRPR-expressing tissues.The four radioconjugates were then evaluated in several cellular studies demonstrating good and selective GRPR uptake and excellent binding affinities in the low nanomolar range, especially for the derivative In-DOTA-Tz radiocomplex. The tumor uptake observed at 15 min post-injection was quickly washed out when compared to the biodistribution data of the classic approach. However, comparing the biodistribution data of pretargeting with the results obtained by only injecting the [111In]In-DOTA-Tz radiocomplex, there is an augmented and significant tumor and pancreas uptake.Our study was based on successive injections of a clickable GRPR antagonist followed by a 4 h interval and successive administration of the [The modest uptake values might be explained by a fast in vivo metabolization of the TCO-containing peptides that are shortly available after the pretargeting interval for the click reaction with the radiocomplex. When compared to monoclonal antibodies, peptides, have very short plasmatic half-lives that might hamper this approach, at least in the experimental conditions that we have described. However, the results presented are promising and demonstrate that it is worth pursuing the optimization of several parameters to improve the in vivo results.For instance, simple measures such as the reduction in the pretargeted interval that might be too long for these types of molecules or the insertion of a moiety capable of extending the plasmatic half-life in the structure of the clickable peptides might improve the outcome of the pretargeting approach using GRPR antagonists. A previous work using a GRPR antagonist conjugated to an albumin-binding domain successfully improved the circulating time of the radioconjugate, but also highlighted a very high kidney uptake, hampering a possible use in radionuclide therapy . The preNonetheless, besides the possibility of exploring the pretargeting approach, we also consider that the use of this strategy based on clickable GRPR antagonists and DOTA/DOTAGA derivatives allowing fine-tuning of their pharmacokinetics and metabolic stability might enable a more straightforward and versatile synthesis of new libraries of (radio)conjugates for the development of theranostic tools toward GRPR-expressing tumors.