Compared with hyperpolarized noble gas MRI, oxygen-enhanced lung imaging is a cost-effective approach to investigate lung function. In this study, we investigated the feasibility of free-breathing phase-resolved oxygen-enhanced pulmonary MRI based on a 3D stack-of-stars ultra-short echo time (UTE) sequence. We conducted both computer simulation and in vivo experiments and calculated percent signal enhancement maps of four different respiratory phases on four healthy volunteers from the end of expiration to the end of inspiration. The phantom experiment was implemented to verify simulation results. The respiratory phase was segmented based on the extracted respiratory signal and sliding window reconstruction, providing phase-resolved pulmonary MRI. Demons registration algorithm was applied to compensate for respiratory motion. The mean percent signal enhancement of the average phase increases from anterior to posterior region, matching previous literature. More details of pulmonary tissues were observed on post-oxygen inhalation images through the phase-resolved technique. Phase-resolved UTE pulmonary MRI shows the potential as a valuable method for oxygen-enhanced MRI that enables the investigation of lung ventilation on middle states of the respiratory cycle. MRI is a non-invasive imaging modality to produce 2D or 3D anatomical images without radiation damage. It has been widely used to image many organs, such as brain, kidney and liver at variable magnetic fields ,2. Howev131/129Xe or 83Kr as gaseous imaging contrast agents are being developed to investigate lung functions in healthy subjects and patients with various lung diseases [129Xe and 3He) is able to generate strong signals because of its extremely high polarization [5 times greater than its thermal equilibrium polarization [In addition to conventional proton MRI, multinuclear techniques that use Fluoride-containing gas, hyperpolarized diseases ,6,7. HypThe oxygen molecules are not only involved in gas exchange in the alveoli but also dissolve into the blood as well, accelerating the longitudinal relaxation of nearby tissues due to the paramagnetism property of oxygen. The oxygen molecules can reduce the T1 value reported firstly by Young et al.  and visuAnother challenge of pulmonary imaging is the motion effects generated from respiration and cardiac motion, especially in Cartesian k-space filling scheme which suffers serious motion artifacts ,19. HoweAlthough the stack-of-stars technique improved image quality, soft blurring artifacts still appear on pulmonary images resulting from respiratory and cardiac motion. To eliminate soft blurring, respiratory gating is often applied during MRI scans ,25,26. Aentation . In this study, we simulated mean PSE changes with flip angle using Bloch equation and further verified the simulation on both homemade T1 phantoms and healthy human. Finally, we investigated signal enhancement at both ends and middle phases of a respiratory cycle at optimized flip angle. Hence, we confirmed the trend of mean PSE change with flip angle from simulation by using in vivo experiment, and then illustrated the feasibility of the proposed self-gated free-breathing phase-resolved oxygen-enhanced pulmonary imaging method. The phase-resolved oxygen-enhanced MRI provided sharp details on both normoxia and hyperoxic images in lung vessels and diaphragm.Four healthy subjects  were enrolled following a protocol approved by the Institutional Review Board and informed written consent was obtained from each subject. Prior to each MRI scan, all subjects underwent pulmonary function test (PFT) to establish baseline global lung function. The spirometry measurements include forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), forced expiratory flow at 75% of FVC (FEF75), forced expiratory flow at 25% and 75% of the pulmonary volume and Peak. Percentage predicted values of these measurements were calculated based on Global Lung Function Initiative (GLI) Network .3 and agarose with varying concentrations [Stack-of-stars center-out golden-angle UTE pulse sequence was implemented in this study. Before the human experiment, the Bloch simulation with variable flip angle (1°~90°) was conducted in MATLAB2020a , assuming T1/T2* values reported in previous literatures ,20,31. Otrations . Before trations . 3, matrix size = 256 × 256 × 30, the number of points in each spokes = 294 , and spatial resolution = 1.94 × 1.94 × 8.3 mm3. For improving SNR, both hyperoxic and normoxic images were reconstructed at 1cm resolution along kz direction by non-uniform fast Fourier transform (NUFFT). Approximately 144,000 projections with golden angle increment (111.246°) were acquired to guarantee enough data for respiratory phase segmentation where 1600 views were used to segment average phase into four motion states.For in vivo experiment, imaging was performed with a free-breathing 3D radial stack-of-stars UTE pulse sequence with a hard RF pulse. Each spoke was acquired in a center-out fashion to achieve an ultrashort echo time, as signal enhancement efficiency increases with shorter echo time . The relIn traditional OE-MRI experiment, the imaging would be conducted twice for air and oxygen datasets acquisition. For golden angle radial technique, each successive spoke goes through the center of k-space and divides the largest remaining angular gap in half, making it possible to reconstruct image by grouping an arbitrary number of consecutive spokes. In our experiment, in order to reduce scan time, we conducted the imaging continuously during the whole experiment. The experiment protocol was shown in Experiments were performed on a whole-body 1.5T clinical scanner  equipped with an eight-channel abdominal coil. Subjects are required to keep supine position and breathe uniformly. The non-rebreather face mask was used to provide high oxygen concentration. Subjects were monitored for the occurrence of adverse events. The golden-angle radial acquisition ensures uniform coverage of k-space. The respiratory signal can be extracted from the k-space data. Following previous literature ,35,36,37Image reconstruction was performed offline in the Matlab programming environment. The aim of our study is to investigate the signal enhancement of the lung images with pure oxygen administration, so the normoxia and hyperoxic images were reconstructed based on the extracted respiratory signal, respectively, and the dynamic process is out of our study. Therefore, there is no need to subtract the oscillation resulting from signal change when extracting respiratory motion signal from the whole data set. Since the golden-angleacquisition scheme allows the retrospective sorting of k-space data, the dataset can be subdivided into arbitrary motion states. The common method for phase segmentation is based on amplitude of extracted respiratory signal, where the uniform binning uses the static threshold to select the motion states, and optimized binning uses an adaptive threshold. This approach performs well on the application of dynamic contrast enhancement (DCE) MRI. Nevertheless, for OE MRI, both air and oxygen volumes need to be processed so that a single threshold is not suitable to both as there may be different spokes for phase reconstruction of two data sets.For improving the accuracy of respiratory phase segmentation, sliding window technique was applied in this study. Technically, the minimum time resolution is new view . Figure https://www.slicer.org/, accessed on 29 October 2021), excluding aorta and blur near the diaphragm. Signal enhancement can be evaluated by PSE or mean PSE (MPSE), which is calculated on pixel level by dividing signal intensity of normoxia image from the signal difference of both images. Before PSE calculation, the high-resolution images were filtered to low resolution (1 cm × 1 cm) in each plane by Gaussian low-pass filter to improve SNR. The dataset was subdivided into two volumes based on oxygen concentration. Non-phase-resolved normoxia and hyperoxic image volumes were reconstructed by 800 spokes at the beginning and last part of the whole dataset, ensuring enough time for oxygen wash-in. Phase-resolved normoxia and hyperoxic images were reconstructed as explained in PSE maps were calculated using Equation (1), where To optimize parameters of UTE sequence, Bloch simulation was conducted before phantom and in vivo experiments. Since lung tissue density is much lower than other organs and air–tissue contact affects negatively SNR, it is important to improve signal intensity when imaging lungs. Considering simulation and phantom experiment results only, the flip angle  where the maximum difference occurred may be the best choice. Nevertheless, Non-phase-resolved Hyperoxic and normoxic images of average phase were reconstructed from the first 800 spokes and last 800 spokes, respectively. Eight planes were selected to cover lung region from anterior to posterior. Two representative subjects’ results are displayed in Over four subjects, the whole lung MPSE varied between 3.56% and 6.03% (4.96 ± 1.06%), consistent with Bloch simulation and results displayed in Repeatability was tested by conducting the experiment on same subjects on different days. Coefficient of Variation (CV) was calculated to evaluate the repeatability of the PSE calculation. States i, iii and iv have relatively better repeatability with lower coefficient of variation , compared with that of the average phase and state ii . We have demonstrated the feasibility of using 3D phase-resolved technique to investigate the oxygen-enhanced lung MRI. 3D radial UTE sequence with the stack-of-stars scheme was used in our study to generate 3D images of both air and pure oxygen breathing in healthy human. Since each spoke collects the center point of k-space, radial acquisition scheme has the inherent average effect, which enables it to be insensitive to respiratory and cardiac motion. Compared to the 2D IR-HASTE sequence, 3D UTE allows free-breathing and interpolation in kz direction allows us to reconstruct more planes with higher resolution . DespiteSince lung density and volume are changing while breathing, it is difficult to lock a specific state of a respiratory cycle. 3D UTE images in the average phase have residual blurring near diaphragm, as displayed in We also investigated a new data acquisition protocol in this study. For traditional oxygen-enhanced MRI experiments, the same image volume should be collected twice, which increases the scan time, especially for phase-resolved imaging which needs more spokes for visualizing separated phases. The sequence was conducted continuously throughout the whole experiment, including the air, wash-in process and 100% oxygen part. Therefore, simplified experiment operation and zero time interval among every part also reduced the experiment time, consequently bringing less bulk motion that is likely exacerbated by relatively long examination times used in traditional protocol. Since radial acquisition allows the reconstruction with arbitrary spoke groups, the continuous protocol with sliding window reconstruction also could be used to investigate the wash-in and wash-out time of oxygen-enhanced MRI, which is beyond the scope of the present work but would be investigated in the future study. Compared with average phase image, the phase-resolved MRI images provide clearer details, displayed in The MPSE of end-respiration is higher than other states, which is consistent with the results reported by previous authors . This coThis feasibility study still has several limitations. First, the volunteer group only includes healthy subjects. More investigations are needed to evaluate the clinical performance on patients with variable lung diseases. Secondly, the work focuses exclusively on the decomposition of respiratory motion as lung volume is affected mainly by respiration. On the other hand, the cardiac motion also influences the signal enhancement when breathing pure oxygen due to the enhancement by oxygenated blood. Fourier decomposition technique could be used in the future to evaluated the ventilation and perfusion influence separately . ThirdlyThe technique of phase-resolved oxygen-enhanced lung imaging could be used to investigate the gas exchange on patients by monitoring PSE map of middle states. Wash-in and wash-out time constants of oxygen respiration are also able to be measured using sliding window reconstruction based on radial acquisition scheme. In this paper, we have demonstrated the feasibility of generating reasonable hyperoxic images and PSE maps at different respiratory phases using self-gated free-breathing stack-of-stars UTE sequence. Compared to traditionally free-breathing images, phase-resolved oxygen-enhanced MRI provided more details on lung vessels and sharp edges on diaphragm. Additionally, the enhancement is easier to be observed on the anatomic image on separated motion states. The modified experiment protocol improved the efficiency of data collection, reducing scan time. Sliding window based reconstruction provided accurately segmented normoxia and hyperoxic image pairs. Therefore, the application of phase-resolved oxygen-enhanced imaging could be useful in tracking diseases related to ventilation. We also statistically confirmed that the MPSE increases from anterior to posterior region. Since this feasibility study only includes healthy volunteers, further study is needed to investigate the PSE map on patients at different respiratory phases.


Can preferred music listening improve following attentional and learning performances? Here we suggest that this may be the case. In Experiment The online version contains supplementary material available at 10.3758/s13423-022-02127-8. Nature paper, Rauscher et al. . All participants gave their written informed consent to participate in the study, which conformed to the standards required by the Declaration of Helsinki and was approved by the Ethics Committee of the University of Turin (Prot. n. 121724 – 01/03/18). Participants were not compensated for taking part in the experiment.Sample size (N = 18) was a priori determined to match the average number of participants involved in previous studies highlighting evoked-response potential (ERP) modulations driven by aesthetic appreciation . These musical pieces were chosen for their low familiarity for a non-expert public. Indeed, none of our participants could recognize or remember them. As assessed by previous studies  following forward versus backward, major versus minor and fast versus slow music listening. Furthermore, to avoid any sequence effect due to specific orders of presentation, musical pieces were played in a random order across the eight blocks. Therefore, the sequence of presentation of the eight musical excerpts was different for each subject.The eight musical stimuli we employed were composed of four classical music excerpts cropped to a 5-min length, and the same four excerpts played backwards. We selected the following classical music pieces see : DebussyEEG Mismatch Negativity Task). Furthermore, we recorded the resting-state electrophysiological activity during the last 2 min of passive forwards or backwards music listening. Resting state EEG data served as input for the analyses in the frequency domain . Subsequently, subjects were asked to express an aesthetic judgment using a Likert scale ranging from 1 to 9 . The software allowed us to select the frequency of the synthetic sounds composing the roving sequence. Low-pitch and high-pitch sounds had a frequency of 600 and 1,200 Hz, respectively. Sound sequences were presented by Eprime V2.0 presentation software . Each sound was played for 50 ms via loudspeakers. Loudness of sounds was set at a comfortable level (≅ 70 dB) and was kept equal across subjects and experiments.The EEG Mismatch Negativity Task is based on a roving auditory paradigm  were equal across pitch types  per run, each lasting 302.4 s (sounds were played at a 1-Hz frequency – block whole duration 288 s + 50 ms duration of each sound). While listening to the sound sequences, participants were asked to remain silent and look straight ahead at a central fixation cross on the computer screen. Differently from traditional oddball paradigms, in roving protocols each stimulus type has the same probability to occur, thus allowing dissociation of genuine effects of Bayesian perceptual learning from rarity-driven modulations. In roving paradigms, different stimuli (high-pitch and low-pitch tones in our case) can represent both Deviant and Standard stimuli Fig.  where thFor a detailed description of the mathematical computations please refer to the We first analysed behavioural results from the aesthetic judgment task. This allowed us to identify the preferred musical pieces for each subject. We divided the eight musical pieces into two categories , for each participant separately based on her/his ratings. The group-level effect of preference was evaluated by comparing the average ratings of the four preferred and non-preferred musical pieces. Thus, we performed a two-tailed t-test (N = 18) between the preferred and the non-preferred musical pieces. Moreover, to exclude that the results from subsequent analyses were influenced by musical stimulus objective features, rather than by subjective preferences, we compared average aesthetic judgements for forward versus backward music, major versus minor music, and fast versus slow tempo music. Therefore, three additional control two-tailed t-tests were performed to investigate whether key, tempo and direction significantly influenced aesthetic judgements.EEG data were collected during the eight runs of the EEG MMN task and during the last 2 min of music listening, with 32 Ag-AgCl electrodes placed on the scalp according to the extended International 10–20 system and referenced to the nose. Electrode impedances were kept below 5 kΩ. The electro-oculogram (EOG) was recorded from two surface electrodes, one placed over the left lower eyelid and the other placed lateral to the outer canthus of the left eye. EEG activity was recorded with a HandyEGG amplifier  and continuously digitized at a sampling rate of 1,024 Hz.Data collected during the EEG MMN Task were off-line pre-processed with Letswave6. Data were segmented into epochs of 1 s, including 200-ms pre-stimulus and 800-ms post-stimulus intervals. Epochs were band-pass filtered (0.5–40 Hz) using a fast Fourier transform filter  for Matlab . The EEG data corresponding to each of the eight musical excerpts were divided into 20 segments of 4 s registered at the end of each excerpt  as revealed by the two-tailed point by point t-test. The comparison of aesthetic ratings corresponding to musical stimuli with different objective features , revealed that, as expected, only the direction of presentation (forward vs. backward) significantly affected aesthetic rating , with higher ratings for forward  compared to backward music . Preferences were neither significantly affected by different keys  nor by different tempo . Therefore, subsequent control analyses run to exclude the possibility that electrophysiological results were affected by mere objective features of musical stimuli rather than by subjective preferences were restricted to the comparison of results following forward and backward music listening.The aesthetic ratings associated with subjectively preferred  versus non-preferred  musical stimuli were significantly different . Crucially, once MMN were grouped according to subjective aesthetic preferences (preferred vs. non-preferred), the same analysis highlighted several clusters of significance: the point-by-point t-test comparing MMN registered after preferred versus non-preferred music listening revealed a large significant cluster extending across frontal electrodes from 100 to 276 ms post-onset and including the MMN negative peak at 120–150 ms post-onset Fig. . The effIn order to test for the presence of a possible enhancement of the encoding of sensory surprise  after preferred music listening, compared to non-preferred music listening, we performed a point-by-point correlation analysis between trial-by-trial fluctuations and Bayesian Surprise, an information-theoretic index of sensory surprise and EEG amplitudes see  and OSM.Additionally, to explore possible differences in resting state brain oscillatory activity following preferred versus non-preferred music listening, we computed, for each participant, the average power spectrum registered during the last 2 min of passive music (or reversed music) listening. For each participant and for each channel, we obtained two curves (preferred vs. non-preferred music) indicating the average power across frequencies . This group of participants was entirely different from the sample of Experiment d = 0.613; α = 0.05; required power = 0.95).Sample size (N = 20) was a priori determined through a power analysis based on the effect size obtained in a pilot experiment identical to this behavioural experiment, involving ten additional participants and comparing HBD accuracies (ACC) between preferred and non-preferred conditions  and high (0.5 Hz) pass filters were applied to the signal to filter out noise. The number of R-wave peaks on the ECG trace for each interval in each block was then measured and recorded. ECG recordings were visually inspected for artifacts and the number of R-wave peaks was recounted manually, when necessary.Interoceptive accuracy was derived according to Schandry  as the d19 = 12.99; p < 0.001; Cohen’s d = 2.906) as revealed by the two-tailed point-by-point t-test. The direction of presentation of musical stimuli also affected AJs, with significantly higher aesthetic ratings for forward music  as compared to backward music . Similarly, mean appreciation was also significantly higher for minor key music  as compared to major key music , suggesting that AJs were also influenced by key. The comparison between AJs corresponding to fast versus slow tempo music revealed no significant difference . Coherently, direction and key were included as control factors in subsequent analyses (to verify whether HBD accuracies were modulated by these features).As in Experiment 19 = 2.87; p = 0.01; Cohen’s d = 0.141). To exclude the possibility that this result was influenced by heart rate variability across preference conditions, we measured the pulse for preferred and non-preferred music. Heart rate was comparable after listening to preferred  and non-preferred  music, with no significant difference between the two .To evaluate the effect of subjective preferences on HBD accuracies, as in Experiment 19 = 0.533; p = 0.596; Cohen’s d = 0.06), nor by the musical key .Additionally, as a further control, to exclude the potential effect of music stimuli basic features, we compared ACC collected after listening to forward versus backward music and after listening to major versus minor key music, independently from subjective aesthetic preferences. Different to the modulatory effect of aesthetic preferences, ACCs in the HBD task were significantly modulated neither by the direction of presentation  MMN responses to frequency deviant sounds were larger; (2) the correlation between single-trial N400 amplitudes and Bayesian Surprise was stronger; (3) resting-state upper alpha power was increased; (4) HBD accuracy was lower.The magnitude of MMN responses is considered to reflect the update of brain predictive representations triggered by surprising sensory inputs  was found to be associated with task performance and cognitive abilities


Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanism of apo- and holo-Tf’s influence on iron release was largely unknown.Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism of apo- and holo-Tf’s influence over iron release. We placed our findings in physiological context by further deciphering how hepcidin played a role in this mechanism as well.We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly binds to ferroportin, whereas apo-Tf directly binds to hephaestin. Only pathological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, and no amount of hepcidin disrupts the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin’s ability to rapidly internalize ferroportin compared to holo-Tf.These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin corporate to suppress iron release. We have established a more thorough understanding of the mechanisms behind iron release regulation with great clinical impact for a variety of neurological conditions in which iron release is dysregulated. Our group and others have shown that endothelial cells (ECs) of the BBB serve as reservoirs for iron before it is subsequently released into the extracellular fluid of the brain. Moreover this release is regulated by levels apo (iron free)- and holo (iron bound)- transferrin (Tf)7 in extracellular fluid. Using both in vitro6 and in vivo7 models, we have shown that increasing the ratio of apo- to holo-Tf, re ecting an iron deficient environment, stimulates iron release from ECs, whereas elevated holo-Tf relative to apo-Tf, reflecting an iron-replete environment, suppresses iron release. This feedback mechanism allows for regional specificity of iron uptake based on regional iron consumption and metabolic needs9.Regulation of iron uptake at the blood-brain barrier (BBB) is crucial for proper brain function. Detrimental alterations in brain iron homeostasis can lead to a variety of neurological conditions, including but not limited to neurodegenerative diseases 11, a ferroxidase that converts ferrous (Fe2+) to ferric (Fe3+) iron. Heph is required for both the stability of Fpn in the plasma membrane and the efflux of iron through Fpn11. Inversely, Fpn can be inhibited by hepcidin12, a pro-inflammatory peptide hormone, primarily secreted by the liver13 and in small amounts by astrocytes14. When hepcidin binds to Fpn, Fpn is ubiquitinated for internalization and subsequent degradation15. Simpson et al. found that, in addition to iron release, holo-Tf also decreases Fpn protein in EC culture models of the BBB6 but the mechanism is unclear. Conversely, it has been proposed that apo-Tf participates in interactions with ferroxidases such as Heph and ceruloplasmin to facilitate iron release18. In the present study, we have determined the differential interactions that apo- and holo-Tf have with Fpn and Heph to control iron release. Moreover, we demonstrate the impact that hepcidin can have on these interactions. By understanding the regulatory mechanism of iron release into the brain, numerous neurological diseases with iron uptake dysregulation can be better studied and potentially treated.Free iron is released from cells, including ECs, through ferroportin (Fpn), the only know iron exporter. Fpn function is aided by a number of proteins, including hephaestin (Heph)20. Brie y, iPSCs were seeded onto a Matrigel-coated plate in E8 medium  containing 10μM ROCK inhibitor  at a density of 15,000 cells/cm2. The iPSCs differentiation was initiated by changing the E8 medium to E6 medium  after 24 hrs seeding. E6 medium was changed daily up to 4 days before switching to human endothelial serum free medium (hESFM)  supplemented with 10nM bFGF  and 10 μM all-trans retinoic acid  and 1% B27 . After 48 hrs of no medium changes, cells were harvested and replated onto Transwell filters coated with collagen IV and fibronectin. Twenty-four hours after replating, bFGF and RA were removed from the medium to induce barrier phenotype. HEK 283 cells were maintained in Dulbecco’s modified Eagle’s medium  and supplemented with 10% FBS and 1% penicillin-streptomycin .Human endothelial-like cells (ECs) were differentiated from ATCC-DYS0100 human iPSCs as described previously21. Chamber slides  were coated with poly-D-lysine 2 hrs before HEK 293 cells were culture on the slides at a density of 15,000 cell/cm2. In order to remove an exogenous Tf, 24 hrs later the media was replaced with DMEM containing no FBS. Cells were exposed to apo- or holo-Tf  for 10 minutes and then washed to procced with PLA. PLA was performed the following day. Primary antibodies used were the following: myelin basic protein 1 , ferritin , Tf , TfR , Tf , Fpn , and Heph . Positive and negative controls used for assay optimization can be found in Supplemental Fig. 2. Imaging and analysis were performed using Revolve R4 microscope (Echo). The integrated density was calculated by summing the pixels from PLA signal and dividing by the field of view area. The integrated density of background from negative controls were subtracted from these values. To determine the integrated density per cell, this was then divided by the number of cells in the field of view. A minimum of three images were taken in different regions of the slides and then averaged for a single biological replicate. Image brightness was uniformly increased for the purposes of publication but not for quantification.PLA is a technique that precisely demonstrates if two proteins directly interact with one another. When two proteins are in close enough proximity to be interacting, the secondary oligomer probes ligate together, allowing for the amplification of the oligomers and resulting in a fluorescent signal. PLA was performed using a Duolink assay kit  according to the manufacturer’s instructions4 cell/cm2 in a 6-well plate. The following day, the cells were transfected with 1μg/well of the HA-tagged Fpn plasmid  using Lipofectamine™ 3000 Transfection Reagent .HEK 293 cells were seeded at a density of 7 × 1022. Briefly, magnetic beads were washed twice with PBS before adding lysates. The bead and lysate solutions were incubated with rotation for 30 minutes at room temperature. After washing with PBS, protein was eluted from beads by resuspending in non-reducing sample buffer and boiling at 90°C for 10 minutes. Magnet was used to isolate the magnetic beads from the protein solution, which was then reduced using 2 M DTT and then loaded for immunoblotting.In order to remove an exogenous Tf, the media was replaced with DMEM containing no FBS 24 hrs before the start of experiments. Cells were exposed to apo- or holo-Tf  for 10 minutes and then washed on ice with cold PBS twice. Chilled 100μl Co-IP lysis buffer  was added to each well. Cells were collected and incubated with rotation for 30 minutes at 4°C. Cell solutions were centrifuged at 14,000 × g for 20 minutes at 4°C. Supernatant was collected, and protein estimation was performed using Pierce BCA Protein Assay Kit . Approximately 1 mg of protein was used for Co-IP using anti-HA magnetic beads  or Protein G magnetic beads  complexed with anti-Heph antibody  according to manufacturer’s instructions23. Cells were gently lifted from the plate and collected in chilled glass mini homogenizers. Once homogenized, samples were spun at 1,500 × g for 10 minutes. The pellet was reserved and the supernatant was spun again at 10,000 × g for 10 minutes. The resulting pellet was combined with the pervious pellet and resuspended in RIPA buffer and 1x protease inhibitor cocktail. After immunoblotting was performed on the samples, the membranes were stained for total protein content using Ponceau S staining solution  to use as a loading control.Cells were washed with PBS three time before incubating with 200μl digitonin buffer 7. Protein was transferred onto a nitrocellulose membrane and probed for Fpn , DMT1 , Heph , TfR , Tf , HA tag , or cyclophilin B  as a loading control. Corresponding secondary antibody conjugated to HRP was used  and bands were visualized using ECL reagents (Perkin-Elmer) on an Amersham Imager 600 (GE Amersham). Cellular lysate samples were normalized to cyclophilin B protein as a loading control, and then subsequently normalized to an untreated control sample within each experiment. Membrane protein samples were stained with Ponceau S and normalized to total protein as a loading control.Samples were loaded onto a 4–20% Criterion TGX Precast Protein Gel (Bio-Rad)Statistical analyses were performed using Prism 9.2 software (Graphpad Software Inc.). Data from at least three independent biological replicates were averaged and are expressed as the mean ± standard error of the mean (SEM). One-way ANOVA with Tukey post-hoc analysis, two-way ANOVA with Sidak’s post hoc analysis, or unpaired t-tests were used to evaluate for statistical significance where appropriate. A p-value < 0.05 was considered significant.In the first series of experiments, we examined the effects of apo- and holo-Tf on the cellular levels of Fpn by incubating iPSC-derived ECs with increasing concentrations of either apo- or holo-Tf in hESFM for 8 hours. ECs were cultured onto Transwell inserts and apo- or holo-Tf was placed in the basal chamber to represent the brain-side. The ECs were collected and probed for various iron transport proteins. Incubations with holo-Tf decreased Fpn protein levels by 50% at concentrations as low as 0.1 μM  in media containing no FBS for 10 minutes prior to co-immunoprecipitation (co-IP). Regardless if the cells were incubated with either apo- or holo-Tf, Tf was co-immunoprecipitated with HA-Fpn , a highly sensitive method of detecting protein-protein interactions. HEK 293 cells were incubated with 0.25 μM of either apo- or holo-Tf in media containing no FBS for 10 minutes. Cells incubated with holo-Tf showed PLA signal when probing for a Tf and Fpn interaction . While cHepcidin is a well-known regulator and binding partner of Fpn, therefore we aimed to understand how the novel interaction between holo-Tf and Fpn could be impacted by physiological conditions that contribute to iron release. To do so, we used PLA to examine if hepcidin competed with holo-Tf for binding to Fpn. HEK 293 cells were co-incubated with 500 nM hepcidin and varying concentrations of holo-Tf –F. Hepci26, 25 nM, had no impact on the holo-Tf-Fpn interaction  to ferric (Fe3+) that can bind to apo-Tf and be utilized by cells. Numerous studies have shown that Heph is required to stabilize Fpn in the plasma membrane and to enable iron export29. We have replicated these findings, by demonstrating that Fpn and Heph can be co-immunoprecipitated from ECs. Furthermore, we demonstrate the novel finding that both apo- and holo-Tf independently are co-immunoprecipitated with Fpn and Heph. These results suggest that apo- and holo-Tf bind to Fpn and Heph in a complex of iron export proteins. In order to narrow down which protein holo-Tf bound to in the membrane that resulted in decreasing Fpn, we employed PLA. We found that holo-Tf directly interacts with Fpn, while apo-Tf does not. On the other hand, apo-Tf interacts with Heph, while holo-Tf does not, a finding that is supported in the literature30. It is hypothesized that apo-Tf binds to Heph to accept the ferric iron that Heph converts from ferrous iron. This stimulates the release of more iron as long as there is apo-Tf to accept it. Taken together these data suggest that apo- and holo-Tf differentially interact with iron export proteins, likely due to their structural differences31. The exact binding sites, conformation changes, and catalysts for these interactions are an exciting unexplored area that could pave the way for clinical manipulation. For example, as has been done experimental7, Tf could be infused to modulate iron accumulation in diseases in which it is dysregulated. Additionally, pharmaceuticals could be designed to facilitate or inhibit the endogenous protein interactions in an effort to correct brain iron accumulation.To complete the process of iron export, Fpn works in a complex with many proteins, including Heph13. Hepcidin is a pro inflammatory hormone peptide primarily secreted by the liver and upregulated in environments of inflammation and high iron levels32. Astrocytes34 and the choroid plexus36 have also been shown to secrete hepcidin, though in much smaller amounts that cannot account for total brain hepcidin levels37, suggesting much of the brain hepcidin comes from systemic levels when pathologically necessary, though this has not yet been proven. A number of groups have shown that astrocytic hepcidin reduces Fpn levels and subsequent iron release39. However, we have previously demonstrated that supraphysiological levels of hepcidin are not capable of blocking iron release from ECs4. These data suggest that hepcidin cannot be the sole regulator of iron release in the brain. In support of this notion, Enculescu et al. modeled iron levels, and when compared to their experimental results, the study found that hepcidin control over iron uptake was necessary, but not sufficient40. Once a secondary regulatory mechanism was added to the model, their experimental results aligned with the model40. Thus, our data directly support that hepcidin is not the sole regulator of iron release and indicate the additional regulators are apo- and holo-Tf.Prior to the discovery that elevated holo-Tf could suppress iron release, hepcidin was the primary focus of iron release regulation26, there was no interruption of the interaction between holo-Tf and Fpn. These findings suggest that hepcidin is only effective at controlling Fpn levels at levels consistent with inflammation or high iron. In observing competition between holo-Tf and hepcidin for Fpn binding, the internalization of Fpn was inhibited to determine if the competition was for binding site availability or rate of internalization. By preventing the internalization of Fpn, hepcidin had no impact on the interaction between holo-Tf and Fpn. This suggests that hepcidin internalizes Fpn faster than holo-Tf, which was confirmed by isolating membrane Fpn. Hepcidin reduces membrane Fpn by nearly 50% in 5 minutes, whereas holo-Tf only starts to reduce membrane Fpn at 60 minutes. On the other hand, no amount of hepcidin impacts the interaction between apo-Tf and Heph. These data offer the intriguing suggestion that if apo-Tf is present, it will bind to Heph even in pathological states and may be an explanation for iron accumulation in neurodegenerative disease. It has been postulated that in Alzheimer’s disease41 and Parkinson’s disease42 the brain may start as functional iron deficient, along with elevated levels of apo-Tf, which triggers increased iron uptake until the excess iron detrimentally damages the BBB and surrounding cells. The question remains however, if the binding of apo-Tf to Heph will continue to stimulate iron release in the presence of hepcidin.Our data offer an opportunity to explore the concept of regulation of iron uptake in general by hepcidin. We found that hepcidin competes with holo-Tf for binding to Fpn at low holo-Tf and high hepcidin concentrations. However, when there was more holo-Tf or less hepcidin present, this effect was reduced. Notably, when hepcidin was only present at physiological baseline levels43. Once endocytosed, the iron is removed and the resulting apo-Tf is released43. The communication of brain iron status via apo- and holo-Tf allows cells to signal their iron needs based on their iron consumption. Numerous studies have shown higher regional iron uptake that correspond to areas with higher iron needs45. Our pervious data suggest that as the apo-to holo-Tf ratio changes in the extracellular fluid, more iron is released locally from the BBB. In support of this notion are data showing CSF from iron deficient monkeys and iron chelated astrocytes increase iron release from cultured bovine retinal ECs (BRECs), while iron loaded biological samples resulted in decreased iron release6. These data have been replicated when cells are exposed to apo- or holo-Tf directly6 or when apo- or holo-Tf is directly infused into the brain7. In all studies mentioned here, apo-Tf increased iron release while holo-Tf decreased iron release.The model of apo- and holo-Tf regulation of iron release from ECs works as a feedback loop. As cells, such as neurons or astrocytes, need iron for metabolic processes, myelin synthesis, or dopamine synthesis, they take up holo-Tf through TfRThe data in this study expand the model for brain iron uptake by suggesting that apo-Tf stimulates iron release by binding to Heph to access exported free iron . Once lo46, thus a local source is needed. The data herein provide insights into a local regulatory process. This study is the first demonstration that apo- and holo-Tf differentially interact with Fpn and Heph to regulate iron release from ECs of the BBB. Moreover, we have identified a physiologically relevant dynamic between hepcidin and holo-Tf and their influence on membrane Fpn levels. Hepcidin interrupts the interaction between holo-Tf and Fpn by internalizing Fpn much faster than holo-Tf. Furthermore, we show that hepcidin does not interrupt the interaction between apo-Tf and hepcidin. These data suggest the mechanism of free iron release from ECs at the BBB and provide opportunity for further studies in neurological disease models to understand how this mechanism may be disrupted in each disease.The regulation of brain iron uptake is not influenced by systemic levels


An effective vaccine against the dengue virus (DENV) should induce a balanced, long-lasting antibody (Ab) response against all four viral serotypes. The burst of plasmablasts in the peripheral blood after vaccination may reflect enriched vaccine-specific Ab secreting cells. Here we characterize the acute plasmablast responses from naïve and DENV-exposed individuals following immunization with the live attenuated tetravalent (LAT) Butantan DENV vaccine (Butantan-DV). The frequency of circulating plasmablasts was determined by flow cytometric analysis of fresh whole blood specimens collected from 40 participants enrolled in the Phase II Butantan-DV clinical trial (NCT01696422) before and after  vaccination. We observed a peak in the number of circulating plasmablast at day 15 after vaccination in both the DENV naïve and the DENV-exposed vaccinees. DENV-exposed vaccinees experienced a significantly higher plasmablast expansion. In the DENV-naïve vaccinees, plasmablasts persisted for approximately three weeks longer than among DENV-exposed volunteers. Our findings indicate that the Butantan-DV can induce plasmablast responses in both DENV-naïve and DENV-exposed individuals and demonstrate the influence of pre-existing DENV immunity on Butantan DV-induced B-cell responses. Dengue is a mosquito-borne viral infection that persists as a major global health problem, with up to 100 million cases every year . Even thAll four dengue virus (DENV) serotypes are endemic in Brazil, triggering cyclic outbreaks every few years . AlthougHumoral and cellular immune responses play important roles in both DENV disease pathogenesis and protection . During After vaccination or infection, recently activated plasmablasts derived from lymphoid organs migrate towards the peripheral blood . This B-The live-attenuated tetravalent (LAT) Butantan DENV vaccine (DV) is a lyophilised version of TV003 vaccine, produced by the Butantan Institute, and they have been shown to be analogous vaccines . TV003 hClinical data and blood samples analyzed in this study were obtained from the first 40 participants from the São Paulo cohort, in Brazil, enrolled in the Phase II randomized, double-blind, placebo-controlled trial of Butantan-DV   performeHealthy male and nonpregnant female volunteers aged between 18 and 59 years old with unremarkable findings in physical examination, blood counts, serum chemistry, and negative serology for hepatitis B, hepatitis C, and HIV, who were randomly assigned into two double-blinded groups at a ratio of approximately 2:1. The vaccine group received one dose of the lyophilized LAT Butantan-DV (n=27). The vaccine consisted of 1,000 PFU of each attenuated serotype virus , rDEN3Δ30/31, and rDEN4Δ30) . PlaceboThese participants were tested for DENV exposure using plaque reduction neutralization test (PRNT)  at recruAll study participants were followed-up after vaccination  for clinical assessments, physical examination and blood collection as shown in The nAb response was measured by PRNT, as described previously  in serumSamples were tested with all four DENV serotypes. At baseline, seropositivity for each DENV serotype was defined as a nAb titer ≥1:10. Seroconversion at follow-up visits was defined as PRNT50 cut off (≥1/10) for DENV-naïve subjects and four-fold or higher increase in PRNT50 titer for DENV exposed subjects.The presence of vaccine viruses was assessed in all participants from 3 to 21 days after vaccine administration. Sera samples were incubated in Vero cells culture and the supernatant was analyzed through real-time RT-PCR, as previously described by Johnson and colleagues  and publ-/low , CD27high  and CD38high  cells) was determined by using a boolean gate on the live  CD3- , CD14-  and CD19+  cell population  or placebo (n=13) in the phase II clinical trial . No statThe median of nAb titers to DENVs 1 to 4 assessed before vaccination as well as at 28, 56 and 91 days following one dose of Butantan-DV are shown in + B cells in some cases. No statistically significant difference in the amount of plasmablast at peak was observed between DENV-naïve and -exposed vaccinees. In pre-exposed vaccinees we observed a similar percentage of plasmablast response regardless of the DENV serotype detected at baseline. Blood from two naïve and five pre-exposed vaccinees contained more than 3 x 103 plasmablasts per ml of blood at day 15 . This corresponds to a two- and three-fold increase, respectively, of their baseline levels. Since most DENV pre-exposed volunteers had experienced asymptomatic dengue, it was not possible to correlate the plasmablast response with the time since previous DENV infection.To characterize the dynamics of the plasmablast response induced by the Butantan-DV, as an indicator of vaccine-induced B-cell activation, we performed flow cytometric analysis of plasmablasts in fresh blood samples collected at several time points after immunization. - CD27+ CD38+ of CD19+ B cells at peak minus baseline) was greater in DENV-exposed participants  compared to DENV-naïve individuals  had higher nAb titers and a greater proportion of the vaccinees seroconverted following the first two vaccine doses; however, the vaccine had lower efficacy in naïve populations , 52. OveAlthough the median peak percentage of the plasmablast response induced by the Butantan-DV in both DENV-naïve and -exposed groups was comparable, pre-vaccination nAbs appeared to be associated with a greater increase in circulating plasmablasts following immunization compared to baseline plasmablast levels. This was notable among individuals who were seropositive for two or more DENV serotypes prior to vaccination. This finding validates the hypothesis that preexisting DENV immunity can influence the magnitude of the plasmablast response  and moduOur results also indicate that preexisting DENV immunity influences the persistence of plasmablasts in the peripheral blood. In DENV-exposed vaccinees, blood plasmablast concentrations returned to baseline levels 22 days after immunization. In contrast, the plasmablast expansion persisted beyond day 22 in DENV-naïve individuals, as previously shown . It is pAssessment of plasmablast responses following infection and vaccination has been used as an early biomarker of serologic responses . The detThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by CAPPesq, Research Projects Ethics Committee, protocols #55308 and #1.213.202 CONEP, protocol #155.880. The patients/participants provided their written informed consent to participate in this study.CS and DM collected, analyzed data and wrote the draft of the manuscript. PC performed experiments and wrote the draft of the manuscript. VA-S performed the statistical analysis. MR performed assays. MT performed experiments and analyzed data. CC and MM wrote sections and revised the manuscript. RG, LF and ZN recruited volunteers and organized the database for the clinical trial. CT and HT processed volunteer samples and organized the database. JK, RP and AP designed the clinical trial. DW and EK contributed to conception of the study and final manuscript revision. All authors contributed to the article and approved the submitted version.This work was supported by funding from the Wallace H. Coulter Center for Translational Research, Miami, FL, the São Paulo Research Foundation (FAPESP) 2015/03933-3, the National Council for Scientific and Technological Development (CNPq), the Brazilian Development Bank (BNDES), and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases .JK and RP are former employees of the Butantan Institute. AP is an employee of the Butantan Institute.The remaining 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.The reviewer (CP) declared a shared affiliation with the author (MT) to the handling editor at the time of review.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.


Cancer remains one of the most common causes of morbidity and mortality worldwide. Multiregional (MRCTs) and single-country clinical trials are two common approaches to support new oncology drug approvals internationally. However, systematic reviews comparing MRCTs with single-country trials for international oncology drug approval are lacking.ClinicalTrials.gov was used to retrieve all pivotal study information. We used an adapted version 2 of the Cochrane risk-of-bias tool for randomized trials (RoB 2) and Risk Of Bias In Non-randomized Studies - of Interventions (ROBINS-I) checklist to assess the risk-of-bias in randomized and non-randomized trials, respectively.We searched health agency websites to retrieve all approved oncology drugs from 2010 to 2022. P < 0.0001), whereas the median time interval for approval was shorter for MRCTs than for single-country standalone studies. The time gap for oncology drugs approved before 2015 was significantly longer than for those approved after 2015. The median timeline for approval in MRCTs involving 3 regions showed the shortest time-to-approval compared with MRCTs involving 4–5 and 1–2 regions. There was no significant difference in the time-to-approval among different tumor types and product types.A total of 48 new drugs and biologics  with initial marketing approval in the United States, European Union, Japan, and China were included. The reporting quality of MRCTs vs. single-country studies was similar. The median time interval for approval was significantly longer for MRCTs than for single-country bridging studies (1,399 vs. 975 days, The median time-to-approval of MRCTs was significantly longer than that of single-country bridging studies but shorter than that of single-country standalone studies, primarily involving 3 regions as the most frequent pattern and the shortest time-to-approval to operate MRCTs as a pivotal trial. Single-country bridging studies still provide essential supplements for international oncology drug approvals if MRCTs do not apply. Future studies should explore how to shorten the time-to-approval for MRCTs.https://www.researchregistry.com/browsethe-registry#registryofsystematicreviewsmeta-analyses/], identifier [1390].[ Cancer remains one of the most common causes of morbidity and mortality worldwide. In 2020, there were an estimated 18.1 million new cancer cases and 9.9 million cancer-related deaths , colorectum (9.4%), liver (8.3%), stomach (7.7%), and breast (6.9%) .United States, China, Japan, and the European Union (EU) are the four largest pharmaceutical markets worldwide for market share . The worThe incidence of new cancer cases is estimated to increase by 59.2% in Asia, 21.0% in Europe, 37.9% in North America, 65.6% in Latin America and the Caribbean, and 89.1% in Africa by 2040, which indicates enormous burden on the global healthcare system. In addition, although the global numbers of treated patients have increased by an average of 4% annually from 2015 to 2020  refers to a drug with no active moiety that the FDA has approved for other applications under section 505(b) of the Federal Food, Drug and Cosmetic Act . NCE andThe US, EU, Japan, and China each have their own accelerated drug approval pathway .Drug review and approval are based on safety, efficacy, and quality through preclinical, clinical, and chemical, manufacturing, and controls studies. The US FDA defines pivotal clinical trials are human clinical trial intended for obtaining regulatory approval or confirming the safety and efficacy of the drug’s intended use . Japan’sPivotal clinical trials may be at any stage—Phase 1 (Ph1), 2, or 3—given the level of evidence as agreed by health agencies to support the drug’s marketing authorization. For any initial marketing approval, there might be single- or multiple pivotal clinical trials as evidence.Multiregional are clinical trials conducted in more than one region under a single protocol that allow data from one country or region to help gain approval in another country or region . MultiplA region refers to a geographical region, country, or regulatory region. A regulatory region comprises countries with a standard set of regulatory requirements for drug approval  . In thisThis refers to clinical trials conducted in a single country, whereas multiple centers may apply for a single-country clinical trial. A single-country clinical trial may be a bridging study for any other pivotal clinical trials or a standalone confirmatory trial conducted in any single country or region. The decision of whether an MRCT or a single-country clinical trial shall apply is based on different factors, which is discussed in the following sections.There are typically four types of global clinical development strategies: simultaneous and synchronization for MRCTs and bridging and standalone for single-country studies .Multiple countries or regions join the same clinical development program at the same time from an early stage until pivotal MRCT.Any country or region that has not joined early stage MRCT but caught up with pivotal MRCT later.An additional study performed in a new region to provide pharmacokinetics (PK), pharmacodynamics, or clinical data on the efficacy, safety, dosage, and dose regimen of an innovative drug in the new region, which will allow extrapolation of foreign clinical data to the population in the new region . A bridgAny single-country clinical trial conducted independently from foreign clinical data. The clinical data obtained from the standalone clinical trial will typically support single-country drug approval only.There are several reasons why this systematic review has become imperative, which are mentioned below:1. Composite success rates in oncology have been treading down since 2015, with only 5.2% in 2021. As a result, the overall productivity of oncology research is among the lowest in the industry . This re2. Clinical trial cost comprises 30–40% of the overall drug research and development cost , and the3. The decisions to conduct MRCTs or single-country trials are often irreversible; therefore, a right-first-time approach is expected for the clinical development plan.4. The final drug approval by health agencies largely depends on sufficient evidence of drug safety, efficacy, and quality from either an MRCT or a single-country pivotal clinical trial.Systematic reviews comparing MRCTs with single-country trials for international oncology drug approvals are lacking. This systematic review is the first one to study the time to global oncology drug approval under MRCTs or single-country studies as pivotal trials.Electronic searches were conducted in the following databases for records from 1 January 2010 to 1 July 2022:–FDA website for approved drugs for oncology (cancer)/hematologic malignancies since 2006.–US FDA website.–EU European Medicines Agency (EMA) website.–Japan Pharmaceuticals and Medical Devices Agency (PMDA) List of Approved Products.–Japan PMDA Review Reports (Drugs).–China National Medical Products Administration (NMPA) website.–China NMPA Center for Drug Evaluation.–Clinical registry platform website.Keywords such as oncology, cancer, hematology, hematologic malignancies, pivotal, pivotal study (studies), pivotal trial(s), and confirmatory were used to retrieve pivotal clinical studies from approved oncology drugs.There were no language restrictions for the search results. The author has kept initial language search results from Japanese and Chinese websites.This review only includes initial marketing authorization approval with the first indication approved in the above countries; it does not include any new indication or line extension applications as subsequent filings. A detailed list of eligibility criteria is presented below.1. Drugs used for treating solid tumors or hematological malignancies that have obtained initial approval in the US, EU, Japan, and mainland China from 1 January 2010 to 1 July 2022 (cutoff date for data search and extraction).2. Pivotal studies that have been initiated for approved oncology drugs in the US, EU, Japan, and China.1.All generics for chemical drugs, biosimilars for therapeutic biologics, and medical devices.2.Solid tumor or hematologic malignancy drugs that have not been approved in the EU, Japan, or mainland China.3.Approved drugs that have been withdrawn from either the US, EU, Japan, or China.4.Approved drugs as line extension  from its first-approved non-oncology indication.5.Studies not confirmed as pivotal trials for approved oncology drugs in the US, EU, Japan, or China.6.Pivotal studies supporting indications that have been withdrawn from the US, EU, Japan, or China after initial approval.7.Pivotal studies that have not yet started.8.Pivotal studies initiated after drug approval as a post-marketing requirement or post-approval commitment studies.Time interval from the pivotal trial initiation to final drug approval in each country or region.Time gap between the last drug approval date in one country and the earliest drug approval date in another country within the US, EU, Japan, or China.ClinicalTrials.gov to conduct a quality assessment. A second reviewer (IO) independently verified the risk-of-bias and cross-checked the extracted data. Disagreements were resolved through discussion.Since this review did not involve any individual patient data listings, one reviewer (MZ) used partial parameters  from RoB 2  to assesFor each included pivotal trial, the following information was extracted:1.Trade name (only US trade name included): Extracted from US approval notifications as a unique identifier. Trade names from other marketed countries were not extracted because they were not directly relevant to the outcome.2.Company name [marketing authorization holder (MAH)]: Extracted from US approval notification. Specific MAH company name was not collected for products marketed in other countries because they were irrelevant to the study outcome.3.INN (identifier for the approved product): Extracted from US approval notification as a unique identifier, which is identical throughout other countries as a unique identifier for the marketed drug.4.Product type : Drug classification was extracted from agency review reports. This assumed the same drug classification applied throughout the US, EU, Japan, and mainland China.5.Date of initial approval (dd-mm-yy), not counting any supplemental application approvals: Extracted from the agency review report.6.Approval type , obtained from agency review report: Extract from agency review report.7.Approved indication : Extracted from agency review report for the approved indication. For multiple indications, information was also extracted from corresponding countries.8.Pivotal trial number [National Clinical Trial (NCT) number]: Used as a unique identifier for any pivotal study retrieved for every approval.9.Pivotal trial start date (dd-mm-yy): Extracted from the “actual study start date” reported in the corresponding NCT# record.10.ClinicalTrials.gov. The number of geographical regions, countries, or regulatory regions was counted.List of countries in the pivotal trial: Retrieved from the “Locations” field within records from 11.Pivotal trial type (MRCT/single-country), dependent on the list of countries involved in the pivotal trial: For a list of countries in the pivotal trial more than one, this was counted as MRCT. Pivotal trials in a single country were counted as a single-country trial.12.Pivotal trial stage (1–4): Retrieved from the “Phase” column from each NCT# record.13.Tumor type : Extracted from #7 approved indication.t-tests (t statistic) were used for two-group comparisons and analyses of variance (F statistic) for multigroup comparisons to evaluate differences between groups when the data were normally distributed and following assumptions regarding the homogeneity of variance; otherwise, non-parametric methods were used [Wilcoxon rank sum tests (Z statistic) for two-group comparisons and Kruskal–Wallis H tests for multigroup comparisons (H statistic)]. For multigroup comparisons with a P-value < 0.05, the p-values in multiple comparison tests were corrected using the Bonferroni method. P < 0.05 was considered statistically significant. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) Version 21.0 .Summary tables were used to present the information from pivotal trials. Descriptive statistics  were used to compute effect estimates. Shapiro–Wilk method was used for the normality test, and the Levene method was used for the homogeneity of variance test. Subsequently, Subgroup analyses were performed based on the following three variables:1.Product type .2.Tumor type .3.Number of different regions under MRCTs.Data analysis was performed by one reviewer (MZ).The search procedure described in A total of 48 new drugs and biologics and 215 pivotal clinical trials were included.A total of two approved drugs and eight pivotal trials are excluded.A total of 48 new oncology drugs were approved in the US, EU, Japan, and China from 2010 to 2022, among which 67% (32/48) were chemical drugs and 33% (16/48) were biologics . RegardiIn the US, 42% (20/48) of the initial approvals were granted accelerated approval by the FDA, which aimed for earlier drug approval to treat severe conditions and fulfilled unmet medical needs based on surrogate endpoints .In the EU, 17% (8/48) of the initial approval were granted conditional approval for life-threatening diseases, which include orphan medicines.In Japan, only one conditional approval was granted among the total 48 approved oncology drugs: lorlatinib (by Pfizer) for treating ALK fusion gene–positive unresectable advanced and recurrent non-small cell lung cancer with resistance or intolerance to ALK tyrosine kinase inhibitor(s).In mainland China, 29% (14/48) of the initial approvals were conditional, including two Hainan Special Access introduced after 2021 .In total, 215 clinical trials were retrieved as pivotal clinical study data for the above 48 oncology drugs approved in the US, EU, Japan, and China.Among the single-region MRCT pivotal trials, 67% (10/15) were conducted in mainland China and other APAC countries . For MRCT with two (geographic) regions, 65% (17/26) were conducted in the US or Canada and EU, 8% (2/26) in the APAC countries and US, and 27% (7/26) in the APAC countries and EU. MRCTs involving 3 (geographic) regions was the most frequent approach and comprised 39% (71/183) of all MRCTs. Among these, 94% (67/71) were conducted in the US, EU, and APAC countries; 5% (3/71) in the US, the EU, and South America; and 1% (1/71) in the US, the EU, and Africa.For all MRCT pivotal trials, simultaneous development strategy was the most frequent approach, applied in 80% of the trials 146/183; .For single-country pivotal trials conducted in China only, 73% (11/15) were bridging studies with PK elements that aimed to achieve extrapolation from global approval, among which 36% (4/11) followed the pre-approval approach  and 64% (7/11) followed the post-approval approach from global first approval. Only four pivotal trials were under the Ph3 stage as standalone development to verify safety and efficacy among the Chinese population, which were indicated for breast cancer, metastatic castration-resistant prostate cancer, multiple myeloma, and acute lymphoblastic leukemia.There were 27/80 (34%) randomized pivotal trials that were identified as low risk-of-bias, 11/80 (14%) as low-to-moderate, 17/80 (21%) as moderate, 15/80 (19%) as moderate-to-high, 7/80 (9%) as high risk, and 3/80 (4%) as unclear risk-of-bias . There wAmong the 80 randomized pivotal trials, 75/80 (94%) were MRCTs and 5/85 (6%) were single-country trials. Among the 49 non-randomized pivotal trials, 24/49 (49%) were MRCTs and 25/49 (51%) were single-country trials.Overall, the reporting quality of between MRCT and single-country was similar, both for randomized controlled trials (RCTs) and non-RCTs. Quality assessment range distribution for MRCTs and single-country trials is summarized in P < 0.0001). However, the difference in the timeline for approval was not statistically significant between MRCTs and single-country  (Bonferroni corrected P = 1.000) and single-country (bridging) and single-country  (Bonferroni corrected P = 0.065) trials , which was significantly longer than that in single-country (bridging) trials  trials .We chose 2015 as the cutoff year to explore the trend of global oncology drug approval gaps. The rationale is mainly due to the regulatory reform for drug and medical devices in mainland China since 2015. The time gap of approval in the US/EU/Japan/China for oncology drugs approved before 2015 was significantly longer than that for drugs approved after 2015. The median gap times in trials approved before 2015 and after 2015 were 2,032 and 1,037 days, respectively .P = 0.0840) or drug types (P = 0.4658)  , 11.P = 0.049). No statistically significant difference was noted between MRCTs involving 1–2 regions and those involving 3 regions (Bonferroni corrected P = 1.000) or between MRCTs involving 1–2 regions and those involving 4–5 regions (Bonferroni corrected P = 0.188)  .The main objective of this review was to explore the patterns of approvals across geographical regions for MRCTs vs. single-country trials. The reporting quality of MRCTs vs. single-country trials was similar. The longer time interval for oncology drugs with MRCTs than with single-country bridging studies was mainly because (1) 69% (22/32) of the single-country trials were initiated after global first approval for oncology drugs and (2) 88% (28/32) of the single-country trials were bridging studies with only PK elements, which were less complex than MRCT pivotal trials with both efficacy and safety elements.The lack of statistical difference between MRCTs and single-country  trials and between single-country (bridging) and single-country  trials may be due to low statistical power because of the small number of available single-country standalone studies. Nevertheless, the median time interval for approval in MRCTs was still shorter than that in single-country standalone studies, which demonstrates the value of MRCTs in accelerating global oncology drug approvals compared with single-country standalone studies. Moreover, clinical evidence gained from MRCTs shall support more regions than single-country standalone studies, which typically will support its country drug approval only.No significant difference was noted in the timeline of approval among different tumor or drug types, indicating that a similar approach to drug development strategy can apply to various types of oncology indications and different modalities of drugs. However, the sponsor shall consider those novel modalities, such as cell therapy products, in terms of their global development strategy and how MRCT shall apply. The only single-country standalone trial for the US was from a cell therapy treatment , which may be driven by the logistics challenge for MRCT conduction for such kind of products .The time gap of approval in the US/EU/Japan/China for oncology drugs approved before 2015 was significantly longer than that for drugs approved after 2015. This was mainly driven by a regulatory reform in China in 2015 when the local health agency allowed China’s participation in global development programs before the first global approval of the drug.The median timeline for approval in MRCTs involving 3 regions was significantly shorter than that for MRCTs involving 4–5 regions and shorter than that for MRCTs involving 1–2 regions, which suggests that MRCTs involving 3 regions might be the most efficient approach. The author assumes that the involvement of more regions within MRCTs shall bring more complexity and take more time than MRCTs involving fewer regions. The shorter median timeline for approval in MRCTs involving 3 regions than those involving 1–2 regions indicates that more variety of ethnic groups of patient enrollments may accelerate global oncology drug approvals. Among the MRCTs involving 3 regions, 94% (67/71) were conducted in the US, EU and APAC countries, covering the top 3 estimated numbers of new oncology cases.The combination of the US, EU, and APAC populations also reflects the global epidemiology profile for high cancer incidence and mortality rates in the APAC, EU, and US regions.The US has the highest number of accelerated approvals (42%), which is because of its earlier launch of drug accelerated development programs since 1992. On the other hand, the higher percentages of conditional approvals granted in China (29%) compared with the EU (17%) and Japan (2%) are mainly attributed to the drug and medical device regulatory reform since 2015 to shorten significant drug lags for globally available treatments that are not accessible in China , as wellThe US, the EU, and Japan have joined the same pivotal study development program more frequently since they are all listed as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) founding members with harmonized drug development standards. China followed through the synchronization approach by either joining an additional APAC-based MRCT involving 1–2 regions or other international MRCTs involving 3–5 regions. The relatively lower number of simultaneous (10/37) and a more significant number of synchronizations MRCTs (27/37) conducted in China were mainly due to the historic lengthy clinical trial approval duration before 2018, which took around 12–15 months to get Clinical Trial Application (CTA) clearance before China can join any MRCT  and EU (n = 21) than in China (n = 6) and Japan (n = 9) suggest that the US and EU have been more proactively seeking earlier clinical development for marketing approval, with the FDA/EMA seeming more supportive in accepting earlier phase of a clinical trial as a pivotal trial for oncology drug approval. The acceptance of earlier phases of pivotal trials will typically lead to accelerated approval in the US or conditional approval in the EU to fulfill unmet cancer treatment needs. On the other hand, the fewer number of earlier phases of a pivotal trial in Japan and China is mainly due to the later implementation of the conditional approval pathway in Japan (2017) and China (2020).Our review result is consistent with the trend of earlier trials from McKinsey’s report , which hWe also compared this review with two other reviews , 20. TheThis systematic review is the first to study the time-to-approval for global oncology drugs under MRCTs or single-country studies as pivotal trials. The search covered a wide range of new oncology drugs throughout the four largest geographic regions as representatives for global pharmaceutical markets in the last decade. The retrieved drugs and pivotal studies provided the most relevant evidence to reflect the current drug modalities  and assessment of pivotal clinical trials as the pillar for international oncology drug approvals by health agencies. In addition, the reporting quality of the included studies was assessed.However, the limitations of this review include the selection of four representative countries and regions that may not reflect the whole international drug approval landscape. As a result, some potentially relevant studies may have been missed during searches. The exclusion of pivotal trials that support subsequent approvals such as additional indications may also limit the extent to which such pivotal studies impact MRCTs and single-country clinical trials for oncology drug approval. The double counting for the same MRCTs for different country marketing approval may also cause confusion, although it does not impact result part and is necessary for data analysis for this review. Moreover, the impact of the COVID-19 pandemic worldwide and uncertainties in geographic conflicts  are not assessed in the current review.Multiregional is the most frequent pattern for pivotal trials to support global oncology drug approvals, regardless of chemical drugs or biologics and solid tumors or hematologic malignancies, based on the search results of this review. With the full implementation of ICH E17 from 2017, it is estimated that pharmaceutical companies shall keep up the trend of applying MRCTs for oncology drugs that aim for global approval. Although the time-to-approval is significantly longer for MRCTs than for single-country bridging studies, the significant drug gaps between first-country approval and the target country are still the major disadvantage of the single-country bridging strategy. Therefore, drug companies may consider single-country bridging studies as a supplementary strategy if target countries cannot join MRCT pivotal trials. There have been only five single-country standalone pivotal trials for global oncology approval since 2010, showing that this development strategy is not typical for global oncology drug approval. The single-country standalone strategy is usually considered if a bridging strategy is not applicable or if a standalone confirmatory trial is required by a health agency for the target country.Our review also shows that MRCTs involving 3 regions are potentially the most efficient approach for pivotal trials that support global oncology drug approvals. Time-to-approval has not shown a significant difference between MRCTs involving 3 regions and MRCTs involving 1–2 regions. However, MRCTs involving 3 regions shall include more diverse ethnic patient groups that support drug approvals for more countries, considering that the US, EU, and APAC regions comprise a majority of MRCTs involving 3 regions. Drug companies shall consider how to streamline clinical operations for MRCTs to shorten the time-to-approval for future global oncology development.Although the quality assessment shows similarities between MRCTs and single-country trials, the overall reporting quality of disclosed pivotal trials needs to be improved by sponsors and applicants for both MRCTs and single-country trials, regardless of randomized or non-randomized approach.From the health agency perspective, they have been accepting foreign clinical data started with adopting ICH E5 standards as the basis for a single-country bridging study to fill the gap between first approval countries and target countries. In addition, implementing ICH E17 standards from 2017 will further promote MRCTs as the best practice for global pivotal clinical trials in oncology. Meanwhile, the collaboration among international regulators may also allow patients with cancer to receive earlier drug access in other countries with the potential delay of drug approvals. For example, the US FDA has introduced Project Orbis through which pharmaceutical companies were asked to submit applications for clinically significant oncology drugs to participating countries simultaneously for concurrent review by their regulatory authorities. The US FDA has also implemented the Real-Time Oncology Review initiative to provide a more efficient review process to ensure earlier access to safe and effective patient treatment.Although the research is only focusing on global oncology drug approvals, the authors assume that the findings shall also apply to non-oncology drug approvals and pivotal clinical trials in general regardless of disease types and product types. The management of trials, namely, clinical trial operation may be the key factor for time to approval variances across different MRCTs. The selection of investigational sites, patient screening and recruitment procedure, site trainings and monitoring are some of the potential holding items that need further investigation in future research.The optimal design of pivotal trials may also be another key holding item. It may be driven by the establishment of more excellent uniformity of new global standards of treatment and a deeper understanding of variations in medical practice across different countries and regions.The median time-to-approval for MRCTs is significantly longer than for single-country bridging studies and shorter than for single-country standalone studies. In addition, there is no difference in the time-to-approval for different product types or tumor types, which indicates that the MRCT development strategy shall apply to all drug modalities for different tumor indications.The reporting quality is similar for MRCTs vs. single-country trials. MRCT involving 3 regions is the most frequent pattern to conduct MRCTs as pivotal trials, and it also demonstrates the shortest time-to-approval compared with MRCTs involving 4–5 and 1–2 regions. The US, EU, and APAC are the most frequent combination of MRCTs involving 3 regions. Single-country clinical trials as bridging studies still provide essential supplements if MRCTs do not apply. Single-country bridging studies and acceptance of foreign clinical data are recommended to shorten drug lags and potentially shorten drug approval time.Despite no significant difference between MRCTs and single-country standalone studies for time-to-approval due to the limited number of single-country standalone trials, MRCTs still show a shorter median time-to-approval than single-country standalone studies. Meanwhile, MRCTs show an advantage in supporting drug approval in multiple regions simultaneously, whereas single-country standalone studies can only support drug approval for their own countries in most cases. There is a trend of an earlier phase of MRCT pivotal trial  to support oncology drug approval internationally, especially in the US and EU. However, the accelerated or conditional approval based on Ph1 or 2 studies are still subject to confirmatory trial for conversion into regular approvals. The reporting quality of future pivotal trials should be improved. Future research should focus on how to accelerate MRCT pivotal trials to support global oncology drugs.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.MZ: protocol development, database searches, data extraction and quality assessment, data-analysis and interpretation, and drafting of the review. IO: protocol development, quality assessment, data-analysis and interpretation, and co-drafting of the review. KR: protocol development, initial database searches, data extraction, and data interpretation. All authors contributed to the article and approved the submitted version.


Increasing evidence demonstrates 3,4-methylenedioxymethamphetamine (MDMA)-assisted therapy (MDMA-AT) may be a safe and effective treatment for post-traumatic stress disorder (PTSD). There is growing interest in MDMA-AT to address a range of other health challenges. Chronic pain and PTSD are frequently comorbid, reciprocally interdependent conditions, though the possible role of MDMA-AT in treating chronic pain remains under-investigated. The present analysis examined the impact of manualized MDMA-AT on chronic pain severity among participants with PTSD who were enrolled in a Phase 2 clinical trial investigating MDMA-AT for PTSD (NCT03282123).n = 32) were drawn from a Phase 2 open-label study sponsored by the Multidisciplinary Association for Psychedelic Studies (MAPS). Multivariable analysis of variance (ANOVA) was utilized to compare pre- vs. post-treatment Chronic Pain Grade Scale (CPGS) values, adjusting for demographics . K-means clustering was then used to group the sample into three clusters to denote high (n = 9), medium (n = 11), and low (n = 12) baseline pain severity, and the same analysis was repeated for each cluster.Exploratory data from a subset of participants who completed chronic pain measures (n = 19) were women, 72% (n = 23) were white, and median age was 38 years [interquartile range (IQR) = 31–47]. Overall, 84% (n = 27) reported having pain, and 75% (n = 24) reported disability associated with their pain. Significant reductions in CPGS subscales for pain intensity and disability score, and overall CPGS severity grade were observed among participants in the highest pain cluster , and for pain intensity in the medium pain cluster  post- vs. pre-treatment.Among the 32 participants included in this analysis, 59% (Findings demonstrate a high prevalence of chronic pain in this sample of people with severe PTSD and that chronic pain scores among medium and high pain subgroups were significantly lower following MDMA-AT. While these data are preliminary, when considered alongside the frequency of comorbid chronic pain and PTSD and promising efficacy of MDMA-AT for treating PTSD, these findings encourage further research exploring the role of MDMA-AT for chronic pain. Increasing evidence demonstrates that MDMA-assisted therapy (MDMA-AT) may be a safe and effective treatment for post-traumatic stress disorder (PTSD) –4. To daChronic pain is a common, complex disease affecting approximately one-fifth of Americans  and up tMDMA-AT is a novel intervention under investigation for PTSD that has been granted Breakthrough Therapy status for this indication by the United States’ Food and Drug Administration in 2017 . In wellChronic pain and PTSD are both challenging to treat, and each contributes to significant dysfunction across all aspects of one’s life despite current available treatments , 20. Chrand psychological treatment, combining the pharmacological effects of MDMA with psychotherapy. However, there remains a lack of research and data on the effect of MDMA-AT on chronic pain conditions. The present study therefore aimed to explore the potential relationship between MDMA-AT and pain outcomes, drawing on exploratory pain data from a Phase 2 trial of MDMA-AT for severe PTSD  conducted by MAPS Public Benefit Corporation (MAPS PBC) (Data (December 2017 to August 2019) were drawn from a subset of participants enrolled in a multi-site Phase 2 open-label study known as MP16 (APS PBC) . This stThe primary outcome measure for the MP16 study was the Clinician Administered PTSD Scale according to DSM-5 (CAPS-5), a semi-structured interview addressing PTSD symptom clusters . ExploraThe primary outcome for the present analysis was chronic pain, as measured by the CPGS questionnaire, which was administered twice, at Preparatory Session 3 (prior to the first MDMA session), and at study termination which occurred 10–14 weeks after the final MDMA session. Participants were asked to base their responses at study termination on the period since the end of treatment. Secondarily, we looked for associations between chronic pain, childhood adversity and PTSD severity at baseline.Pain intensity and disability score subscales are combined with disability points score to assign a chronic pain severity grade (0-IV); individual subscales as well as overall severity grade have demonstrated validity and reliability (severity grade (0–IV), see The CPGS questionnaire is a 7-item self-report instrument that measures two dimensions of overall pain severity: pain intensity and pain-related disability, and is valid and reliable for measuring change in severity of chronic pain over time . Data eniability  . Bivariate linear and ordinal regression were used to estimate pre-treatment associations between CPGS values, and ACE score, CAPS-5 total severity, and demographic variables.n = 9), medium , and lowest pain  within the sample as measured using the CPGS subscale values of pain intensity, disability score, and the overall pain severity grade  using t-testing for bivariate association and two-way ANOVA for multivariable analysis, adjusting for demographics . We hypothesized that only participants reporting medium or high within-sample chronic pain at baseline would experience significant improvement post-treatment, therefore we repeated the same analysis for each CPGS cluster.To explore the association between MDMA-AT and chronic pain, we compared pre- vs. post-treatment CPGS n = 19) were female, 72% (n = 23) were white, and the median age was 38 [interquartile range (IQR) = 31–47] (n = 27) reported having pain, and 75% (n = 24) reported disability associated with their pain. Participants median ACE score was 4 (IQR = 3–7). No strong associations were found between pre-treatment CPGS values and ACE score, CAPS-5 total severity, or demographics  were significantly reduced post-intervention vs. baseline   and there was a trend to reduction for disability score and severity grade subscales in both bivariate and multivariable analysis (In bivariate  < 0.05) –7. A siganalysis , 8.pain intensity, disability score, and composite chronic pain severity grade were observed following MDMA-AT among participants in the highest pain cluster (n = 9), and for pain intensity only in the medium pain cluster (n = 11), suggesting that MDMA-AT may be associated with reduction in chronic pain and pain-related disability. Significant reductions in pain and pain-related disability among participants in the highest pain cluster may be due to higher baseline pain values allowing greater margin for improvement; there may also be a mechanism related to an amygdala-based threat response to higher levels of pain being positively impacted by MDMA-AT, since overlapping brain areas have been shown to be active in both pain related threat perception, and anxiety and fear-based threat perception as typified by PTSD  and are consistent with previous research in the intersecting fields of chronic pain and trauma, that indicate an association with adverse childhood experiences (ACE) , 62. Strt-testing and ANOVA for the whole sample and then separately for each of these stable clusters. The small sample size (n = 32) limited the power of these analyses, especially for the high pain cluster (n = 9) that demonstrated significant post-treatment reductions in pain values. Analysis was not conducted according to a hierarchical testing strategy, introducing the possibility for Type 1 error. Despite these limitations, participants in this sample were not subject to expectation bias regarding any positive impact of MDMA-AT on pain, since participants were recruited to the original study for experimental treatment of PTSD, whereas CPGS was administered and collected as exploratory data only. These data encourage further research with a larger sample in randomized, controlled trials in which the intervention is specifically investigated as a treatment for chronic pain.K-means clustering provided a simple and efficient method to define groups according to pre-treatment CPGS values representing high, medium, and low within-sample pain values, which allowed for analysis using regression, In this sample, MDMA was administered 3 times according to a flexible dosing schedule; however, the analysis was not powered to detect differences in total MDMA dose given that the vast majority of participants received the escalation dose (120 mg HCl for the second and/or third dosing session) and supplemental dose of 40 or 60 mg HCl. Future studies could address exposure-response analyses on change in chronic pain.10 = Extreme Change, rather than 10 = Unable to Carry on Any Activities in response to the question In the past 6 months how much has pain interfered with your daily activities rated on 0-10 scale? Therefore, while participants were aware the 10-point scale asked for a rating of severity of pain interference with daily activities, they might have interpreted extreme change as still allowing for minimal activity, rather than absolute inability to carry on any activities. This could theoretically lead to false elevation of baseline and post-intervention disability score, which could likewise falsely elevate total severity score.While the CPGS is a valid and reliable instrument, recall bias may limit accuracy of self-report responses. Further, the CPGS version administered to participants included one question (#5) that differed from the published version of the measure: on the 10-point scale, 10 was defined for this analysis as Associations between MDMA-AT and chronic pain may be influenced by confounding variables not examined in the present study, such as concurrent analgesic medications. Furthermore, participants in study MP16 were not selected for experiencing chronic pain, rather they were selected for having PTSD symptoms for at least 6 months, therefore some participants may have completed the questionnaire in reference to pain experienced for a shorter period. This limits generalizability of these exploratory data to true chronic pain conditions that are not comorbid with PTSD. In addition, different etiologies for pain could lead to differing pain or disability perceptions, or different secondary responses to the MDMA-AT for PTSD intervention. Within study population and within cluster distribution of pain by etiology could lead to further information and enhance future research protocols investigating MDMA-AT for chronic pain that include a mixed sample.This study included predominantly white participants, which limits the generalizability of the findings to other populations. Not all people are affected by chronic pain equally; most data indicate higher prevalence in racialized and marginalized populations including African American and Indigenous people, women, and individuals from lower socioeconomic backgrounds . FurtherThese findings demonstrate a high prevalence of pain in this sample of participants with severe PTSD, and that pain intensity, disability, and a composite pain severity index grade among those with the highest pain were significantly lower following MDMA-AT. While these data are limited and primarily hypothesis generating, they suggest that MDMA-AT may be associated with reductions in pain and pain-related disability among individuals with PTSD. Despite being derived from an exploratory endpoint, these findings when considered alongside the promising efficacy of MDMA-AT for treating PTSD and the transdiagnostic similarities between PTSD and chronic pain, support advancement of research exploring the role of MDMA-AT as a novel intervention for chronic pain and PTSD/chronic pain co-morbidity.http://maps.org/datause. All requests for raw and analyzed data are promptly reviewed by MAPS PBC to verify if the request is subject to any confidentiality obligations. Patient-related data not included in this publication were generated as part of clinical trials and may be subject to patient confidentiality. Any data that can be shared may be released via a data use agreement.The datasets analyzed for this study were accessed by request to the MAPS Public Benefit Corporation (MAPS PBC). Restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are, however, available upon reasonable request and with the permission of MAPS at The MP16 clinical trial was reviewed and approved by the Western Copernicus Group Independent IRB , Western IRB , University of California San Francisco Human Resource Protection Program IRB, University of Washington Human Subjects Division IRB, and University of British Columbia Providence Health Care Research Ethics Board. Participants provided their written and informed consent for participation in MP16.BY-K: substantial contributions to the conception and design of study MP16. DC and EA: data analysis, plan conception, and design. DC: initial draft manuscript preparation. EN, DC, and EA: analysis and interpretation of the results. PK, WS, DL, and BY-K: review of data analysis and interpretation. DC, EA, PK, WS, DL, and BY-K: critical and final review of the manuscript. All authors contributed to the article and approved the submitted version.