GA mitochondrial matrix inhibitor), a first-in-class anticancer agent that couples the Heat Shock Protein-90 (Hsp90) inhibitor 17-allylamino-geldanamycin (17-AAG) to the mitochondrial-targeting moiety, triphenylphosphonium. Formulated as a stable (≥24 weeks at −20°C) injectable suspension produced by microfluidization (<200 nm particle size), Gamitrinib (>99.5% purity) is heavily bound to plasma proteins (>99%), has intrinsic clearance from liver microsomes of 3.30 mL/min/g and minimally penetrates a Caco-2 intestinal monolayer. Compared to 17-AAG, Gamitrinib has slower clearance (85.6 ± 5.8 mL/min/kg), longer t1/2 (12.2 ± 1.55 h), mean AUC0-t of 783.1 ± 71.3 h∙ng/mL, and unique metabolism without generation of 17-AG. Concentrations of Gamitrinib that trigger tumor cell killing (IC50 ~1-4 µM) do not affect cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2B6, CYP2C8 or ion channel conductance . Twice weekly IV administration of Gamitrinib to Sprague-Dawley rats or beagle dogs for up to 36 d is feasible. At dose levels of up to 5 (rats)- and 12 (dogs)-fold higher than therapeutically effective doses in mice (10 mg/kg), Gamitrinib treatment is unremarkable in dogs with no alterations in clinical-chemistry parameters, heart function, or tissue histology, and causes occasional inflammation at the infusion site and mild elevation of serum urea nitrogen in rats (≥10 mg/kg/dose). Therefore, targeting mitochondria for cancer therapy is feasible and well tolerated. A publicly funded, first-in-human phase I clinical trial of Gamitrinib in patients with advanced cancer is ongoing Mitochondria are key tumor drivers, but their suitability as a therapeutic target is unknown. Here, we report on the preclinical characterization of Gamitrinib ( As a result, multiple mitochondrial-targeted anticancer agents, or mitocans,9 have been developed, and mitochondrial protease ClpP agonist, ONC201,10 Complex I inhibitor metformin/ME-344,11 antagonist of mitochondrial translation, tigecycline,12 and PDH/KDH blocker CPI-61313 have entered clinical trials in humans.The rewiring of metabolic pathways, including in mitochondria,14 One example is the ubiquitous dependence or ‘addiction’ of tumor mitochondria to a heightened protein folding environment,15 essential to buffer the proteotoxic stress invariably associated with tumor growth, in vivo.16 Mechanistically, this is accomplished by the selective accumulation of molecular chaperones, including Heat Shock Protein-90 (Hsp90) and its homolog TNF Receptor-Associated Protein 1 (TRAP1),17 as well as AAA+ proteases18 in tumor mitochondria, compared to normal tissues. In turn, Hsp90 chaperoning stabilizes the multifunctional mitochondrial proteome in cancer,20 including key metabolic regulators,21 lowers ROS,22 and prevents cell death.24In addition, there is evidence that mitochondria may be uniquely ‘wired’ in cancer compared to normal tissues, potentially enabling a broader therapeutic window.26 pharmacologically, this pathway escapes inhibition by small-molecule Hsp90 antagonists with geldanamycin (GA) or non-GA backbones27 as these agents fail to accumulate in mitochondria.28 To overcome this conundrum, we generated Gamitrinib , a first-in-class, mitochondrial-targeted inhibitor of organelle protein folding25 that links the GA Hsp90 inhibitor 17-allylamino-geldanamyicn 27 to an efficient mitochondrial-import carrier, triphenylphosphonium (TPP).29 Due to its unique chemical structure, Gamitrinib selectively accumulates in mitochondria28 with a 106-fold enrichment compared to cytosol by mass spectrometry of isolated subcellular fractions.30 Once in mitochondria, Gamitrinib triggers acute proteotoxic stress,23 shuts off multipleorganelle functions, including bioenergetics,19 and delivers potent anticancer activity with IC50 of 0.16–29 µM in an NCI 60 cell-line screen, including cell lines representative of common malignancies, such as colon adenocarcinoma , breast adenocarcinoma , and melanoma .32 Strong anticancer activity was also seen in a combination of regimens of Gamitrinib plus molecular therapy33–35 in models of epithelial and hematopoietic malignancies.Although targeting chaperone-directed proteostasis in mitochondria shows promising antitumor activity,Here, we report the preclinical characterization of Gamitrinib as the first, subcellularly directed antagonist of mitochondrial proteostasis. Based on these findings, a publicly funded, first-in-human phase I clinical trial of Gamitrinib in patients with advanced cancer is currently ongoing .2 and plated on 35 mm dishes at least 24 h prior to the experiment.Caco-2 and HEK293 cells were obtained from the American Type Culture Collection  and grown in culture according to the supplier’s specifications. In some experiments, HEK293 cells were stably transfected with hERG cDNA, and polyclonal cultures were maintained in the presence of 250 µg/ml geneticin (G418). Cells were maintained in a medium containing 10% fetal calf serum at 37°C in 5% COGA mitochondrial matrix inhibitor) have been described previously.28 The structure of Gamitrinib is combinatorial and contains the Hsp90 inhibitor 17-AAG linked to triphenylphosphonium as a mitochondrial-targeting carrier. The bulk Gamitrinib powder is stored at −20°C in the dark.The complete chemical synthesis, HPLC profile, and mass spectrometry of Gamitrinib  working solutions of Gamitrinib (5 mg/mL) for preclinical studies. The formulation workflow is as follows: Step 1 – Solubilization of Gamitrinib powder in DMSO (2.5%); Step 2 – Dilution in 1.25% (w/v) Polysorbate 80, 0.31% (w/v) Lecithin (Lipoid S100) and 12.5% (w/v) Sucrose (10%) in sterile water for injection; Step 3 – Dilution in 5% dextrose (87.5%). Therefore, the final Gamitrinib formulation is ~5 mg/mL Gamitrinib, 2.5% DMSO, 0.125% Polysorbate 80, 0.031% Lecithin, 1.25% Sucrose, and 4.375% Dextrose. For Good Manufacturing Practice (GMP) studies, a Gamitrinib Injectable Suspension (GIS) was prepared by microfluidization. Gamitrinib stock solutions prepared as above were passed through a microfluidizer  with the rate of flow set at low, medium, and high. At the end of GIS processing, microfluidization was continued at reduced pressure (~2000 psi) for 1–2 min. The parameters for GIS microfluidization as are follows: ratio of organic to aqueous phase (DMSO: aqueous vehicle 1:40 v/v); filter membrane materials ; microfluidizer pressure during mixing organic and aqueous phase ; post-mixing pressure in microfluidizer (2000 psi); temperature inside interaction chamber (0 to −10°C before initiating microfluidization). The final GIS after microfluidization is 4.86 mg/mL, with average particle size of 154 nm, D(0.9) size of 229 nm, pH 6.0.2EDTA as an anticoagulant at 0.083, 0.25, 0.5, 1, 2, 4, and 24 h post-dose, chilled on ice, and centrifuged at 5000 rpm for 10 min. Aliquots (0.2 mL) of each plasma sample were stored frozen at −20°C. For analysis, 50 µL aliquots of the study samples were mixed with 50 µL of ACN/H2O (50:50), extracted by protein precipitation using ACN containing the internal standard Gamitrinib-d15 (100 ng/mL), and the aliquots of the supernatant were mixed with deionized water for Gamitrinib determination. Batch samples under analysis included a calibration curve, a matrix blank (blank rat plasma), a reagent blank, a control zero , and duplicate QC samples at three concentration levels  in addition to the study samples. Within each batch, the study samples were bracketed by calibration standards or QC samples. The lowest calibration standard served to evaluate system suitability at the beginning of each batch. In all batches, the system suitability samples displayed adequate separation and acceptable peak shapes, retention times, and signal-to-noise ratios. Overall, the concentration of Gamitrinib was measured in 21 rat plasma samples in 1 analytical batch using LC-MS/MS data acquisition on a Shimadzu Nexera LC system coupled with an AB Sciex Triple Quad 5500 mass spectrometer. Chromatograms were integrated using Analyst 1.6.2 software. A weighted  linear regression was used to generate the calibration curve for Gamitrinib. The concentration of Gamitrinib was calculated using the peak area ratio of analyte to internal standard based on the standard curve. The mean, standard deviation, precision, accuracy, and assay variability were calculated using Microsoft Excel.Sprague-Dawley rats (n = 3) were administered with a single intravenous (IV) dose of 1 mL Gamitrinib (5 mg/kg) formulated as described above without added microfluidization. Blood samples were collected via lateral tail vein using Kad libitum for 6 d prior to study initiation. Environmental controls were set to maintain a temperature range of 20 to 26°C, a relative humidity range of 30 to 70%, eight or greater air changes/h, and a 12-h light/dark cycle. Animals were infused with sterile isotonic (0.9%) saline for a minimum of 4 d  during the predose phase. At initiation of dosing, animals were 8 to 9 weeks old, and body weights ranged from 283 to 341 g for males and 188 to 238 g for females. Gamitrinib formulated as indicated above was administered by IV infusion on d 4, 8, 11, 15, 18, 22, 25, and 29 of the dosing phase at 1, 10, 25 mg/kg/dose and dose volume of 5 mL/kg. The vehicle control administered with the same schedule contained 2.5% DMSO, 0.125% polysorbate 80, 0.031% lecithin, 1.25% sucrose and 4.375% dextrose. Animals were checked twice daily throughout the duration of the study for mortality, abnormalities, and signs of pain or distress. Detailed observations were conducted for each animal up to two times during the predose phase and for each toxicity animal prior to dosing on d 1, 8, 15, 22, and 29 of the dosing phase and on d 1, 7, and 14 of the recovery phase. Blood samples were collected on d. 1, 4 and 29 of the dosing phase 5 min, 1 h and 24 h post-dose, maintained on chilled cryoracks and centrifuged within 1 h of collection. Tissue samples harvested from each animal were embedded in paraffin, sectioned, and slides were prepared and stained with hematoxylin and eosin.Ninety-six male and female Sprague-Dawley rats (6–7 weeks old) were received from Charles River Laboratories  catheterized via a femoral vein prior to arrival. Animals were acclimated in individual stainless-steel cages with access to water and Certified Rodent Diet #2014 C (Envigo RMS) ad libitum and Certified Canine Diet #5007  for 4 to 5 h each day. At study initiation, animal body weights ranged from 8.6 to 11.5 kg for males and 6.0 to 9.4 kg for females. At least 1 week prior to initiation of dosing, animals were fasted overnight, anesthetized, and a catheter attached to a subcutaneous vascular access port was surgically implanted into a jugular vein. Animals were acclimated to infusion jackets prior to catheter implantation surgery. Animals were infused with sterile saline at a dose rate of 5 mL/h when the vascular access ports were accessed and when animals were connected to the infusion system (except when dosed with the test or vehicle control article). The patency of each catheter was checked as needed. Prednisolone tablets were administered orally at 2 mg/kg the night prior to dosing. Diphenhydramine  was administered prior to dosing and post the start of infusion (d 1 only). Male and female dogs received dose levels of Gamitrinib of 1.25, 3.33 and 6.25 mg/kg/dose formulated as indicated above during a 1 h infusion on d 1, 8, 11, 15, 18, 22, 25, 29, 32, and 36 at a volume of 2 mL/kg. The vehicle control was as above. During dosing on d 1, multiple animals, including controls, were noted with clinical observations of swollen head/body, hypoactivity, twitching, red skin, and/or vocalization due to vehicle components. Dosing was stopped, and 23 of 24 animals were administered additional diphenhydramine, 11 were administered Flunixin meglumine and buprenorphine, and 6 were administered acepromazine. Symptoms subsided in all animals after completion of these interventions. As a result of these observations, the polysorbate levels, which were increased from 0.025% (w/v) to 0.125% (w/v) to minimize precipitation, were returned to the original concentrations; 2 mg/kg prednisone was administered orally the night prior to dosing; and the diphenhydramine pretreatment dose was updated to 5 mg/kg 15 to 30 min prior to dosing. Detailed observations were conducted for each animal up to seven times during the predose phase, prior to dosing on d 1, 8, 15, 22, 29, and 36 of the dosing phase, and on d 1, 7, and 14 of the recovery phase. Blood samples (1 mL) were collected via the cephalic vein 5, 15, and 30 min and 1, 4, 8, and 24 h post the end of infusion on d 8 and 36 of the dosing phase. Formalin-fixed and paraffin-embedded tissue samples collected at the end of the study (d 39) were processed for histologic examination.Eighteen male and female (5–6 mo old) purebred beagle dogs  were acclimated for 47 d  prior to study initiation. Animals were given water Levene’s test was used to test for equality of variances between groups. Where Levene’s test was not significant (P > .05), ANOVA was conducted; where Levene’s test was significant (P ≤ .05), a rank transformation was applied before the ANOVA was conducted. Where the group effect from the ANOVA was significant (P ≤ .05), comparisons between each treated group and the control were made using Dunnett’s t-test. If the ANOVA was not significant (P > .05), no further analyses were conducted.28 Clinical-grade (GMP compliant) Gamitrinib synthesized as described in Supplementary Figure S1A28 has the chemical formula C52H65F6N3O8P2 (>99.5% purity by UPLC), is purple solid (TM.795) and crystalline by X-ray powder diffraction, with a molecular weight of 1036.03. A 500 MHz1H NMR spectrum (DMSO-d6), 125 MHz13C NMR spectrum (DMSO-d6) and 282 MHz19F NMR spectrum (DMSO-d6) are all consistent with structure. The water content is 0.7% , and the residual solvent concentrations  are all below limit of quantification . Gamitrinib is formulated using a three-step dilution process described in the Materials and Methods section. A sterile, Gamitrinib Injectable Suspension (GIS) is prepared for clinical use using microfluidization with the schematic flowchart shown in Supplementary Figure S1B. The resulting GIS has average particle size of 154 nm and D(0.9) size of 229 nm, pH 6.0. When stored frozen at −20°C, the GIS shows no significant changes in stability or particle size distribution upon analysis at 1, 2, 4, 8, 12, and 24 weeks after manufacturing 30 did not inhibit cytochrome P450 isoforms CYP1A2 , CYP2A6 , CYP2B6 , and CYP2C8  . Conversely, Gamitrinib inhibited CYP2C9  and CYP3A4  .Concentrations of Gamitrinib that trigger tumor cell killing in culture (IC50 of hERG inhibition of 3.5 µM (terfenadine IC50 21.7 nM)  inhibited Nav1.5 currents by 22.3 ± 5.3% , Kv4.3/KChIP2 by 6.8 ± 2.2% , Cav1.2 by 12.2 ± 1.5% , Kv1.5 by 6.6 ± 1.3% , KCNQ1/mink by 22.5 ± 1.1% , hERG by 37.9 ± 1.7% , HCN4 by −0.2 ± 3.7% , and Kir2.1 by −7.3 ± 3.4%  ). The po21.7 nM) ).FigureTo further characterize the potential cardiac toxicity of Gamitrinib, electrocardiography studies were carried out on beagle dogs administered IV Gamitrinib at dose levels of 1.25, 3.3, and 6.25 mg/kg/dose twice weekly for 36 d plus a 14-d recovery period . In this analysis, one out of five male dogs administered Gamitrinib at 6.25 mg/kg/dose exhibited a small (7%) prolongation of QTc interval (17 msec) on d 32 of the dosing phase, which reversed during the recovery phase. No Gamitrinib-related prolongation of QTc interval was observed in female dogs administered 6.25 mg/kg/dose or in both sexes administered 1.25 or 3.33 mg/kg/dose . No Gamitrinib-related ECG changes in PR interval, QRS duration, QT interval, or heart rate were observed on d 32 of the dosing phase in animals administered 1.25, 3.33, or 6.25 mg/kg/dose or on d 11 of the recovery phase in animals administered 6.25 mg/kg/dose. No other rhythm abnormalities or qualitative ECG changes were observed .max was 1175.807 ng/mL (ss) of 65.471 L/kg, medium to slow clearance at 85.656 ± 5.856 ml/min/kg, and mean terminal-phase half-life (t1/2) of 12.25 ± 1.55 h  to Sprague-Dawley rats (n = 3), the mean Gamitrinib C07 ng/mL ), with a± 1.55 h . Mean AUectively . Gamitriectively , Supplem decline ). CLSS vapp’) of 1.90 nm/s in the A-to-B direction and 10.94 nm/s in the B-to-A direction with a Papp Efflux Ratio (ER) of 5.77 (Supplementary Table S1).Gamitrinib was heavily bound to plasma proteins (99.3 ± 0.07%) with an average free fraction of 0.7 ± 0.07%, comparable to control warfarin . The stability of Gamitrinib in human plasma was 91.4% with an average recovery of 82.8 ± 3% . At a concentration of 0.5 µM, the elimination rate constant (k) of Gamitrinib in phase I, cytochrome P450-mediated human liver microsome metabolism was 0.041  with half-life (t½) of 16.7 min  and intrinsic clearance (CLint) of 3.30 mL/min/g . Gamitrinib showed negligible penetration across a monolayer of Caco-2 intestinal cells with an apparent permeability coefficient  is 10 mg/kg/dose, corresponding to Cmax and AUC0–24 values of 311 ng/mL and 1300 ng∙h/mL, respectively, on d 29.Male rats administered Gamitrinib IV (1 h infusion) at dose levels of 1, 10, or 25 mg/kg/dose twice weekly on d 1, 4, 8, 11, 15, 18, 22, 25, and 29 (dosing phase) exhibited a small, fully recoverable and not adverse reduction in mean body weights at 10 (−5.5%) or 25 (−5.7%) mg/kg/dose . Gamitrimax, 560 ± 404 ng/mL; AUC0–24, 1740 ± 713 ng∙h/mL on d 36 of dosing, both sexes). In the absence of effects on the overt well-being of the animals and evidence of reversibility of Gamitrinib-related findings, 6.25 mg/kg/dose is considered the highest non-severely toxic dose (HNSTD). This dose level corresponds to Cmax and AUC0–24 values of 1260 ± 556 ng/mL and 3290 ± 1090 ng∙h/mL, respectively (both sexes), on d 36 of the dosing phase.Male and female beagle dogs administered IV Gamitrinib at dose levels of 1.25, 3.33, and 6.25 mg/kg/dose on d 1, 8, 15, 22, 29, and 36 of the dosing phase showed no alterations in body weight or other clinical observations (Supplementary Table S3). Bone marrow and liver parameters were unremarkable in all group levels, and only a trend of increased serum urea nitrogen and creatinine was observed in animals (both sexes) receiving the highest dose level of Gamitrinib of 6.25 mg/kg/dose . This comitocan inhibitor of mitochondrial protein folding, can be synthesized as a clinical-grade material, formulated as a sterile, stable injectable suspension and administered IV to two animal species for up to 36 d. In line with its unique mechanism of action of subcellular organelle targeting, Gamitrinib drug-like properties differ from those of non-mitochondrial-targeted 17-AAG (Tanespimycin) with slower clearance, longer half-life, and unique metabolism without generation of 17-AG. Finally, prolonged IV administration of Gamitrinib is essentially unremarkable in beagle dogs and causes occasional inflammation at the infusion site and modest alterations of kidney function in Sprague-Dawley rats.In this study, we have shown that Gamitrinib, a first-in-class 1 and mitochondria provide an actionable, multifunctional therapeutic target,8 the (pre)clinical development of mitocans is mostly in infancy.9 The most visible conclusion of these studies is the encouraging preclinical safety of Gamitrinib, the first mitocan to disrupt a Hsp90 mitochondrial proteome in cancer.19 When administered to dogs, concentrations of Gamitrinib up to 12.8-fold higher than therapeutically active doses in mice (10 mg/kg) did not elicit the extensive alterations of bone marrow, liver, and gastrointestinal tract functions seen with non-mitochondrial targeted 17-AAG37 and its derivatives, 17-DMAG,38 and IPI-504.39 The only reportable observation in our study was that the highest dose level of Gamitrinib of 6.25 mg/kg caused only modest elevation of urea nitrogen and creatinine concentrations, with microscopic evidence of kidney tubule degeneration/regeneration, which was reversed during the 14-d recovery phase. The potential cardiac liability of Gamitrinib, prompted by the role of Hsp90 in hERG protein folding40 was also unremarkable. Gamitrinib IC50 for hERG conductance in patch-clamp studies (3.5 µM) was up to 20-fold times higher than the IC50 of inhibition of tumor growth observed in an NCI 60-cell line screen,28 and only 1 out of 10 animals exhibited a small, 7% prolongation of QTc interval (17 msec) fully reversed during the recovery phase. In the rat study, Gamitrinib concentrations (25 mg/kg) up to 5-fold higher than the efficacious antitumor dose in mice, produced only occasional inflammation at the infusion/catheter site, accompanied by mild elevation of urea nitrogen and evidence of kidney tubule degeneration/regeneration.Although rewiring of metabolic pathways is a key tumor driver,37–39 remains to be elucidated. Previous data have suggested that Hsp90 may have greater affinity in tumors compared to normal tissues,14 which when combined with the selective accumulation of TRAP1 and Hsp90 in tumor mitochondria,17 may make the therapeutic inhibition of mitochondrial chaperones15 more efficient in cancer than normal tissues. It is also possible that the rapid intracellular transfer of Gamitrinib followed by its stable sequestration in mitochondria30 prevents a meaningful inhibition of cytosolic Hsp90, likely responsible for the toxicity of non-mitochondrial-targeted Hsp90 antagonists.37–39 This is consistent with other data showing that Gamitrinib treatment does not induce downregulation of Hsp90 client proteins,31 or a heat shock response, i.e. elevation of Hsp70, two hallmarks of Hsp90 inhibition in the cytosol.27The molecular basis for the significantly more favorable safety of Gamitrinib compared to non-mitochondrial-targeted Hsp90 inhibitors1/2, 12.25 ± 1.55 h), and no generation of 17-AG, a key metabolite of 17-AAG processing.36 The structural basis for the increased Gamitrinib stability in vivo is currently not known. However, these data are reminiscent of the greater stability of 17-DMAG compared to 17-AAG,41 and it is possible that the presence of the triphenylphosphonium side chain reduces the oxidative metabolism of Gamitrinib,41 causing slower drug clearance and longer t1/2.In addition to improved safety, Gamitrinib exhibited unique drug-like properties compared to 17-AAG. These included slower clearance (85.65 ± 5.85 mL/min/kg), much longer terminal-phase half-life . In addition to validating mitochondrial proteostasis and Hsp90 as actionable therapeutic targets, the clinical development of Gamitrinib suggests that other mitochondrial functions exploited in cancer, such as one-carbon metabolismClick here for additional data file.


Figure 8 and Figure 8, the numbering of figure legends was incorrect. In In the original article, there was a mistake in the legend for FIGURE 8 | Representative images of BMSCs with the alizarin red staining to determine the mineralized nodules. (A) Control group; (B) TFDR low dosage group; (C) TFDR medium dosage group; (D) TFDR high dosage group; (E) The mineralized nodules at each time point of the control group, TFDR low dosage group, TFDR medium dosage group, and TFDR high dosage group were evaluated. The data are expressed as the mean ± SEM of three independent experiments. #p < 0.05 vs. control group, Δp < 0.05 vs. TFDR low dosage group, ▲p < 0.05 vs. TFDR high dosage group.”“p < 0.05 vs. control group, #p < 0.05 vs. the TFDR low dosage group, TFDR high dosage group. (B) (a) p38 MAPK, p-p38 MAPK, BMP-2, RUNX-2, VEGF, and HIF-1αprotein expression on BMSCs detected by western blot analysis. (b)–(g) were statistical analysis of (a). The data are expressed as the mean ± SEM of three independent experiments. *p < 0.01 vs. the Control group; #p < 0.01 vs. the TFDR low dosage group.”“In addition, there was a mistake in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Antiaris toxicaria, can effectively and selectively suppress proliferation and cause apoptosis in glioma cells, which has an IC50 that is 100 times lower than that in mouse normal neural stem cells. Importantly, cell sphere formation assay and real time-quantitative analysis reveal that anticarin β inhibits cancer stemness by modulating related stemness gene expression. Additionally, anticarin β induces DNA damage to regulate the oncogene expression of signal transducer and activator of transcription 3 (STAT3), Akt, mitogen-activated protein kinases (MAPKs), and eventually leading to apoptosis. Furthermore, anticarin β effectively inhibits glioma growth and prolongs the lifts pan of tumor-bearing mice without systemic toxicity in the orthotopic xenograft mice model. These results suggest that anticarin β is a promising candidate inhibitor for malignant glioma.Glioma is the most common form of malignant brain cancer. It is very difficult to cure malignant glioma because of the presence of glioma stem cells, which are a barrier to cure, have high tumorigenesis, associated with drug resistance, and responsible for relapse by regulating stemness genes. In this study, our results demonstrated that anticarin β, a natural compound from  Glioma is the most common and lethal kind of brain tumor, accounting for about 50% of malignant brain tumors. The average survival time from diagnosis to death for glioma patients is less than 15 months, and the 5-year survival rate is about 4.7% . TherefoAntiaris toxicaria. Anticarin β is a coumarin derivative with a special structure that has been reported previously , and High-Performance Liquid Chromatography (HPLC). The extract was divided into several peaks by C18 column according to absorbance at 365 nm (18 reverse-phase (HPLC) , which performs authentication on its cell lines. Human cell lines were cultured in DMEM/F12 supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37˚C and 5% CO2.Normal mouse neural stem cell line (mNSC) was obtained from Sigma-Aldrich . Neural stem cells were cultured in glioma stem cell medium that consisted of DMEM/F12, 1 x B27, 50 ng/mL bFGF, and 50 ng/mL EGF supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. Culture dishes were pre-coated with 10 µg/mL laminin for 8 h at 37˚C in a humidified incubator before neural stem cells were seeded and maintained at 37˚C in a humidified incubator with 5% COThe morphology of 10 µM anticarin β-treated cell lines  at 24 h after treatment was examined and images were observed by phase-contrast microscopy .4 cell lines  in 150 µl per well were seeded into 96-well plates and treated with 50 µl anticarin β at 10 μM, 5 μM, 2.5 μM, 1.25 μM, 625 nM, 312.5 nM, 156.25 nM, 78.125 nM, 39.0625 nM, respectively. Cells treated with the same volume of phosphate buffer saline  were used as normal control. All groups of cells were incubated for 24 h at 37˚C with 5% CO2 after treatment with anticarin β. MTS reagent was diluted 1:10 with fresh complete medium and added 100 μL per well. After incubating for 1 hour, the absorbance was measured at 490 nm. The inhibition of cells treated with anticarin β was compared with the control group and expressed as a percentage. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 6.0 (GraphPad Software).A total of 2 x 10Human glioma cells, U87-MG and T98G, were plated in 6-well plates (100 cells each well) and incubated for 7 days with treatments of anticarin β (0.5 μM). The control groups were treated with the same volume of PBS. The cells were then fixed with 4% formaldehyde for 20 min and stained with 0.25% crystal violet for 15 min. The number of colonies, defined as > 50 cells/colony, was counted and quantified. The data were analyzed using ImageJ.4 cells were mixed with 0.8% soft agar to 0.4% and plated 1 mL per well in a 6-well plate, cooled, and added 1 mL medium each well. The plate was cultured in a humidified incubator at 37˚C with 5% CO2 and the medium was supplemented every 3 days. After the clonal spheres had reached 20 µm, they were treated with 1 μM anticarin β or the same volume of PBS every 3 days. After two weeks, the clonal spheres were collected, washed, and fixed with 4% paraformaldehyde containing 0.005% crystal violet for 2 h at room temperature. Images were obtained and the clonal spheres were counted.3.2% soft agar was made by 0.4 g low gelling agar, mixed with 13 mL water, and sterilized by high-pressure steam sterilization. Then, 3.2% soft agar was diluted to 0.8% with medium and plated 1 mL per well in a 6-well plate as the bottom layer of agar. After colling at room temperature, 2 x 10via Transwell assay (8-μm filter). For the migration assay, 5 x 104 cells resuspended in 200 μL culture medium with treatments of anticarin β (0.5 μM) without FBS were inoculated into the top chambers, the control groups were treated with the same volume of PBS; the bottom chambers were supplemented with 500 μL medium supplemented with 10% FBS. After 48 h, the non-migrating cells were removed, and the cells migrating through the membrane were fixed with methanol for 30 min and stained with 0.1% crystal violet for 15 min. The migrating cells were counted and the data were analyzed using GraphPad Prism 6.0 (GraphPad Software). For the invasion assay, the same number of cells was seeded into the top chambers that were precoated with Matrigel solution (BD Diagnostics), and the following experimental steps were the same as the migration assay.Cell migration and invasion abilities were detected 5 cells/per well. Anticarin β (0.5 μM) was added and the control wells were treated with the same volume of PBS. Cells were treated for 12 h at 37˚C in a humidified incubator. Then, cells were treated with EdU (10 µM) for 2 h at 37˚C in a humidified incubator with 5% CO2. Following incubation, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS containing 3% BSA (PBS - BSA) for 20 min. Then, the cells were immersed in Click reaction mixture for 30 min and in Hoechst 33324  for 10 min at room temperature protected from light. Before the observation, the cells were washed twice with PBS and mounted onto a glass slide. Images were obtained using a fluorescent microscope at the magnifications 20x.Cell proliferation assay was performed using EdU Cell Proliferation Kit with Alexa Fluor 594  and following the protocol described by the manufacturer. Cells were seeded onto a 24-well plate with coverslips, with 1x10GAPDH for normalization. The primers used are provided as follows. Oct4: forward: 5’-TAG TCC CTT CGC AAG CCC T-3’, Reverse: 5’-CGA GAA GGC GAA ATC CGA AG-3’; Nanog: forward: 5’-CAA TGG TGT GAC GCA GGG AT-3’, Reverse: 5’-GGA CTG GAT GTT CTG GGT CTG-3’; Klf4: forward: 5’-ATG CTC ACC CCA CCT TCT TC-3’, Reverse: 5’-TTC TCA CCT GTG TGG GTT CG-3’; Sox2: forward: 5’-ACC AGC GCA TGG ACA GTT AC-3’, Reverse: 5’-CCG TTC ATG TAG GTC TGC GA-3’; Stat3: forward: 5’-ACC ATT GAC CTG CCG ATG TC-3’, Reverse: 5’-TAG GCG CCT CAG TCG TAT CT-3’; Cd133: forward: 5’-AGT CGG AAA CTG GCA GAT AGC-3’, Reverse: 5’-GGT AGT GTT GTA CTG GGC CAA T-3’; GAPDH: forward: 5’-GAA AGC CTG CCG GTG ACT AA-3’, Reverse: 5’-GCA TCA CCC GGA GGA GAA AT-3’.The total RNA extraction of cells was performed using Trizol reagent . 1 μg of total RNA was reverse transcribed using FastKing gDNA Dispelling RT SuperMix (Tiangen Biotech). The action was carried out at 42°C for 15 min and terminated by deactivation of the enzyme at 95°C for 5 min. PCR was conducted using EvaGreen 2X qPCR MasterMix  in StepOne™ Real-Time PCR System  using the manufacturer’s default settings for relative quantification of standard Ct. Baseline and threshold values were automatically detected using the StepOne™ Software v2.3  with default settings. Gene expression was calculated using the comparative ΔΔCT method with the housekeeping genes 5 cells/mL in PBS. Combine the cell suspension with 1% molten low melting point agarose and immediately pipette it onto a slide. After placing slides at 4°C for 10 min, immerse slides in lysis solution overnight. Then, slides were immersed in freshly Neutral electrophoresis solution (pH = 9.0) for 30 min. Electrophoresis was carried out at the rate of 1.0 V/cm for 45 min at 4°C. After electrophoresis, the slides were immersed with DNA precipitation solution for 30 min and ice-cold 70% ethanol for 30 min. Subsequently, the slides were dried at room temperature, and DNA was stained with 50 μL of SYBR Green I dye  for 15 min and immediately analyzed using a fluorescence microscope. Data were analyzed using CometScore .DNA damage was assessed by single-cell gel electrophoresis assay under neutral conditions as described . Human gApoptosis assay was performed using GreenNuc™ Caspase-3 Assay Kit for Live Cells  and fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for tissue sections.5 cells/well) were plated in a 6 cm glass-bottom dish. Cells were treated with 1 μM anticarin β for 24 h. Each dish was incubated with 5 μM GreenNuc™ Caspase-3 Substrate and Hoechst 33324  for 30 min in dark at room temperature. Before observing, the dishes were washed with PBS. Images were obtained using a fluorescent microscope at the magnification of 20x and 40x.For cells, human glioma cell lines U87-MG  at 95°C for 5 min. Tunel assay was performed using TUNEL FITC Apoptosis Detection Kit  according to the manufacturer’s protocol. The nuclei were counterstained with DAPI for 5 min. Images were obtained using a fluorescent microscope, and the staining was scored using ImageJ.Human glioma cell lines U87-MG, treated with or without 1 μM anticarin β for a different time, and tumor tissues were collected and lysed in RIPA buffer , containing Protease Inhibitor Cocktail  and Phosphatase Inhibitor Cocktail II . Lysates were sonicated, clarified by centrifugation, and protein concentrations were measured using the BCA protein assay . Proteins were separated on 10% acrylamide SDS–PAGE gels, transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies at 4°C overnight and then incubated with secondary antibodies at room temperature for 1 h. The signal detection was performed using enhanced chemiluminescence ECL substrate . Band images were acquired using a LAS 4000 mini system . Each experiment was independently repeated in triplicate. The antibodies used as followed: Primary antibodies Phospho-p38 (Thr180/Tyr182) , p38 , phospho-Erk1/2 (Thr202/Tyr204) , Erk1/2 , phospho-JNK (Thr183/Tyr185) , JNK , phospho-Akt (Ser473) ,Akt (pan), p-STAT3 (Ser727) , STAT3 , E-cadherin , N-cadherin , β-catenin , vimentin , CD44 , Nanog , Sox2 , Oct4 , CD133 , Bcl-2 , Bax , clv-caspase3 , clv-PARP , GAPDH ; secondary goat anti-mouse HRP-IgG  and goat anti-rabbit HRP-IgG  antibodies were used for immunoblotting.5 U87-MG cells in the right hippocampus. The injection position of each mouse was relatively consistent, and the injection depth was 3 mm. 3 days post-inject, the mice were randomly divided into three groups (6 mice per group). The intracranial administration of PBS or anticarin β (1 mg/kg) using Alzet Osmotic Pumps was started on animals each day, and the intragastric administration of temozolomide  was started on animals once. All mice were used in vivo research after death. The critical organs  and brains were harvested. The brain is divided into two parts according to the location of injection, and the tumor area was measured. Half of tissues were formalin-fixed, cut into small pieces, paraffin-embedded, sectioned, and stained with haematoxylin & eosin (H&E) or antibodies. The effect of anticarin β was evaluated on specimens in at least three different sections. Half of tissues were frozen at -80°C for other experiments.Male NOD/SCID mice  were purchased from Beijing Vital River Laboratory Animal Technology Company. Before the experiments, mice were performed to adapt to the environment for a week. A total of 18 male NOD/SCID mice were injected with 2 x 10Flow cytometry was used to analyze the percentage of cancer stem cells in T98G and U87-MG. Tumor cells were collected, rinsed, and fixed in 4% polyoxymethylene for 30 min. Then, cells were incubated with anti-CD44 antibodies for 60 min at room temperature in the dark. After washing with PBS, cells were incubated with FITC-labled secondary antibodies for 60 min at room temperature in the dark. The cells were resuspended in PBS and analyzed using an LSRFortessa flow cytometer . The data were analyzed using FlowJo software (Beckman Coulter). Each experiment was carried out in triplicates.t-test. Ordinary one-way ANOVA with multiple comparisons tests was used for examining differences among multiple groups. The overall survival of mice was analyzed by the Kaplan-Meier method and compared with the Log-rank test. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software), and p-values were calculated, significance is represented as follows, * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).The quantitative data are represented as the mean ± s.d. of triplicate experiments. The difference between two groups was compared with Student’s Antiaris toxicaria. Anticarin β was purified and characterized (50) ~1.01 ± 0.10 and 1.20 ± 0.11 μM at 24 h, respectively. For the mNSCs, the IC50 value was more than 500 μM. Furthermore, the colony formation assay was performed to determine the effect of anticarin β on cell proliferative potential of human glioma cells. The colonies formed after anticarin β treatment was reduced 25% in U87-MG and 35% in T98G compare with the control  was suppressed by anticarin β treatment for increasing time. Taken together, these results demonstrate that anticarin β destroy the self-renewal capacity of glioma cancer stem cells.The stemness of cancer plays an essential role in tumorigenic, drug resistance, and relapse. The stem cells contain self-renewal capacity and give rise to the various cell lineages that comprise the tumor; they have been postulated as responsible for recurrence, and drug resistance of glioma . Thus, hIn cancer therapies, DNA damage is a countermeasure to control cancer cell fate, death, or survival, to limit cancer progression. Upon DNA damage, cells initiate several response pathways to activate DNA repair or apoptosis . Previouvia affecting Akt, MAPKs pathways, and apoptotic-related proteins.To further explore the underlying molecular mechanism, western blot analysis was used to investigate signaling pathways. Akt, p38, extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), Bax, B-cell lymphoma-2 (Bcl-2), caspase3, and PARP both play important roles in regulating cancer cell proliferation and apoptosis , 25. Thein vivo using an orthotopic xenograft NOD/SCID mice tumor model. Human glioma cell line U87-MG (2 x 105) was injected into the right hippocampus of mice. Mice were randomly divided into 3 groups (negative group (PBS), positive group , and anticarin β group. Consistent with our in vitro results, anticarin β significantly suppressed the growth of glioma cells  in a dose-dependent manner and showed no significant cytotoxicity to mouse neural stem cells. Additionally, our study indicated that anticarin β could suppress the migration, invasion, and proliferation of human glioma. In vivo, anticarin β had no obvious systemic toxicity, dramatically reduced tumor growth, and prolong overall survival duration. However, as for its specific adverse effects on mice, further study is still necessary for the future.Anticarin β, a coumarin derivative isolated from the bark of tivities . HoweverStemness, initially defined by the expression of stem cell genes, is a property shared by embryonic stem cells and adult stem cells , 29. GliApoptosis is modulated by death receptors and death factors, to control cell proliferation and a reaction to cell injury. Tumorigenesis includes the occurrence of an imbalance between cell proliferation and apoptotic cell death, leading to the escape of apoptosis and over-proliferation. DNA damage can result in genomic instability, apoptosis, cell cycle checkpoint alteration, or postmitotic death , 34, 35.In conclusion, our findings above suggested that anticarin β effectively and selectively suppress the growth, proliferation, and stemness of human glioma cells by inducing apoptosis and DNA damage, which likely through modulation of STAT3, Akt, MAPKs pathways, and apoptotic-related proteins. In the human glioma orthotopic xenograft models, administration of anticarin β exhibited obvious anti-cancer activity. Furthermore, anticarin β with low cytotoxicity has the potential to be a promising compound to prevent human glioma tumorigenesis. The anti-tumor effect of anticarin β against glioma could be combined with other FDA-approved DNA-damaging anti-cancer drugs in future studies.https://doi.org/10.17632/fhw7ff6gd3.1).The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The original data is available at Mendeley Data .Conception and design: MZ, GW, and RL. Development of methodology: MZ, XZ, GW, and RL. Acquisition of data : MZ, ZD, GW, and RL. Analysis and interpretation of data : MZ, DZ, and GW. Writing, review, and/or revision of the manuscript: MZ, GW, and RL. Administrative, technical, or material support : MZ, GW, and RL. Study supervision: GW and RL. All authors contributed to the article and approved the submitted version.This work was supported by funding from National Natural Science Foundation of China (81903666), Science and Technology Department of Yunnan Province .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.


Plasmodium falciparum malaria and endemic Burkitt's lymphoma (eBL) – an aggressive Epstein-Barr virus (EBV)-associated B-cell tumour occurring almost exclusively in the tropics – strongly suggests a link between the two diseases. It is suspected that the polyclonal B-cell activation in P. falciparum malaria may precipitate a breakdown in homeostatic T-cell control of EBV-immortalized B-cell proliferation. Previous studies have suggested that a particular T-cell subset, characterized by expression of Vδ1+ γδ T-cell receptors, is important for maintaining B-cell homeostasis, both in P. falciparum- exposed populations and in individuals subject to polyclonal B-cell activation of other aetiology. The objective of the present study was, therefore, to characterize lymphocyte phenotypes and to investigate possible differences in T-cell subset composition and activation status in P. falciparum-exposed Ghanaian children with and without eBL.The overlap in geographical distribution of γδ, Vδ1, Vδ3, Vγ9 and B-cells) and acquired on a flow cytometer.Venous blood samples in heparin from 21 eBL patients , referred to the Burkitt's Tumour Centre at Korle-Bu Teaching Hospital, Accra and 15 healthy, age and sex matched children, were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, R-phycoerythrin (RPE)- and RPE-Cy5-conjugated antibodies (CD3, CD4, CD8, CD25, CD69, CD95, HLA-DR, TCR-+ cells in eBL patients, due mainly to perturbations among TCR-γδ+ cells was observed. In contrast, the proportions of CD4+ or CD8+ cells were relatively unaffected, as were the mean numbers of peripheral blood mononuclear cells.A reduction in the proportion of CD3γδ+ cells occurs in Ghanaian children with eBL, a pattern which is similar to P. falciparum-induced changes. The data supports the hypothesis of a regulatory role for Vδ1+ TcR-γδ T-cells in maintaining B-cell homeostasis and provides insights into the pathogenesis of eBL.Selective changes in numbers and activation status of TCR- Plasmodium falciparum malaria and eBL strikingly overlap, and both diseases mainly affect children, who are also most susceptible to malaria in areas of intense P. falciparum transmission  for eBL against 68.0 [57.8 to 75.0] for healthy controls, p = 0.11 or CD8+ cells, 27.8 [26.6 to 31.7] for eBL against 20.9 [16.6 to 29.7] healthy controls, p = 0.08, were relatively unaffected. .The mean frequency of peripheral blood CD3γδ+ CD25 and HLA-DR+ were significantly higher in eBL patients compared to healthy controls, p = 0.007 and 0.003 respectively. There were no differences between the two groups with respect to TCR-γδ+ CD69+ and TCR-γδ+ CD95+  compared to the healthy controls (13.5 [9.3 to 16.9]), p = 0.04.The frequencies of TCR-γδ+ cells are selectively increased in P. falciparum-exposed healthy Ghanaians in general, and in children in particular [P. falciparum malaria among such children led to marked perturbations in lymphocyte subset composition, which particularly affected TCR-γδ+ cells [P. falciparum malaria .In a previous study, it was reported that frequencies of TCR-rticular . The finδ+ cells ,8. Interia Table . Thus, als Table . In conted Table . The meaP. falciparum malaria, the selective depletion of T-cells and especially of TCR-γδ+ cells [γδ+ cells – as an important factor behind the observed changes in lymphocyte subset composition in eBL patients (Table in P. falciparum malaria [In children with δ+ cells ,11 appeaδ+ cells ,13. Whet malaria , lends fγδ+ cell compartment, the perturbations in relation to P. falciparum malaria among children living in endemic areas are mainly caused by a dramatic increase in the proportion of γδ T-cells expressing a T-cell receptor composed of a Vδ1 chain in combination with γ-chains other than Vγ9 [δ1+ cells were not directly examined here, the pronounced reduction in the proportions of the complementary Vγ9+ and Vδ2+ subsets in eBL patients (Table δ1+ cells is increased in eBL patients as it is in children with P. falciparum malaria.Within the TCR-than Vγ9 . AlthougP. falciparum malaria, which may reflect a causal link between the two diseases. The characteristic massive B-cell activation in P. falciparum malaria [δ1+ cells, as it has been shown that T-cells belonging to this subset can recognize and kill activated B-cells [Taken together, the data presented here show a remarkably similar impact on peripheral lymphocyte composition of eBL and  malaria ,18 appea malaria . It is t B-cells -22.P. falciparum malaria and eBL.Malaria-induced B-cell activation may thus precipitate malignant transformation of EBV-infected B-cells. Incomplete elimination of malignant T-cells due to defects in auto-regulatory T-cells may subsequently lead to eBL. This study provides insights regarding the pathogenesis of eBL and suggests a plausible link between the pathogenesis of GF: designed the study, carried out laboratory work, data analysis and interpretation and drafted the manuscript.JW assisted with design of study, selection, clinical examination and management of BL patients, as well as revision of manuscript for intellectual content.J KAT assisted with flow cytometry and data analysis.LH designed the study, carried out data analysis and extensive revision of manuscript for intellectual contentBDA designed study, assisted with data analysis and extensively revised manuscript for intellectual content.


Parathyroid hormone (PTH) is essential for calcium homeostasis and its action is mediated by the PTH type 1 receptor (PTHR1), a class B G-protein-coupled receptor. Hypoparathyroidism and osteoporosis can be treated with PTH injections; however, no orally effective PTH analogue is available. Here we show that PCO371 is a novel, orally active small molecule that acts as a full agonist of PTHR1. PCO371 does not affect the PTH type 2 receptor (PTHR2), and analysis using PTHR1–PTHR2 chimeric receptors indicated that Proline 415 of PTHR1 is critical for PCO371-mediated PTHR1 activation. Oral administration of PCO371 to osteopenic rats provokes a significant increase in bone turnover with limited increase in bone mass. In hypocalcemic rats, PCO371 restores serum calcium levels without increasing urinary calcium, and with stronger and longer-lasting effects than PTH injections. These results strongly suggest that PCO371 can provide a new treatment option for PTH-related disorders, including hypoparathyroidism. Hypoparathyroidism and osteoporosis can be treated with parathyroid hormone, but frequent injections are required. Here the authors develop a small-molecule agonist for the parathyroid hormone type I receptor that can be administered orally, and demonstrate its efficacy in rats. PTH is the principal regulator of blood Ca concentration, acting primarily on bones and kidneys1Lack of functional PTH results in hypoparathyroidism, which is a rare disease characterized by hypocalcemia. The clinical manifestations of hypoparathyroidism include muscle cramping and potentially life-threatening complications, such as seizure or laryngeal spasm88101281412131717In this study, we identified a nonpeptidyl small-molecule PTHR1 agonist, PCO371, through a cell-based functional screening assay. The compound activates human PTHR1 (hPTHR1) but not the human PTH type 2 receptor (hPTHR2). Analysis using hPTHR1–hPTHR2 chimeric receptors demonstrated that Proline 415 in transmembrane segment 6 (TM6) of hPTHR1 is a key residue for the receptor selectivity and activation. Orally administered PCO371 exhibits PTH-like biological activity in osteopenic ovariectomized (OVX) rats and in hypocalcemic thyroparathyroidectomized (TPTX) rats. In the former model, intravenously administered PCO371 increases bone mineral density (BMD) and bone strength as effectively as PTH; however, when administered orally, only a limited increase in BMD and bone strength is observed. In the latter model, 4-week oral administration shows ability to restore the normal serum Ca level without increasing urinary Ca. To our knowledge, PCO371 is the first example of a small-molecule PTHR1 agonist with good oral bioavailability and efficacy in animal models. The safety and pharmacokinetics of PCO371 are currently being evaluated in humans.50=2.4 μmol l−1, 50=17 μmol l−1) as did hPTH(1–34)  similar to that of cells expressing full-length hPTHR1. On the other hand, the ability of hPTH(1–34) to induce cAMP production was significantly lower in cells transfected with hPTHR1-delNT than in those with the full-length hPTHR1  –PTH(1–15)], which interacts mainly with the receptor's transmembrane domain, as a radiolabelled tracer−1 inhibited the binding of 125I-–PTH(1–15) almost completely (PCO371  was idenTH(1–34) . PCO371 TH(1–34) , with anh hPTHR1 . We thermpletely , suggest−1, whereas the cognate ligands and relevant reference compounds showed significant activity in these assays  that express endogenous rat PTHR1. Although the potency was much weaker than that of hPTH(1–34), PCO371 induced cAMP production as did hPTH(1–34), suggesting that PCO371 acts as a full agonist of PTHR1 .0), and the other is G-protein-coupled conformation (RG)0 and exhibits only a transient response22230 and produce prolonged calcemic responses in vivo. The peptides that bind R0 with high affinity are able to produce cAMP signalling responses in PTHR1-expressing cells for a certain amount of time after initially binding to PTHR1. A previous study also suggested that there is a good correlation between R0 binding affinity and the duration of the cAMP response induced by a given PTH ligand after initial binding to PTHR1 , osteocalcin (encoded by Bglap) and receptor activator of nuclear factor-κB ligand , and dose-dependently decreased expression of mRNAs for osteoprotegerin (encoded by Tnfrsf11b) and sclerostin (encoded by Sost), as did hPTH(1–34)  for Fos (encoded by TH(1–34) . In theshe cells . This an−1 or more had the ability to stimulate 45Ca release from prelabeled bones as did hPTH(1–34)  .To investigate the mechanisms underlying the selectivity of PCO371 for hPTHR1 over hPTHR2, we performed cAMP production assays using transfected COS-7 cells expressing hPTHR1 mutants in which amino acid residue(s) were replaced by the corresponding hPTHR2 amino acid residue(s) . hPTH1–. Since tCmax) of PCO371 were attained at 1–1.5 h, and the terminal half-lives (T1/2) were 1.5–1.7 h. Oral bioavailability of PCO371 was 34% at a dose of 2 mg kg−1. After a single subcutaneous injection of hPTH(1–34), the concentration in serum was elevated within 15 min in a dose-dependent manner, and the T1/2 was 15 min.Pharmacokinetics profiles of PCO371 and hPTH(1–34) after single administration to normal rats are summarized in −1) or subcutaneous hPTH(1–34) injections caused significant increase in BMD and bone strength in the lumbar vertebrae of OVX rats compared with OVX rats dosed with vehicle (−1) and intravenous PCO371 (3 mg kg−1) did not affect BMD or bone strength in the lumbar spine (−1) partially increased BMD in the proximal femur (−1) PCO371 induced a significant increase in serum osteocalcin and urinary collagen type 1 cross-linked C-telopeptide (CTX)/creatinine  in TPTX rats. After a single oral administration, PCO371 dose-dependently increased serum Ca and decreased Pi, with greater efficacy and longer-lasting effects than those of hPTH(1–84) (T1/2 and Tmax than hPTH(1–84) (−1)10−1 or more dose-dependently increased serum Ca to within the target therapeutic range, and maintained the Ca level within this range for at least 6 h after each administration from the first administration until the end of the study (−1) was observed at 0, 6 and 10 h after administrations of PCO371 on days 1, 7, 14, 22 and 29. Urinary Ca excretion after 4-week treatment was not increased by treatment with PCO371 up to 9 mg kg−1, even when the serum Ca had reached the target therapeutic range 2D3] levels increased significantly in rats treated with 9 mg kg−1 PCO371 (We next examined the effect of PCO371 on serum Ca and inorganic phosphate (PTH(1–84)  or hPTH(TH(1–84) . PharmacTH(1–84) . We thenhe study . In thisic range , possibl1 PCO371 , similar1 PCO371 . There w1 PCO371 .3 while maintaining serum Ca levels, and this has been clinically demonstrated by combining hPTH(1–84) with conventional vitamin D and Ca therapy−1 normalized the serum Ca to within or above the target therapeutic range, but was accompanied by considerable urinary Ca excretion 2D3, which then stimulates intestinal absorption of Ca; whereas in bone, PTH stimulates bone turnover and mobilizes Ca into the circulation112As a principal regulator of Ca homeostasis, PTH increases tubular reabsorption of Ca in the kidney and promotes renal excretion of phosphate. In blood, PTH stimulates activity of 25-hydroxyvitamin D2D3 production, and Ca release from the bone , oral PCO371 exhibited more robust and longer-lasting calcemic effects than did subcutaneous hPTH(1–34) or hPTH(1–84) in vivo. The duration of cAMP-signalling response induced by PCO371 in UMR-106 cells was not prolonged compared with PTH(1–34) or LA-PTH the bone . PCO371 the bone  and phosthe bone  in vivo,r LA-PTH . These r−1; In the present study, we used TPTX rats as an animal model for hypoparathyroidism, and demonstrated that complementing alfacalcidol therapy with PCO371 increased serum Ca to a level comparable with that produced by twice the dose of alfacalcidol without elevating urinary Ca excretion. Our results suggest that replacing conventional therapy for hypoparathyroidism with PCO371 is expected to provide three advantages: robust serum Ca control, low risk of hypercalciuria and a decrease in pill burden. However, we should bear in mind that rat skeleton is different from human skeleton, thus non-rodent animal models for studying human bone metabolism are needed to bridge the gap between animal studies and clinical trials. We therefore examined the effects of PCO371 on normal dogs, since dogs have been used to study the human skeleton because of their extensive basic multicellular unit-based remodelling. In a pharmacological study with normal dogs, serum Ca was significantly increased by single oral administrations of PCO371 (3.0−30 mg kg−1) showed a spiked pharmacokinetics profile and significantly increased both BMD and bone strength to levels comparable with those produced by hPTH(1–34) (0.9 nmol kg−1), although markers of bone formation and resorption were more increased by PCO371 than they were by hPTH(1–34).In the treatment of osteoporosis, in contrast to hypoparathyroidism, a sharp, transient increase in PTH levels is warranted to obtain a desirable anabolic effect on bone16The results obtained in the present study, however, do not support the benefits of oral PCO371 as an anti-osteoporosis drug; further studies at different doses and intervals or studies in other animals, such as dog and monkey, are needed to better evaluate the potential of the compound for the treatment of osteoporosis.Supra-therapeutic doses of hPTH(1–34) and hPTH(1–84) have been shown to increase the incidence of osteosarcoma in rat carcinogenicity studies. Because of this potential risk, hPTH(1–34) currently carries a black-box warning for the treatment of osteoporosis, although there appears to be no evidence of an increased risk of osteosarcoma in human subjects receiving the PTH peptideClass B GPCRs serve as receptors and mediators of various endocrine peptide hormones, such as PTH, calcitonin and GLP-1 refs , 33. Alt1718342 adrenergic receptor-Gs protein complexA general mechanism of peptide binding for class B GPCRs has emerged, termed the two-domain model. In this mechanism, the C-terminal region of the ligand binds to the N-terminal extracellular domain of the receptor, and the N-terminal region of the ligand interacts with the transmembrane domain to activate the receptor and induce intracellular signalling1920In conclusion, the small-molecule PTHR1 agonist PCO371 is a compound with potential to be developed and used as an orally available drug to treat hypoparathyroidism. PCO371 is also useful to more fully understand the mechanism of activation of class B GPCRs.Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification . Silica δ units). The following NMR abbreviations are used: s=singlet, d=doublet, q=quartet, m=multiplet, dd=doublet of doublets, brs=broad singlet. High-resolution mass spectrometry (HRMS) was performed on a Xevo G2-S Tof instrument (Waters).Chemical shifts are shown in parts per million  , 4-trifluoromethoxybenzoic acid , and diisopropylethylamine (DIPEA)  in anhydrous DMF (150 ml) was added 2--1,1,3,3-tetramethylisouronium hexafluorophosphate(V)  at 0 °C. The mixture was stirred at room temperature overnight, quenched by 0.5 mol l−1 HCl aq., and the product was extracted with EtOAc. The organic layer was washed with H2O and sat. aq. NaCl, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude solid was washed with EtOAc-hexane to afford tert-Butyl 4-Cyano-4-[4-(trifluoromethoxy)benzamido]piperidine-1-carboxylate (2)  as a white solid. 1H-NMR  δ: 7.84–7.81 , 7.29 , 6.25 , 4.14–3.97 , 3.34–3.24 , 2.61–2.47 , 1.90–1.81 , 1.47  δ: 165.54, 154.31, 152.15 , 131.39, 129.16, 120.79, 120.27 , 118.27, 80.57, 50.97, 40.00, 34.82, 28.37 −, 412.1484; found 412.1493.To a stirred mixture of 7 9H, s; . 13C-NMR2, 28.37 . HRMS (etert-butyl 4-cyano-4-[4-(trifluoromethoxy)benzamido]piperidine-1-carboxylate (2)  in EtOH (150 ml) was added 2 mol l−1 NaOH aq. (30.4 ml) followed by 30% H2O2 aq. (15.5 ml) at 0 °C. The mixture was stirred at room temperature for 1 h. Then 5 mol l−1 NaOH aq. (30.5 ml) was added at room temperature. The mixture was stirred at 50 °C for 2 h. This mixture was concentrated in vacuo and EtOAc and NH4Cl were added. The organic layer was washed with H2O and sat. aq. NaCl and dried over anhydrous Na2SO4. The organic layer was concentrated in vacuo to afford tert-butyl 2-[4-(trifluoromethoxy)phenyl]-4-oxo-1,3,8-triazaspiro[4.5]dec-1-ene-8-carboxylate (3)  as a white solid. 1H-NMR  δ: 10.61 , 8.02 , 7.37 , 4.18–3.96 , 3.54–3.37 , 2.05–1.89 , 1.60–1.53 , 1.51  δ: 188.89, 156.52, 154.83, 151.83, 128.80, 126.97, 121.13, 120.34 , 79.78, 70.49, 39.81, 32.54, 28.50 −, 412.1484; found 412.1502.To a stirred mixture of  9H, sl; . 13C-NMR4, 28.50 . HRMS (Etert-butyl 2-[4-(trifluoromethoxy)phenyl]-4-oxo-1,3,8-triazaspiro[4.5]dec-1-ene-8-carboxylate (3)  and 4 mol l−1 HCl in dioxane  in CH2Cl2 (68 ml) was stirred at room temperature for 3 h. This mixture was concentrated in vacuo, and 1 mol l−1 K3PO4 aq. was added. The product was extracted with EtOAc (300 ml)–EtOH . The organic layer was dried over anhydrous Na2SO4, and concentrated in vacuo to afford 2-[4-(trifluoromethoxy)phenyl]-1,3,8-triazaspiro[4.5]dec-1-en-4-one (4)  as a white solid. 1H-NMR  δ: 8.08 , 7.45 , 3.32–3.17 , 2.05–1.95 , 1.75–1.66  δ: 189.71, 153.33, 153.31, 130.81, 128.88, 122.19, 121.84 , 69.09, 42.00, 32.90 +, 314.1116; found 314.1118.A mixture of 6 2H, m; . 13C-NMR0, 32.90 . HRMS (E4)  and Et3N  in anhydrous CH2Cl2 (210 ml), 2-chloroethane-1-sulfonyl chloride  was added at 0 °C. The mixture was stirred at room temperature for 1 h under N2. This mixture was quenched by H2O, and the product was extracted with CH2Cl2. The organic layer was washed with H2O and sat. aq. NaCl, dried over anhydrous Na2SO4, and evaporated in vacuo. The crude residue was purified by column chromatography  to afford 2-[4-(trifluoromethoxy)phenyl]-8-(vinylsulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (5)  as a white solid. 1H-NMR  δ: 10.78 , 8.02 , 7.38 , 6.54 , 6.31 , 6.09 , 3.81–3.74 , 3.35–3.27 , 2.23–2.14 , 1.71–1.64  δ: 188.61, 156.96, 152.00, 132.99, 128.85, 128.49, 126.66, 121.15, 120.33 , 69.53, 41.81, 32.49 +, 404.0892; found 404.0893.To a stirred mixture of 2-[4-(trifluoromethoxy)phenyl]-1,3,8-triazaspiro[4.5]dec-1-en-4-one  , 1--5,5-dimethylimidazolidine-2,4-dione (9) , Pd(dba)2 , methyl dicyclohexylamine , and tri-tert-butylphosphine tetrafluoroboric acid adduct  in anhydrous NMP (7.6 ml) was stirred at 100 °C for 2 h under N2. The reaction mixture was diluted with EtOAc. The organic layer was washed with H2O, dried over anhydrous MgSO4 and evaporated in vacuo. The crude residue was purified by column chromatography  to afford (E)-1-{3,5-dimethyl-4-[2-({4-oxo-2-[4-(trifluoromethoxy)lphenyl]-1,3,8-triazaspiro[4.5]dec-1-en-8-yl}sulfonyl)vinyl]phenyl}-5,5-dimethylimidazolidine-2,4-dione (6)  as a colourless foam. 1H-NMR (DMSO-d6) δ: 11.75 , 11.17 , 8.09 , 7.55 , 7.49 , 7.15 , 7.01 , 3.67–3.59 , 3.29–3.19 , 2.39 , 1.96–1.86 , 1.70–1.62 , 1.35  δ: 186.31, 177.15, 157.59, 154.55, 150.50, 139.82, 137.25, 134.58, 131.87, 129.07, 128.21, 128.10, 127.65, 121.11, 119.84 , 67.65, 63.96, 41.65, 31.93, 23.36, 20.48 +, 634.1947; found 634.1969.A mixture of 2-[4-(trifluoromethoxyl)phenyl]-8-(vinylsulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one -1-{3,5-Dimethyl-4-[2-({4-oxo-2-[4-(trifluoromethoxyl)phenyl]-1,3,8-triazaspiro[4.5]dec-1-en-8-yl}sulfonyl)vinyl]phenyl}-5,5-dimethylimidazolidine-2,4-dione (6)  in DMF (15 ml), MeOH (100 ml), and MeCN (100 ml) was added 20% Pd(OH)2 on carbon (50% wet)  under N2, and this mixture was stirred at room temperature for 1 h under H2. The mixture was filtered and concentrated in vacuo, and dissolved in EtOAc. The organic layer was washed with H2O, dried over anhydrous MgSO4, and evaporated in vacuo. The residue was purified by column chromatography  to afford 1-{3,5-dimethyl-4-[2-({4-oxo-2-[4-(trifluoromethoxy)phenyl]-1,3,8-triazaspiro[4.5]dec-1-en-8-yl}sulfonyl)ethyl]phenyl}-5,5-dimethylimidazolidine-2,4-dione (PCO371)  as a white solid. 1H-NMR  δ: 9.37 , 7.93 , 7.73 , 7.35 , 6.94 , 3.88–3.81 , 3.53–3.45 , 3.25–3.19 , 3.07–3.00 , 2.40 , 2.19–2.10 , 1.78–1.70 , 1.47  δ: 187.73, 176.67, 156.89, 154.62, 151.95, 137.99, 135.80, 131.83, 128.98, 128.80, 126.63, 121.10, 120.30 , 69.27, 65.21, 47.43, 41.97, 32.72, 23.68, 23.00, 19.91 + 636.2103; found 636.2106.To a stirred solution of  , DIPEA  and 2-bromoisobutyric acid  in anhydrous DMI (13 ml) was stirred at 100 °C for 1 h under N2  was added, and the mixture was stirred at 100 °C for 1 h under N2. The mixture was cooled to room temperature, and MeOH (52 ml) and 5 mol l−1 NaOH aq.  were added. This mixture was stirred at 75 °C for 1.5 h and acidified with 1 mol l−1 KHSO4 aq. (pH 5) at 0 °C, diluted with H2O, and the product was extracted with EtOAc. The organic layer was washed with H2O, dried over anhydrous MgSO4 and evaporated in vacuo to afford compound 8 . This crude mixture was used in the next step without further purification.A mixture of 4-bromo-3,5-dimethylaniline (under N2 . Then ad2Cl2 (62 ml) and AcOH (62 ml), NaOCN  was added at room temperature. The mixture was stirred at room temperature for 3 h and poured into sat. aq. NaHCO3 (400 ml). The reaction mixture was basified with 5 mol l−1 NaOH aq. (pH 7–8), and the product was extracted with EtOAc. The organic layer was dried over anhydrous MgSO4 and evaporated in vacuo. The crude solid was washed with EtOAc-hexane and CH2Cl2-hexane, consecutively, and dried to afford 1--5,5-dimethylimidazolidine-2,4-dione (9) . 1H-NMR  δ: 8.64 , 6.96 , 2.44 , 1.45  δ: 176.50, 154.57, 139.91, 131.92, 128.47, 128.12, 65.11, 24.03, 23.68. −, 309.0239; found 309.0240To a stirred solution of the above crude mixture (5.79 g) in CH5 6H, s; . 13C-NMR, 23.68.  HRMS (ESin vitro and in vivo activities similar to that of hPTH(1–34). This compound was further optimized to improve oral bioavailability to yield a clinical candidate compound, PCO371.To identify small-molecule agonists of PTHR1, we used a cell-based high-throughput screening method employing LLC-PK1 cells (ATCC) expressing hPTHR1 (HKRK-B7 cells) and using urokinase-type plasminogen activator expression as a readout4 cells per 100 μl per well) and incubated overnight. The next day, the COS-7 cells were transfected by adding 10 μl of a mixture of hPTHR1, hPTHR2 or mutant hPTHR1 plasmid, rat PTHR1 or rat PTHR2 plasmid, or empty vector (0.1 μg per well), FuGENE HD Transfection Reagent , and Opti-MEM I Reduced Serum Medium  to each well. On the third day after cell seeding, the culture medium in the wells was discarded, and 50 μl of PCO371 free base and hPTH(1–34)  serially diluted in assay medium , and 2 mmol l−1 HEPES/McCoy's 5A medium, Life Technologies) was added to each well. The plate was placed in a 37 °C constant-temperature water bath for 20 min to allow the reaction to proceed. After incubation, the liquid in the wells was discarded, the wells were washed with the assay medium (100 μl per well), and the plate was frozen on dry ice. Next, 40 μl of 50 mmol l−1 HCl was added to each well, and the plate was stored in a freezer set at ≤−20 °C. The cAMP concentrations were measured by using a cAMP EIA kit . EC50 values were determined from three independent experiments in duplicate by a non-linear regression Emax model . The geometric mean of the EC50 values obtained in three experiments in duplicate was calculated using Microsoft Office Excel 2007. UMR-106 cells (ATCC) were cultured for 1 day, and the cells were treated with PCO371 (10 nmol l−1 to 0.1 mmol l−1) or hPTH(1–34) (0.1 pmol l−1 to 9 μmol l−1) in the same assay medium as that used for the COS-7 cells for 20 min at 37 °C, and incubations were terminated with 50 mmol l−1. The cAMP concentrations were determined using a cAMP EIA kit .COS-7 cells (ATCC) were seeded into a 96-well flat-bottomed plate (1.4 × 104 cells per 100 μl per well) and incubated overnight. The next day, the COS-7 cells were transfected by adding 10 μl of a mixture of hPTHR1 plasmid or empty vector (0.1 μg per well), FuGENE HD Transfection Reagent , and Opti-MEM I Reduced Serum Medium  to each well. On the second day after the cells were seeded into the plate, the culture medium in the wells was discarded, and 100 μl of 3H-myoinositol/0.1% BSA/DMEM adjusted to 111 kBq ml−1  was added to each well. On the third day after the cells were seeded into the plate, the culture medium in the wells was discarded, each well was washed with 100 μl of binding buffer , and 50 μl of PCO371 free base (0.1 μmol l−1 to 0.1 mmol l−1) or hPTH(1–34) (10 pmol l−1 to 0.1 mmol l−1) serially diluted with binding buffer (+LiCl) was added to each well. The plate was placed in a CO2 incubator set at 37 °C for 40 min to allow the reaction to proceed. After incubation, the liquid in the wells was discarded, and each well was washed with 100 μl of binding buffer (+LiCl). A 100-μl aliquot of 5% trichloroacetic acid (TCA) solution was added to each well, and the plate was left to stand for about 3 h on ice. The TCA solution in each well was recovered into a microtube, and then another 100 μl of 5% TCA solution was added to each well and recovered into the same microtube. Next, 400 μl water-saturated ether was added to each tube, the contents of the tube were thoroughly mixed, and the ether was aspirated. These three steps were repeated, and then 20 μl Tris-NaOH was added to each tube. A POLY PREP chromatography column  was packed with AG1-X8 Resin  and washed with 10 ml of 10 mmol l−1 myoinositol, and then the above sample was added. The column was washed with 10 ml of 10 mmol l−1 myoinositol, and then lipids were washed out by adding 8 ml of 5 mmol l−1 Borax/60 mmol l−1 NH4COOH. Inositol phosphates were eluted using 3 ml of 1 mol l−1 NH4COOH/100 mmol l−1 HCOOH, and then 10 ml of ULTIMA-FLO AF  was added. After that, the 3H radioactivity was measured using a liquid scintillation analyzer. EC50 values were determined from three independent experiments in triplicate by a non-linear regression Emax model . In addition, EC50 of PCO371 free base was defined as the concentration of PCO371 free base that achieves the EC50 for hPTH(1–34). The geometric means of the EC50 values obtained in three experiments in duplicate were calculated using Microsoft Office Excel 2007.COS-7 cells (ATCC) were seeded into a 96-well flat-bottomed plate (1.4 × 10Mutant hPTHR1 (hPTHR1-delNT) was constructed by removing N-terminal residues 23–181 and inserting a nine-amino acid influenza hemagglutinin (HA) tag joined to a tetraglycine linker (YPYDVPDYA-GGGG-) between Ala-22 and Glu-182.125I-labelled goat anti-rabbit IgG antibody (PerkinElmer) or by Cell-ELISA using Anti-HA mouse antibody  and Goat Anti-Mouse IgG HRP Secondary Antibody (Abgent).Wild-type human or mutant PTHR1s  were generated by PCR-mutagenesis, and the sequences were confirmed by replacement of non-functional N-terminal residues 93–101 (EDKEAPTGS) in the E2 region with a nine-amino acid HA tag (YPYDVPDYA) for HA binding. We confirmed that the cell-surface expression of mutant receptors on transfected COS-7 cells was comparable to that of HA-tagged wild-type hPTHR1 by means of an HA antibody binding assay using Anti-HA rabbit antibody (Sigma-Aldrich) and secondary 6 cells per dish) and incubated overnight. The next day, the COS-7 cells were transfected by adding 500 μl of a mixture of hPTHR1 plasmid (10 μg per dish), FuGENE HD Transfection Reagent  and Opti-MEM I Reduced Serum Medium  to each dish. On the third day after the cells were seeded into the dishes, the culture medium in the dishes was discarded, each dish was washed with 5 ml of Buffer A  without protease inhibitor cocktail , and then 750 μl of Buffer A  was added to each dish. The cells were recovered, and then homogenized on ice. The recovered cells were centrifuged , and the supernatant was recovered. The supernatant was centrifuged again , and the supernatant was removed leaving the membrane fraction, which was suspended in membrane buffer . The protein concentration was determined using Protein Assay Dye Reagent Concentrate . The membrane protein solution was stored in a freezer set at ≤−80 °C.COS-7 cells (ATCC) were seeded into 10-cm dishes  as the competing entity. Membrane protein solution was prepared by dilution with membrane buffer (+PI). PCO371 free base solution (0.1 μmol l−1 to 0.1 mmol l−1) or hPTH(1–34) solution (0.1 pmol l−1 to 1 μmol l−1) serially diluted with membrane buffer , membrane protein solution (10 μl) and tracer (10 μl) was added to a Multiscreen HTS 96 HV Opaque plate. After reaction for 1.5 h at room temperature, the reaction liquid was aspirated and the filter was washed with 200 μl of membrane buffer. The dried filter was recovered, and the 125I radioactivity was measured using a gamma counter. The experiment was performed independently three times in triplicate. Total 125I count rates (cpm) measured by the gamma counter were used as the primary data. Binding to hPTHR1 was evaluated by calculating the per cent specific binding from the following formula using Microsoft Office Excel 2007.The binding of hPTH(1–34) and PCO371 free base to hPTHR1 was evaluated by competition assay using Percentage specific binding was calculated by the following formula: equation (1)−1 hPTH(1–34) added was entered as the nonspecific binding (NSB), the mean count rate for unspiked samples was entered as the total count, and the total count minus the NSB was entered as B0.The mean count rate for the samples with 1 μmol lFos, Rn02396759_m1), osteocalcin , receptor activator of nuclear factor-κB ligand , osteoprotegerin  and sclerostin .UMR-106 cells (ATCC) were incubated with DMEM with 0.2% BSA for 4 h and were then treated with hPTH(1–34) or PCO371 for 1 or 6 h. At 1 or 6 h after adding test compounds, total RNA was isolated by using an RNeasy Plus Kit (Qiagen). cDNA was transcribed from the isolated total RNA by using TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific). mRNA levels were quantified by a real-time PCR system (Thermo Fisher Scientific). The relative quantity of transcripts was calculated by using the comparative threshold cycle (Ct) method and normalized to 18S ribosomal RNA as an internal control. The following cDNA-specific primers and probes were used: Fos  alone  or with ligands (1 μmol l−1 for hPTH(1–34), 0.1 μmol l−1 for LA-PTH and 0.1 mmol l−1 for PCO371) in the assay medium for 10 min, washed (t=0), incubated for 0, 30, 60 and 120 min (washout phase), and then further incubated for 5 min in the assay medium containing IBMX (2 mmol l−1). Incubations were terminated with 50 mmol l−1 HCl. The cAMP concentrations were determined using a cAMP EIA kit .The duration of cAMP signalling capacity of PCO371, hPTH(1–34) and LA-PTH  was assessed in a time-course experiment in UMR-106 cells (cAMP washout assay). The cells were treated with assay medium , βTC6 (GLP-1), HT-29 , CHO , and HEK-293 . Their antagonistic activity was measured in the presence of the control agonists shown in 50 and IC50 values of control agonists or antagonists were calculated by non-linear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve-fitting using the formula: Selectivity assays against class B GPCRs were performed by Cerep (France). Agonistic and antagonistic effects of PCO371 against a panel of 12 class B GPCRs  were examined at PCO371 concentrations of 1 and 10 μmol l50=EC50 or IC50, nH=slope factor. Agonistic activity of PCO371 at 1 or 10 μmol l−1 was calculated as a per cent of the control agonist response; intracellular cAMP accumulation by PCO371 × 100/intracellular cAMP accumulation by the control agonist at the EC50 concentration. Antagonistic activity was expressed as a per cent of the control agonist inhibition response , a value between 25 and 50% as weak to moderate, and a value<25% was considered as negative.http://www.aaalac.org). The studies were also carried out in compliance with the ‘Act on Welfare and Management of Animals' in Japan.In accordance with the Guidelines for the Care and Use of Laboratory Animals at Chugai Pharmaceutical, all animal studies were performed under the approval of the company's Institutional Animal Care and Use Committee. The company is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International  was subcutaneously injected into a pregnant Crl:CD(SD) rat (Charles River Laboratories) on its 18th day of gestation. At 48 h after the injection, the pregnant rat was euthanized, and the ulnae and radial bones were isolated from euthanized fetal rats. The long bones were placed on nylon mesh and covered with paraffin paper, and a floating culture in BGJb medium (Life Technologies) containing antibiotic–antimycotic  was carried out for 3 days at 37 °C in 95% air and 5% CO2 atmosphere. On the 4th day, the medium was replaced with fresh BGJb medium containing hPTH(1–34) (1 nmol l−1 to 1 μmol l−1) or PCO371 (0.1 nmol l−1 to 3 μmol l−1) or vehicle  and cultured for a further 4 days at 37 °C. Bone samples were then treated with 10% TCA (500 μl/bone) overnight at 37 °C. Radioactivity of the bone and the medium samples was measured45Ca release (%) was calculated by the following formula  using a DCS-600EX-IIIR bone densitometer , and the rats were divided into five groups, each with a similar mean value of BMD and body weight. Sample sizes were determined based on previous experiments with hPTHs and a published study with ibandronate and eldecalcitriol in our laboratoriesn=7–10). Then, OVX rats were treated once daily intravenously (vehicle or PCO371 at 3 or 10 mg kg−1) or orally (vehicle or PCO371 at 30 mg kg−1) or subcutaneously [vehicle or hPTH(1–34) at 0.9 nmol kg−1] for 12 weeks. Ten to twelve OVX rats were assigned to each dose group. Nine rats were assigned to the sham-operated control group and were treated with vehicle for PCO371. Ten per cent DMSO /10% Kolliphor EL (Sigma-Aldrich) in 10% hydroxypropyl-β-cyclodextrin /0.752% glycine  buffer was used as vehicle for PCO371, and phosphate-citrate buffer (pH 6.0) was used as vehicle for hPTH(1–34). To label bone-forming surfaces, tetracycline  was subcutaneously injected on Day 78 and calcein  was subcutaneously injected on day 83. Under isoflurane anaesthesia, jugular vein blood was collected immediately before and at 1, 2, 6, 10 and 24 h after administration on day 82 to measure plasma concentration of PCO371 or on day 84 to measure serum Ca levels . Urine was collected for 24 h from metabolic cages on days 84–85 to measure urinary collagen type 1 cross-linked C-telopeptide (CTX) by using the RatLaps ELISA System (Immunodiagnostic Systems). Under isoflurane anaesthesia, blood was collected on day 85 from the abdominal aorta to measure serum osteocalcin level by using the Rat Osteocalcin ELISA System . The lumbar spine and bilateral femurs were excised after 12 weeks of treatment. The first lumbar vertebra (L1), 3rd to 5th lumbar vertebrae (L3–L5), the right femur, and the right tibia were preserved in 70% ethanol to measure bone histomorphometry and BMD . Measurement of bone histomorphometry was performed by the Ito Bone Histomorphometry Institute . The second lumbar vertebra (L2) was wrapped in saline-soaked gauze and stored at −80 °C until measurement. Ultimate compressive strength (N) of the L2 vertebra was measured by using a mechanical testing machine .OVX was carried out in 32-week-old female Crl:CD (SD) rats (Charles River Laboratories). Twelve weeks after surgery, lumbar vertebral BMD (L3–L5) was measured −1 at 5 days after surgery were used for the experiment. The rats were divided into six groups, each with a similar mean value of serum Ca levels and body weight. TPTX rats were treated once orally  or subcutaneously (vehicle or hPTH(1–84)  at 9 nmol kg−1). Six TPTX rats were assigned to each dose group. The sample size was determined based on previous experiments and a published study with hPTHs and LA-PTH in our laboratoriesn=5–6). Vehicle for PCO371 was the same as in the OVX rat study, and phosphate-citrate buffer (pH 6.0) was used as vehicle for hPTH(1–84). Under isoflurane anaesthesia, jugular vein blood was collected immediately before and at 1, 2, 6, 10 and 24 h after administrations to measure serum Ca  and Pi levels .TPTX was carried out in 6-week-old female Crl:CD(SD) rats (Charles River Laboratories). Rats with serum Ca levels of <8.0 mg dli in TPTX rats (6-week-old) were evaluated at the dose of 3, 9.5 or 30 mg kg−1  or 9 nmol kg−1 (hPTH(1–34), s.c.) according to the same method as that described above.In another experiment, the effects of a single treatment with PCO371 or hPTH(1–34) on serum Ca and P−1 at 5 days after surgery were used in the following experiments. The rats were divided into four groups both in study 1 and 2, each with a similar mean value of serum Ca levels and body weight. Sample sizes were determined as described in the above single administration study. To evaluate the calcemic effects of PCO371 and hPTHs, we defined a target therapeutic range for serum Ca of 7.6–11.2 mg dl−1, which corresponds to the low normal to normal range of serum Ca levels10TPTX was carried out in 6-week-old female Crl:CD (SD) rats (Charles River Laboratories). Rats with serum Ca levels of <8.0 mg dl−1) or subcutaneously [hPTH(1–34) or vehicle at 9 nmol kg−1] for 4 weeks. Four rats were assigned to sham control, and treated with vehicle for PCO371. Vehicles for PCO371 and hPTH(1–34) were the same as in the OVX rat study. Under isoflurane anaesthesia, jugular vein blood was collected immediately before and at 6 and 10 h (for PCO371) or at 1 and 10 h (for hPTH) after administrations to measure the serum Ca levels on days 1 (the first dosing day), 7, 14, 22 and 29. Urine was collected for 24 h by using metabolic cages on days 29–30, and Ca excretion was calculated from urine volume and Ca level. Under isoflurane anaesthesia, jugular vein blood was collected 10 h after administrations to measure serum 1,25(OH)2D3 levels on Day 29 by using a 1,25(OH)2D3 RIA kit (Fujirebio). Four or five TPTX rats were assigned to each dose group. Serum and urine Ca levels were measured in the same way as in the single-dosing study. In another experiment, TPTX rats were treated subcutaneously once daily with hPTH(1–84) at 9 nmol kg−1, for 4 weeks. Serum Ca was measured immediately before and at 2, 6 and 10 h after administration on day 1 (the first dosing day), 7, 14, 21 and 28 using the same method as described above. Urine was collected for 24 h by using metabolic cages on days 28 and 29, and Ca excretion was calculated from urine volume and Ca level.TPTX rats were treated twice daily (at around 09:00 and 19:00 hours) orally  for 4 weeks. Five or six TPTX rats were assigned to each dose group. The vehicle for alfacalcidol was medium-chain triglyceride (Chugai Pharma Manufacturing). Under isoflurane anaesthesia, jugular vein blood was collected immediately before and at 6 and 10 h after administration to measure serum Ca levels on day 1 (the first dosing day), 8, 14, 22 and 28. Urine was collected for 24 h by using metabolic cages on days 28 and 29. Serum and urine Ca levels were measured in the same way as in the single-dosing study.TPTX rats were orally treated once daily  at 38 or 75 ng kg−1). Six dogs were assigned to each group. Vehicle for PCO371 was the same as in the rat studies. The sample size was determined based on previous experiments with hPTHs in our laboratories (n=6). Blood was collected from the cephalic vein immediately before and at 1, 2, 4, 6, 8, 10 and 24 h after administration to measure serum Ca levels .Marshall Beagle dogs  were treated once orally , or at a dose of 2, 6 or 18 mg kg−1 orally to 7-week-old female TPTX rats  or at 30 mg kg−1 orally or 3, 10 mg kg−1 intravenously to 55-week-old OVX rats as described above. Blood samples were collected at 15 and 30 min, and 1, 2, 4, 8 and 24 h after administration. Blood samples were also collected 2 min after intravenous administration in mature OVX rats. The concentrations of plasma PCO371 were determined by liquid chromatography–tandem mass spectrometry [LC-MS/MS (API-3200 (AB SCIEX), detection limit: 1 ng ml−1). Pharmacokinetics parameters  were calculated by a non-compartmental model using WinNonlin 6.4 (Pharsight). Oral bioavailability (BA) was calculated with AUCinf after oral and intravenous administration at a dose of 2 mg kg−1 by using the following equation PCO371 was administered at a dose of 2, 6 or 18 mg kg−1 to normal female 10-week-old Crl:CD(SD) rats (Charles River Laboratories). Four rats were assigned to each group. Blood samples were collected at pre-dose and at 2, 5, 10, 15, 30 and 45 min, and 1 and 2 h after administration. The concentrations of plasma hPTH(1–34) were determined by an enzyme immuno assay (EIA) system .hPTH(1–34) was subcutaneously injected at a dose of 3 nmol kg−1 in TPTX female 8-week-old Crl:CD(SD) rats (Charles River Laboratories). Blood samples were collected from the jugular vein at pre-dose and at 5, 10, 15, 20, 30 and 45 min and at 1, 2, 4 and 24 h after administration. Five TPTX rats were assigned to each dose group. The concentrations of plasma hPTH(1–84) were determined by an EIA system .hPTH(1–84) was subcutaneously injected at a dose of 30, 100 or 300 nmol kgP<0.05). The data displayed normal variance. The dose-dependent effect was tested with Williams's test. Dunnett's test was performed to determine which data differed from the control. Student's t test was performed to test for significant difference between two groups. SAS Preclinical Package (SAS Institute) software was used for statistical analyses for all other experiments. Quantitative results are represented as the mean±s.d. or s.e.m., unless otherwise noted.Statistical significance was assumed at the two-sided 5% level .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-19 and Supplementary Tables 1-4