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Statistical Analysis | death, bleeding, PAD, stroke, peripheral artery disease, CHF, CAD, nonadherence, diabetes | CHF, BLEEDING, PERIPHERAL ARTERY DISEASE, CONGESTIVE HEART FAILURE (CHF), CARDIOVASCULAR DISEASE, HYPERLIPIDEMIA, STROKE, PAD, SECONDARY, ATRIAL FIBRILLATION, HYPERTENSION, ASCVD, CORONARY ARTERY DISEASE, CAD, DIABETES | The statistical analysis plan was prespecified before conducting the analyses but after database lock. Baseline characteristics, medical history, and selected concomitant medications are presented separately for participants who took enteric-coated or uncoated aspirin by randomly assigned aspirin dose. Discrete variables are presented as counts and percentages, and continuous variables as medians and IQRs. The χCumulative incidence at median follow-up for primary and secondary effectiveness end points and the primary safety end point was compared between participants taking enteric-coated or uncoated aspirin using unadjusted and multivariable Cox proportional hazards models. Cumulative incidence was calculated at median follow-up (26.2 months; IQR, 19.8-35.4 months) using the Kalbfleisch and Prentice cumulative incidence function estimator and comparisons reported as hazard ratios (HRs) with 95% CI. The Fine-Gray method was used to account for the competing risk of death for nonlethal secondary effectiveness and primary safety outcomes. Primary effectiveness end point, MI, all-cause mortality, and revascularization adjustment measures were randomly assigned treatment, age, current smoking, randomized follow-up strata, no internet use at randomization, race, ethnicity, history of coronary artery disease (CAD), MI, CABG, PCI, ASCVD, hypertension, hyperlipidemia, atrial fibrillation, congestive heart failure (CHF), peripheral artery disease (PAD), prior aspirin use, body mass index (BMI), diabetes, history of bleeding, and baseline P2YTo assess whether the association of aspirin formulation was consistent across randomly assigned aspirin dose for selected end points (primary effectiveness end point, all-cause mortality, and primary safety end point), we included an interaction term in the multivariable Cox proportional hazards models and report tests of the interactions terms. Counts and cumulative incidence at median follow-up with 95% CI are reported by randomized aspirin dose and aspirin formulation. Comparisons of aspirin formulation group, by randomly assigned aspirin dose, are reported as aspirin dose-specific HRs with 95% CI. Cumulative incidence figures reporting the primary effectiveness end point and the primary safety end point are provided by randomly assigned aspirin dose and aspirin formulation. The interaction models were adjusted for covariates prespecified as potential confounders and included age, sex, ethnicity, randomly assigned follow-up stratum (follow-up every 3 or 6 months), race, prior aspirin use, P2YTo account for nonadherence to the randomly assigned aspirin dose, we conducted a sensitivity analysis to assess if aspirin formulation modified the association of time-varying participant-reported aspirin dose with selected study end points (primary effectiveness end point, all-cause death, and primary safety end point). We constructed a Cox proportional hazards model with actual aspirin dose (self-reported at 81 mg, 325 mg, not taking, or other) as a time-varying exposure, an interaction term of time-varying aspirin dose and aspirin formulation reported at baseline, and selected covariates. The HRs were interpreted as the association of current aspirin dose with outcomes. The key assumption for this model was that the decision to switch doses was made for reasons unrelated to the outcome of interest (ie, the primary end point of death, MI, or stroke), beyond the baseline adjustment measures. The adjusted models included age, race, ethnicity, prior aspirin dose, prior MI, prior PCI, history of atrial fibrillation, no internet use at randomization, history of bleeding, and baseline P2YIn an exploratory analysis, we also tested whether there was an interaction with an acid reducing medication (ARM) by formulation of aspirin used. Multivariable Cox proportional hazards models were used to assess whether the association of enteric coating with prespecified end points was modified by the use of ARM. In this analysis, primary end point, nonfatal MI, all-cause mortality, and revascularization adjustment measures were randomly assigned treatment, proton pump inhibitor medication, age, current smoking, randomized follow-up strata (3-month or 6-month intervals), no internet use at randomization, race, ethnicity, history of CAD, MI, CABG, PCI, cardiovascular disease, hypertension, hyperlipidemia, atrial fibrillation, CHF, PAD, prior aspirin use, BMI, and baseline P2YAll hypothesis tests were 2-sided, and | PMC10551818 |
Results | PMC10551818 | |||
Demographic Characteristics, Medication Use, and Medical History | Of 15 076 participants enrolled in the ADAPTABLE study, 10 678 (70.1%) reported the aspirin formulation used (median [IQR] age, 68.0 [61.3-73.7] years; 7285 [68.2%] men and 3393 [31.8%] women; 900 [8.4%] Black or African American, 8965 [84.0%] White, 482 [4.5%] other race (Asian, American Indian or Alaska Native, multiple race, or other), and 331 missing race data or preferred not to say; 270 [2.5%] Hispanic, 10 051 [94.2%] non-Hispanic, and 354 [3.3%] did not respond to ethnicity query) ( | PMC10551818 | ||
Participant Demographic and Presenting Characteristics, Medical History, and Medications | bleeding, PAD, ICH, peripheral artery disease, CHF, COPD | BLEEDING, MYOCARDIAL INFARCTION, PERIPHERAL ARTERY DISEASE, CARDIOVASCULAR DISEASE, INTRACEREBRAL HEMORRHAGE, GI TRACT BLEED, CVD, CHRONIC OBSTRUCTIVE PULMONARY DISEASE, COPD, PAD, BLEEDING DISORDER, CONGESTIVE HEART FAILURE, CHF | Abbreviations: ARM, acid reducing medication; BMI, body mass index (calculated as weight in kilograms divided by height in meters squared); CABG, coronary artery bypass graft; CHF, congestive heart failure; COPD, chronic obstructive pulmonary disease; CVD, cardiovascular disease; EHR, electronic health record; GI, gastrointestinal; ICH, intracerebral hemorrhage; MI, myocardial infarction; PAD, peripheral artery disease; PCI, percutaneous coronary intervention.Other includes Asian, American Indian or Alaska Native, multiple race, or other.History of bleeding includes GI tract bleed, ICH, and bleeding disorder. | PMC10551818 |
Diagram of Patient Flow in the Analysis | Participants were randomly assigned to aspirin dose but not to aspirin formulation, which was at the discretion of the participant. For several analyses, participants were grouped by aspirin formulation regardless of aspirin dose (shown at the bottom of the figure). Regardless of aspirin dose, there were 7366 total participants taking enteric-coated aspirin and 3312 taking uncoated aspirin. | PMC10551818 | ||
Effectiveness Outcomes | Within the enteric-coated aspirin cohort, the primary effectiveness end point occurred in 297 participants (cumulative incidence at median follow-up 6.6%) in the 81-mg dose group and 246 participants (7.2%) in the 325-mg dose group (adjusted hazard ratio [AHR], 1.13; 95% CI, 0.88-1.45) ( | PMC10551818 | ||
Cumulative Incidence of Study End Points by Aspirin Formulation Type and Randomly Assigned Dose of Aspirin | MYOCARDIAL INFARCTION | Abbreviations: GI, gastrointestinal; MI, myocardial infarction.Cumulative incidence is reported at median follow-up, 26.2 months from randomization.Adjustment variables include age, sex, ethnicity, strata, race, prior aspirin use, P2Y | PMC10551818 | |
Cumulative Incidence of the Primary End Point (All-Cause Death, Myocardial Infarction [MI], or Stroke) by Randomly Assigned Aspirin Dose (81 or 325 mg) and by Self-Selected Aspirin Formulation (Enteric-Coated or Uncoated) From Participant Self-Reported, Electronic Health Record, and Claims Data | AHR indicates adjusted hazard ratio. | PMC10551818 | ||
Safety Outcomes | bleeding | BLEEDING | Overall bleeding requiring blood product transfusion (major bleeding) was low in ADAPTABLE. Across the 4 cohorts, there was no significant interaction of major bleeding by aspirin dose or formulation ( | PMC10551818 |
Cumulative Incidence of Bleeding Accounting for the Competing Risk of Death by Randomly Assigned Aspirin Dose (81 or 325 mg) and Self-Selected Aspirin Formulation (Enteric-Coated or Uncoated) From Participant Self-Reported, Electronic Health Record, and Claims Data | bleeding | BLEEDING | Major bleeding was defined as bleeding requiring blood product transfusion. AHR indicates adjusted hazard ratio. | PMC10551818 |
Sensitivity Analyses | death | EVENT | In a sensitivity analysis of time-varying self-reported aspirin dose, reflecting the association of aspirin dose at the time of the event, aspirin formulation, with or without covariate adjustment, did not modify the association of self-reported aspirin dose with the primary effectiveness outcome, all cause death, or the primary safety outcome (eTable 4 in | PMC10551818 |
Discussion | GI tract bleeding, bleeding | EVENTS, SECONDARY, ASCVD, BLEEDING | In this post hoc secondary analysis of ADAPTABLE, a large multicenter, pragmatic, randomized clinical trial, we evaluated the association of aspirin formulation (enteric-coated vs uncoated aspirin) with the effectiveness and safety of aspirin in secondary prevention of ASCVD. The results of this analysis did not show any difference in the effectiveness or safety outcomes analyzed by formulation of aspirin consumed regardless of the dose of aspirin participants were randomly assigned. While prior pharmacodynamic studiesDue to reduced aspirin bioavailability and the possibility of limited cardiovascular protection, the value of enteric-coated aspirin has been called into question during the last few decades. Cox et alAcid-reducing medications, such as proton pump inhibitors and histamine type 2 receptor antagonists, used to buffer aspirin within the stomach, have been shown to affect dissolution of the enteric formulations of aspirin by altering the pH, composition, and ionic strength in the stomach.In terms of the safety profile, enteric coating has been postulated to have better protection against GI tract bleeding and other major bleeding events.When major bleeding was evaluated within formulation cohorts, there was no association with aspirin dose among participants using uncoated aspirin and a small but significant association among patients using enteric-coated aspirin at 325 mg compared with 81 mg. The lack of bleeding difference in the participants using uncoated aspirin may account for the apparent lack of difference in major bleeding seen in the overall ADAPTABLE trial. The results of the present study for the enteric-coated aspirin cohort differed from those of other trials in which no difference in major bleeding was noted; however, in those trials, formulation of aspirin used was not mentioned.It has been proposed that the coadministration of ARM with enteric-coated aspirin would decrease the frequency of bleeding. However, we were unable to demonstrate a clinical interaction between ARM and the presence of enteric coating associated with either major bleeding or GI tract bleeding in this analysis. | PMC10551818 |
Limitations | bleeding, ASCVD | MINOR, BLEEDING, ASCVD | This study has limitations. First, formulation of aspirin was participant determined, and participants reported only at baseline, with only 70.1% of the participants reporting the formulation of the aspirin they were using. The determination of whether a participant took the reported medication or was adherent to it was not captured after baseline. This assumption of which formulation of medication the participants were taking is an act of trust, as patients could have switched during the study on their own or with the advice of their physician. This would lead to a false conclusion if a large number of participants switched formulations of medication during the study. Second, the use of enteric-coated aspirin was not part of the randomization stratification; thus the data must be treated as though from an observational study. Because the participants decided which formulation of aspirin they would use, there may be unknown confounders leading to the choice of aspirin formulation used by the participant. For instance, a well-known leading brand of aspirin has a large number of their aspirin products, particularly cardiovascular products, packaged with the phrase “safety coating” included. However, adjustments were applied to the data to attempt to control for known confounders although unknown confounders may still affect the results. Third, there are no pharmacokinetic or pharmacodynamic data demonstrating whether enteric coating had any association with serum aspirin levels or platelet inhibition. Fourth, we did not collect detailed data on bleeding outcomes. It is unknown whether minor GI tract or other bleeding was reduced by either enteric coating or the dose of aspirin consumed. Finally, despite attempts to enroll a diverse population of patients with ASCVD, the enrollment of women and historically underrepresented minority groups lagged, so generalizability of the results remains questionable. | PMC10551818 |
Conclusions | bleeding, ASCVD | SECONDARY, ASCVD, BLEEDING | In this post hoc secondary analysis of data from the ADAPTABLE randomized clinical trial, there were no significant differences in the primary effectiveness or safety end points between enteric-coated aspirin and uncoated aspirin among participants with established ASCVD although a reduction in bleeding with enteric-coated aspirin cannot be reliably excluded. More research is needed to confirm whether enteric-coated aspirin formulations or newer formulations will improve ischemic and bleeding outcomes among patients with ASCVD. | PMC10551818 |
Background/Objective | centre-involving diabetic macular oedema | PROLIFERATIVE DIABETIC RETINOPATHY (PDR) | The prokinetic levosulpiride elevates vasoinhibin levels in the vitreous of patients with proliferative diabetic retinopathy (PDR) suggesting clinical benefits due to the anti-vasopermeability and anti-angiogenic properties of vasoinhibin. We investigated the biological activity of levosulpiride in centre-involving diabetic macular oedema (DME). | PMC10858020 |
Patients/Methods | centre-involving | Prospective, randomized, double-blinded, dual-centre, phase 2 trial in patients with centre-involving DME orally treated with placebo ( | PMC10858020 | |
Results | Levosulpiride improved changes from baseline in best-corrected visual acuity ( | PMC10858020 | ||
Conclusions | centre-involving | Oral levosulpiride for 8 weeks improved visual and structural outcomes in patients with centre-involving DME by mechanisms that may include intraocular upregulation of vasoinhibin and downregulation of VEGF and PlGF. Larger clinical trials evaluating long-term efficacy and safety are warranted. | PMC10858020 | |
Subject terms | PMC10858020 | |||
Introduction | Diabetic retinopathy, centre-involving, visual impairment, microvascular complications, vasopermeability, vision loss, loss of vision, diabetes | LEAKAGE, DIABETIC GASTROPARESIS, DIABETIC RETINOPATHY, DIABETIC MACULAR OEDEMA, LOSS OF VISION, DIABETES | Diabetic retinopathy (DR) and diabetic macular oedema (DME) are microvascular complications causing visual impairment in diabetes. Leakage from retinal capillaries in DME produces the accumulation of extracellular fluid and proteins that alter the structure and function of the macula and may lead to permanent loss of vision if untreated. Vascular endothelial growth factor (VEGF) isoforms, including placental growth factor (PlGF), are major vasopermeability factors in DME, and intravitreal agents blocking VEGF and PlGF have become a first-line of treatment for centre-involving DME with vision loss [Vasoinhibin, a fragment of the pituitary hormone prolactin (PRL), inhibits the permeability and growth of blood vessels [Levosulpiride is a well-tolerated, dopamine D2 receptor blocker used as a prokinetic drug to treat diabetic gastroparesis [ | PMC10858020 |
Patients and methods | MAY, RETINA, DEL | This is a phase 2, prospective, double-blinded, randomized clinical trial conducted from May 2017 to November 2022 at two sites in Querétaro, México (Instituto Mexicano de Oftalmología and Instituto de la Retina del Bajío) and sponsored by the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT, grants 247164, 289568, and A1-S-9620B) and the Universidad Nacional Autónoma de México (UNAM, grant 405PC). The study adhered to the guidelines of the Declaration of Helsinki, and the protocol and consent forms were approved by the Bioethics Committees of the Instituto de Neurobiología, UNAM and the Instituto Mexicano de Oftalmología. Each subject provided written informed consent and the study was supervised by an independent data and safety monitoring committee. The study was registered at | PMC10858020 | |
DME study | PMC10858020 | |||
Protocol | hyperprolactinemia, Diabetic Retinopathy, glaucoma, centre-involving | DIABETIC RETINOPATHY, BLIND, GLAUCOMA, HYPERPROLACTINEMIA, TYPE 2 DIABETES, INTRAOCULAR PRESSURE | From 176 screened mestizo patients with type 2 diabetes, there were 55 eligible centre-involving DME participants (aged 40–69 years, best-corrected visual acuity (BCVA) between 58 to 16 Early Treatment Diabetic Retinopathy Study (ETDRS) letters at 4 m (20/16 to 20/100 Snellen equivalent), and central foveal thickness (CFT) of ≥224 μm). After participants provided their written informed consent, blood samples were withdrawn to evaluate basal PRL levels and safety parameters [thyroid stimulating hormone (TSH), glycated haemoglobin (HbA1c), and creatinine]. Twelve patients were excluded [4 due to hyperprolactinemia (>20 ng/ml), 3 because of glomerular filtration rates <28 ml/min, 4 due to hard exudates in the fovea region, and 1 for receiving glaucoma treatment]. The remaining 43 subjects were randomized by the study coordinator based on a computer-generated list of random numbers in a 1:1 allocation ratio to receive levosulpiride (DISLEP®, Ferrer Therapeutics; 25-mg orally TID) or placebo (lactose pill orally TID) during a follow-up period of 8 weeks and study visits every 2 weeks. From the 43 subjects, 9 dropped out [5 did not comply with levosulpiride treatment confirmed by the circulating levels of PRL, 2 were hesitant to continue with study medication (one placebo and one levosulpiride), and 2 were unable to maintain visit appointments]. Thirty-four study subjects (12 females and 22 males) completed the study (17 treated with placebo and 17 with levosulpiride) and were evaluated [medical history, physical examination, BCVA, optical coherence tomography (OCT), blood and intraocular pressure, fundoscopy, and laboratory analysis of blood samples] at baseline and at study visits every 2 weeks. Determination of TSH was only done at baseline and HbA1c and fluorescein angiography were done at baseline and at week 8. Patients and investigators assessing outcomes were blind to treatment assignment. | PMC10858020 |
Primary endpoints | BCVA, OCT, and fluorescein angiography were assessed by certified examiners using standardised protocols as reported [ | PMC10858020 | ||
Secondary endpoint | PRL levels in serum confirmed adherence to levosulpiride treatment (also monitored by counting drug tablet return) and were quantified using the IMMULITE 2000 XPi immunoassay system (Siemens, Munich, Germany). The intra-assay and inter-assay coefficients of variation were less than 1%. | PMC10858020 | ||
Safety assessment | ADVERSE EFFECTS | Medical history, physical examination, ocular pressure, vital signs (sitting systolic and diastolic blood pressure), and laboratory tests in blood (HbA1c and creatinine) evaluated the safety of the study medication (levosulpiride vs. placebo). The occurrence of adverse effects was also sought by nondirective questioning of the patient at each visit or between visits. | PMC10858020 | |
PDR study | PMC10858020 | |||
Protocol | PDR, type 2 diabetes, hyperprolactinemia | HYPERPROLACTINEMIA, TYPE 2 DIABETES | Briefly, from 175 screened PDR mestizo patients with type 2 diabetes undergoing elective primary pars plana vitrectomy, 41 eligible patients signed the informed consent, and their blood was withdrawn to evaluate PRL, HbA1c, and creatinine levels. From these 41 patients, 2 were excluded due to basal hyperprolactinemia (>20 ng/ml) and 3 because of vitrectomy being re-scheduled. All 36 patients (19 females and 17 males) met the inclusion criteria (aged 40–69 years, no history of prior vitrectomy, PRL serum levels ≤20 ng/ml, and glomerular filtration rate >28 ml/min) and were randomized 1:1 to receive placebo (orally TID, | PMC10858020 |
Primary endpoints | VEGF and PlGF were measured in the same vitreous sample by enzyme-linked immunosorbent assay (ELISA) using the Quantikine Human VEGF and PlGF kits (R&D System, Minneapolis, MN) performed according to the manufacturer’s instructions. The intra-assay and inter-assay coefficients of variation for VEGF were 5.4% and 7.3%, respectively; and 5.4% and 11.2% for PlGF, respectively. | PMC10858020 | ||
Secondary endpoint | PRL levels in serum at baseline and at vitrectomy were measured as indicated above. | PMC10858020 | ||
Statistics | GraphPad Prism Software Inc. version 6.01 was used. Statistical differences between two groups were determined by Student’s | PMC10858020 | ||
Results | PMC10858020 | |||
BCVA outcome | As early as 4 weeks after initiating treatment with levosulpiride, the mean longitudinal change in BCVA over baseline was significantly higher than after placebo (Fig. | PMC10858020 | ||
Levosulpiride improved the change from baseline in visual and structural parameters. | Changes from baseline in best-corrected visual acuity (BCVA) ( | PMC10858020 | ||
CFT and MMV outcomes | The overtime BCVA improvement with levosulpiride was paralleled by longitudinal upgrades in CFT and MMV. The mean CFT longitudinal change of study eyes declined relative to baseline at weeks 4 to 8 after levosulpiride vs. placebo and accounted for a mean loss of 46.6 μm at the end of treatment (Fig. | PMC10858020 | ||
Overall visual and anatomic outcomes | RETINA | Finally, the overall change from baseline to week 8 of all primary endpoints (BCVA, CFT, MMV, OCT macular image, fundoscopy, and fluorescein angiography) was evaluated by six independent retina specialists blinded to treatment through a scale that ranged from −4 to +4, where −4 was highest worsening, 0 no change, and +4 highest improvement. Figure | PMC10858020 | |
Levosulpiride improved the overall grading of primary endpoints. | Three examples of levosulpiride- ( | PMC10858020 | ||
Safety | Somnolence, tachycardia, somnolence | ADVERSE EFFECTS, SIDE EFFECT | Only two DME patients reported adverse effects. One patient in the placebo group informed tachycardia 2 weeks after treatment and abandoned the study, whereas one levosulpiride case reported somnolence, which could relate to the study medication as it is a known side effect of levosulpiride. Somnolence occurred 6 weeks after initiating treatment and the patient withdrew from the study. There were no significant differences in mean ocular pressure, mean serum levels of HbA1c, mean glomerular filtration rate, and mean blood pressure between the placebo- and levosulpiride-treated DME patients (Table | PMC10858020 |
Vitreous levels of VEGF and PlGF | PDR | The vitreous concentrations of VEGF and PlGF were significantly reduced in PDR patients treated with levosulpiride (Fig. | PMC10858020 | |
Levosulpiride reduced the vitreous levels of VEGF and PlGF by mechanisms that may involve the prolactin/vasoinhibin axis. | Vascular endothelial growth factor (VEGF) ( | PMC10858020 | ||
Discussion | vascular retinopathies, centre-involving, diabetic, hyperprolactinemia, ocular complications, vision loss, gain of vision | HYPERPROLACTINEMIA, MACULAR OEDEMA, DIABETIC GASTROPARESIS, COMPLICATION | Levosulpiride is a prokinetic [The study was designed to assess the efficacy of levosulpiride in previously untreated DME patients with the caveat of deferring for the end of the study any other treatment. Accordingly, the protocol was short-termed (8 weeks) and carried out in DME patients with moderate vision loss (defined as ≥16 ETDRS letters at 4 m; Snellen equivalent ~20/100) that were carefully evaluated every 2 weeks. Levosulpiride showed meaningful responses compared with the natural history of the process (demonstrated by the placebo group). Mean changes in visual and anatomical parameters improved early (4 weeks) after initiating levosulpiride treatment. At week 8, gain and losses from baseline in BCVA (+6 letters), CFT (−46.6 μm), and MMV (−0.49 mmLevosulpiride efficacy rate in gain of vision (41%) was lower than efficacy rates in CFT (55%) and MMV (65%). Improvements in anatomical parameters are indicative of resolution of macular oedema and may [Beneficial outcomes are outstanding when considering that levosulpiride is a non-invasive orally active medication that contrasts with the standard care of DME using intravitreal drug delivery, which poses a risk of ocular complications [As expected, levosulpiride did not pose any significant safety concern during the 8 weeks of treatment. We used the oral dose of levosulpiride (25 mg TID) employed for the treatment of diabetic gastroparesis, a complication found in 5% of diabetic patients [This study had several strengths including a powerful assessment of adherence to treatment monitored by the high efficacy of levosulpiride-induced hyperprolactinemia [In summary, this interventional, randomized, placebo-controlled phase 2 study, in which levosulpiride was administered orally according to indications of use in daily practice, showed the effectiveness and safety of this prokinetic agent for improving visual and structural outcomes in patients with centre-involving DME. Its mechanism of action may involve the PRL/vasoinhibin axis [Our study repositions levosulpiride outside its prokinetic scope as a safe, affordable, and early treatment for DME, DR, and other vascular retinopathies. Larger and longer studies are needed to solidify these findings. | PMC10858020 |
Summary | PMC10858020 | |||
What was known before | diabetic retinopathy, hyperprolactinemia, diabetic macular oedema | DIABETIC RETINOPATHY, PROLIFERATIVE DIABETIC RETINOPATHY, DIABETIC MACULAR OEDEMA, RETINA, HYPERPROLACTINEMIA |
Elevation of systemic prolactin leads to the intraocular accumulation of vasoinhibin, a proteolytic fragment of prolactin that inhibits the excessive permeability and growth of blood vessels in the retina.The prokinetic oral medication, levosulpiride, induces hyperprolactinemia and elevates the levels of vasoinhibin in the retina of diabetic rats and in the vitreous of patients with proliferative diabetic retinopathy.Levosulpiride may have beneficial outcomes in diabetic macular oedema and diabetic retinopathy due to the vascular properties of vasoinhibin. | PMC10858020 |
Acknowledgements | The authors acknowledge the substantial contribution of Nancy Sánchez Martínez and Graciela Ibarra Vargas for the assessment and interpretation of optometry, optical coherence tomography, and fluorescein angiography data; Fernando López Barrera for statistical analysis, and Jessica Gonzalez Norris for editing the manuscript. | PMC10858020 | ||
Author contributions | TB | CDN-A, MLR-O, RG-F, PR-N, EL-S, TB, GME, JT, and CC conceived and designed the study; CDN-A, ML, EA-C, RG-F, MG-R, YV-G, PR-N, NP, and JFR-M acquired data; CDN-A, ML, EA-C, MLR-O, RG-F, MG-R, YV-G, PR-N, JFR-M, JS, GR-H, LS-M, ND-L, TB, GME, JT, and CC analysed and interpreted the data; CC wrote the manuscript and obtained funding; All authors critically revised and approved the final version. | PMC10858020 | |
Funding | This work was supported by the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) (grants 247164, 289568, and A1-S-9620B) and Universidad Nacional Autónoma de México (UNAM) (grant 405PC) to CC. The funding organisations had no role in the design or conduct of this research. | PMC10858020 | ||
Data availability | All original data generated or analysed during this study are included in this published article. | PMC10858020 | ||
Competing interests | TB | The authors declare the following broadly competing interest: CC, GME, JT, and TB are inventors of a submitted patent application (WO/2021/098996). The Universidad Nacional Autónoma de México (UNAM) and the authors JT and TB are owners of the pending patent. | PMC10858020 | |
References | PMC10858020 | |||
Keywords | pain, Osteoarthritis, OA, OA symptoms | DISEASE, DISEASE PROGRESSION, DEGENERATIVE, OSTEOARTHRITIS | Osteoarthritis (OA) is a degenerative multifactorial disease with concomitant structural, inflammatory, and metabolic changes that fluctuate in a temporal and patient-specific manner. This complexity has contributed to refractory responses to various treatments. MSCs have shown promise as multimodal therapeutics in mitigating OA symptoms and disease progression. Here, we evaluated 15 randomized controlled clinical trials (RCTs) and 11 nonrandomized RCTs using culture-expanded MSCs in the treatment of knee OA, and we found net positive effects of MSCs on mitigating pain and symptoms (improving function in 12/15 RCTs relative to baseline and in 11/15 RCTs relative to control groups at study endpoints) and on cartilage protection and/or repair (18/21 clinical studies). We examined MSC dose, tissue of origin, and autologous vs. allogeneic origins as well as patient clinical phenotype, endotype, age, sex and level of OA severity as key parameters in parsing MSC clinical effectiveness. The relatively small sample size of 610 patients limited the drawing of definitive conclusions. Nonetheless, we noted trends toward moderate to higher doses of MSCs in select OA patient clinical phenotypes mitigating pain and leading to structural improvements or cartilage preservation. Evidence from preclinical studies is supportive of MSC anti-inflammatory and immunomodulatory effects, but additional investigations on immunomodulatory, chondroprotective and other clinical mechanisms of action are needed. We hypothesize that MSC basal immunomodulatory “fitness” correlates with OA treatment efficacy, but this hypothesis needs to be validated in future studies. We conclude with a roadmap articulating the need to match an OA patient subset defined by molecular endotype and clinical phenotype with basally immunomodulatory “fit” or engineered-to-be-fit-for-OA MSCs in well-designed, data-intensive clinical trials to advance the field. | PMC10229578 |
Subject terms | PMC10229578 | |||
Introduction | PMC10229578 | |||
Overview of osteoarthritis | TNF-α, Osteoarthritis, OA, tumor necrosis, arthritis | OSTEOARTHRITIS, TUMOR NECROSIS, DISEASE, ARTHRITIS, INFLAMMATION, PATHOGENESIS | Osteoarthritis (OA) is the most common form of arthritis, affecting an estimated 650 million people (654.1 [95% CI, 565.6–745.6]) aged 40 and older worldwide as of 2020, with an incidence of 203 per 10,000 person-years [95% CI, 206–331] [OA imposes socioeconomic burdens due to its morbidity, which results in reduced daily activity and productivity. Currently, arthritis costs an estimated US$ 24 billion annually in Canada, with projected increases to US$ 49 billion annually by 2031 [OA is a multifactorial disease affecting articular cartilage, subchondral bone, menisci (in the knee), synovial tissue (a membrane lining the joint and heterogeneously composed of fibroblasts, MΦs, and endothelial and stromal cells), tendon/ligaments and muscle [OA is highly heterogeneous in terms of clinical features and responses to available treatments, as well as through the contribution of different biochemical factors that contribute to the progressive and gradual loss of articular cartilage [Inflammation plays a critical role in OA, and proinflammatory cytokines such as interleukin-1-beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α) are implicated in OA pathogenesis [ | PMC10229578 |
Mesenchymal stromal cells (MSCs) for the treatment of OA | OA, OA [ | MSCs are attractive therapeutic candidates for OA due to their multimodal mechanisms of action, including their immunomodulatory properties. MSCs modulate the inflammatory cytokine milieu and immune cell responses in the joint via the release of secreted paracrine factors and extracellular vesicles (EVs) and through host macrophage-mediated efferocytosis [In this review, we will examine a curated set of peer-reviewed RCTs and discuss clinical evidence of efficacy from these controlled trials, focusing only on culture-expanded MSCs in treating OA [ | PMC10229578 | |
Culture-expanded MSCs and randomized, controlled OA clinical trial results | PMC10229578 | |||
RCT patient populations | pain | DISEASE | To further understand the differential responses to MSC treatments, we further investigated the heterogeneous OA patient populations enrolled in these trials. Some trials, including those of Freitag et al. [Fourteen out of fifteen RCTs reviewed in this section classified patients by KL grade, typically enrolling patients with KL grade 2–3 knee OA. However, within this classification, there is considerable heterogeneity in clinical phenotype (and disease endotype). We tried to parse differences in clinical phenotype looking at baseline pain scores (Table | PMC10229578 |
MSC dose comparisons in RCTs | The studies included in RCT analyses used a range of doses from 3.9 × 10Differential median baseline WOMAC total scores for the 10 × 10Lamo-Espinosa et al. conducted a subsequent 4-year follow-up study with 27 of the 30 patients [Similarly, Chen and Hu et al. demonstrated improvements in overall WOMAC scores at 48 weeks posttreatment in the group receiving the highest dose (64 × 10Conversely, two studies, Gupta et al. [Kuah et al. [Taken together, the limited RCT data on dose escalations provide no evidence of a clear winner in terms of patient-reported outcomes. While both the Gupta et al. [ | PMC10229578 | ||
Repeat MSC injection RCTs | There are only three published RCTs in which repeat MSC intra-articular injections were administered. Freitag et al. used cryopreserved autologous MSC(AT) at doses of 100 × 10Interestingly, Freitag’s group initially included a fourth arm of 40 × 10Conversely, the design of the Matas et al. trial included a control group (The Lu et al. trial employed a unique dosing method in which four injections were given a week apart for both the MSC and control groups. The control group received four 2.5 mL injections of HA, while the treatment group received 50 × 10Taken together, all three trials reported benefits of dual injections, although only the Matas and Lu trials showed differences in PROMs at 12 months, while the Freitag and Lu trials showed gains in cartilage volume or a lack of cartilage volume losses at 12 months with dual injections. | PMC10229578 | ||
Autologous vs. allogeneic MSCs in RCTs | RCTs investigating autologous MSCs made up the majority (9/15) of the studies reviewed, while the remaining (6/15) studies investigated MSCs of allogeneic origin. The autologous tissue of origin was split between bone marrow (5/9) and adipose tissue (4/9), while allogeneic sources were split between adipose tissue (2/6) and bone marrow (2/6). The remaining allogeneic trials used MSCs from Wharton’s jelly (1/6) or the placenta (1/6). Most of the dose escalation trials (3/4) used allogeneic MSC sources, while the three repeat injection trials were divided two-to-one between autologous and allogeneic MSCs [In 5/6 of the MSC allogeneic trials, there was a significant improvement in PROMs (WOMAC, KOOS, and KSCRS scores) relative to the control groups. This was true regardless of doses ranging as low as 3.9 × 10Similarly, in 8/9 RCTs using autologous MSCs, there was significant improvement in PROMs [In summary, the 15 RCTs with 610 treated patients showed positive effects of MSCs relative to control treatments at 12-month timepoints in terms of PROMs. However, clear distinctions are not apparent in regard to dose dependencies, MSC tissue of origin, and autologous or allogeneic MSC sources. We did note that in 7/9 autologous MSC trials, moderate to high (10 × 10 | PMC10229578 | ||
MSC mechanisms of action in knee OA | DISEASE, ACUTE GVHD | Despite the completion of 15 peer-reviewed RCTs, there is limited evidence on the clinical mechanism of action of MSCs in OA. An improved understanding of the mechanism of action would be beneficial in the following ways: (i) it would facilitate the design of improved MSC therapies with properties tailored to achieve the desired effects in OA; (ii) it would facilitate the selection of critical quality attributes (CQAs) for MSC investigational products that are relevant to the mechanism of action and could be used as potential release criteria; (iii) in patient stratification or precision medicine-based approaches, it could facilitate the selection of patients with a higher response likelihood to MSC interventions based on disease status biomarkers relevant to the therapeutic mechanism of action; and (iv) it would facilitate the selection of relevant readouts or biomarkers that inform clinical responses based on the expected clinical effects of an MSC intervention.To underscore the importance of understanding the mechanism of action, a previous biologic license application for Remestemcel-L™, an allogeneic MSC(M) product for the treatment of steroid-refractory acute GVHD, was rejected by the United States Food and Drug Administration (FDA), partially due to the inadequacy of the CQAs employed by the sponsor; the researchers did not demonstrate a clear relationship of the MSC CQAs to the clinical potency of the product [The following subsections provide an overview of current insights into MSC mechanisms of action in OA, with emphasis on immunomodulatory mechanisms. We provide an overview of in vitro and preclinical studies that provide insights into MSC-mediated immunomodulation as an important mechanism of action in OA. We further detail the clinical evidence (beyond the 15 RCTs in the previous section) where available, discussing immunomodulatory, chondroprotective, regenerative and other effects of MSCs in the treatment of knee OA. | PMC10229578 | |
MSC anti-inflammatory and immunomodulatory effects on OA | inflammation, OA | INFLAMMATION, DISEASE, KNEE OSTEOARTHRITIS, PATHOPHYSIOLOGY, SECONDARY | MSC-mediated anti-inflammatory and immunomodulatory effects on OA inflammation are also known to play a central role in OA, with both innate and adaptive mediators involved in the pathophysiology [Evidence of the anti-inflammatory and immunomodulatory functionality of MSCs has been extensively reviewed in various preclinical studies [The hypothesis that immunomodulation and inflammation play a central role in MSC-mediated OA repair and tissue remodeling is further supported by data from our laboratory showing that iron nanoparticle-labeled syngeneic murine MSC(M) injected into immunocompetent C57Bl/6 mice with surgically induced OA were retained within the synovium up to 4 weeks postsurgery; importantly, the presence of the iron-labeled MSC(M) was confirmed with dual Prussian Blue and SCA-1Cell tracking studies in equine [Clinical and preclinical evidence of mesenchymal stromal cell mechanisms of action on immunomodulation and chondroprotection in knee osteoarthritis. Solid arrows are used to demonstrate mechanisms for which there is supporting clinical evidence: dashed arrows indicate the relationship between immunomodulatory and chondroprotective mechanisms supported by preclinical evidence. MSC mesenchymal stromal cell, CQAs critical quality attributes, OA osteoarthritisNow, it should be noted that the role of inflammation in OA is not fully understood, and it is unclear whether inflammation represents a primary etiology of the disease or whether it arises secondary to other processes, such as joint damage and altered mechanical signaling within the cartilage [ | PMC10229578 |
Clinical evidence of MSC immunomodulation in OA | While the findings of preclinical studies (extensively reviewed [Systemic and local levels of inflammatory biomarkers after intra-articular MSC injections have also been investigated in recent knee OA trials. A study by Li et al. demonstrated systemic anti-inflammatory effects of MSC(M) injections and platelet lysate (three monthly injections with MSCs and platelet lysate injected three days apart) relative to arthroscopic debridement and HA injections in knee OA patients (KL grade 0–2, In contrast to the above, Bastos et al. have shown that there were no significant differences in synovial fluid levels of various inflammatory cytokines (IL-17A, IFN-γ, TNF -α, IL-10, IL-6, IL-4, and IL-2) between knee OA (KL grade 1–4) patient groups treated with autologous MSC(M) (40 × 10 | PMC10229578 | ||
Other potential mechanisms | synovitis, bone marrow lesions, fibrosis | CRITICAL LIMB ISCHEMIA, SYNOVITIS, DISEASE, FIBROSIS, PATHOPHYSIOLOGY, IDIOPATHIC PULMONARY FIBROSIS, OSTEOPHYTE | In addition to having immunomodulatory and chondroprotective effects in OA, MSCs may act through a variety of other mechanisms that target multiple tissues within the joint. These mechanisms include antifibrotic and angiogenic effects, which are interrelated with immunomodulation. In particular, the pro-angiogenic mechanisms of MSCs have been widely documented and are of clinical relevance to a variety of conditions, such as critical limb ischemia [There is a complex interplay between inflammatory and angiogenic processes, with immune cells playing a critical role in the temporal regulation of vessel remodeling through both pro- and anti-angiogenic mechanisms [Antifibrotic effects of MSC therapies have been documented in several clinical indications for which fibrosis is a disease hallmark, including for idiopathic pulmonary fibrosis [In addition to the immunomodulatory, angiogenic, and antifibrotic properties of MSCs that may impact multiple tissues within the joint, MSCs may have tissue-specific effects on the subchondral bone, menisci, and ligaments. MRI data on these tissues from clinical studies involving MSCs in OA are sparse and limited by the sensitivity of the technique. However, Chen et al. have shown that autologous MSC(AT) injection significantly reduced the MOAKS for bone marrow lesions and cysts, meniscus pathology, and periarticular features at 48 weeks relative to baseline, while no significant differences were observed for osteophyte, synovitis, ligament tears or tendon abnormality scores [The menisci and ligaments of the joint play important roles in mechanical support, and damage to these tissues can precede radiographic OA; however, their roles in OA progression and pathophysiology are poorly understood [ | PMC10229578 |
Influence of MSC immunomodulatory basal fitness on their mechanisms of action | alloantibody | PROLIFERATION, RECRUITMENT | Several factors can influence the fitness and overall phenotype of MSCs that may in turn influence their mechanisms of action, as discussed here with emphasis on effects mediating immunomodulatory fitness. Donor heterogeneity represents a major variable and includes a multitude of factors, such as donor health status, age, BMI and sex, that can influence the in vitro clonogenic potential, paracrine functions, and proclivity toward senescence of MSCs [In support of the immunomodulatory mechanism of action of MSCs in OA, we and others have investigated the relationships between the baseline immunomodulatory fitness of donor MSCs in vitro and their clinical efficacy. In our clinical trial, we measured the levels of IFN‐γ-induced expression of immunomodulatory genes (prostaglandin-endoperoxide synthase 2, Delivery strategies are also likely to influence mechanisms of action, and the administration of freshly thawed cryopreserved MSCs can influence their cell viability and apoptotic status [The tissue sources for MSCs represent an important variable that could also influence MSC mechanisms of action. To date, bone marrow, adipose tissue and umbilical tissues represent the most commonly used MSC sources in OA clinical trials. An elegant study by Ménard et al. investigated matched human MSC(AT) and MSC(M) from 14 donors and demonstrated distinct transcriptional profiles that were imprinted by the tissue of origin in each MSC population, with different sets of immunomodulatory genes and chemokines upregulated in MSC(AT) and MSC(M). MSC(AT) displayed reduced HLADR expression, as well as augmented neutrophil recruitment and suppression of T-cell proliferation in vitro, whereas MSC(M) were better able to suppress natural killer (NK) cell proliferation in experiments with paired donors [The choice of autologous versus allogeneic MSCs is another factor that may impact mechanisms of action. An advantage of allogeneic MSC sources is that they offer the ability to select healthy donors and screen for MSCs with high fitness levels, as we have postulated [In contrast, autologous sourcing would circumvent alloantibody production, but donor site morbidity and variable basal MSC therapeutic fitness, which can be impacted by donor factors including age, sex, BMI, physical activity and health status, must be factored in with its use. For example, MSCs obtained from older donors may be more prone to senescence, which may limit the cell numbers required for specific dosing, skew their secretory phenotype and diminish their functional immunomodulatory properties [ | PMC10229578 |
Summary of current knowledge on MSC mechanisms of action in OA | There is limited clinical evidence on MSC mechanisms of action in OA. Recent clinical data have supported immunomodulation as a relevant mechanism of action for MSCs in OA [ | PMC10229578 | ||
Roadmap for successful MSC OA trials | PMC10229578 | |||
Selecting the right OA patient endotype for MSC therapies | OA, inflammation, synovial inflammation, pain, heterogeneous disease | INFLAMMATION | OA is a heterogeneous disease, and at least six clinical phenotype subsets have been identified [Categorizing OA patients with the inflammatory phenotype (based on synovial inflammation) may be most relevant to MSC therapies with their proposed anti-inflammatory mechanisms of action. Indeed, the current most effective therapeutic treatments for OA symptoms and pain are nonsteroidal anti-inflammatory drugs (NSAIDs) and intra-articular corticosteroid injections that work through anti-inflammatory effects on synovial inflammation with large effect sizes [Part of the conundrum is that it is not clear that an inflammatory phenotype is a true OA clinical phenotype [US imaging has been postulated to be one such measurement modality for identifying patients with increased inflammation [Ultimately, the use of combinatorial tools, including US and MRI, serum biomarkers, and synovial fluid levels of immune cell subsets and proinflammatory mediators, will help identify true inflammatory patient endotypes (vs. clinical phenotypes) that might be most receptive to MSC treatments based on their proposed anti-inflammatory and immunomodulatory effects, particularly on host MΦs. Flow cytometry evaluations would thus be valuable to profile cellular responses within local joint biopsy samples, including immune cell profiling of synovial fluid as we have previously shown [The identification of circulating miR biomarkers [ | PMC10229578 |
Designing the right MSC therapeutic product for OA | alloreactions | HYPOXIC, DISEASE | As discussed above, the immunomodulatory basal fitness of MSCs, which can be modulated by age, sex, BMI, comorbidities, manufacturing parameters and cryopreservation and can be quantified by a range of quantitative CQAs [Functional high-content in vitro potency assays that are relevant to the mechanisms of action may also be useful for characterizing MSCs to better understand the influence of basal fitness levels on treatment efficacy [As discussed above, dosing, carrier choice and selection are also not fully understood, and in many cases medium to high (≥40 × 10Limited short-term clinical evidence supports repeat dosing of autologous and allogeneic MSCs, albeit without a full understanding of the potential implications of alloreactions to repeat allogeneic MSC dosing. Clinical trials using allogeneic MSCs should incorporate measurements of humoral and cellular responses to better discern the correlation, if any, between alloreactions and reduced clinical efficacy. Importantly, data on longer-term structural changes as well as symptom and disease modification are not sufficient to recommend repeat injections, but they merit investigation in future clinical trials.Ultimately, there are conflicting data regarding dosing from the RCTs and non-RCTs, and this seems to be driven in part by the baseline patient characteristics or clinical phenotyping; from our analyses, it appears that often the patients receiving lower doses of MSC products seem to have lower baseline pain/symptom scores, which confounds the analysis on dosing. Of the 15 RCTs, we noted differences in dose ranges tested for autologous (4.11–100 × 10Next-generation MSCs, with improved immunomodulatory basal fitness and designed to be fit-for-purpose (for OA treatment), are also being evaluated preclinically and clinically. Small-diameter MSC(CB) grown in hypoxic conditions in calcium-rich medium with improved basal fitness, including the ability to polarize MΦs to proresolving subtypes [Induced-pluripotent stem cell (iPSC)-derived MSCs (iMSCs) represent another next generation MSC product currently under clinical evaluation for the treatment of tibiofemoral knee OA in a Phase III double-blinded, randomized, controlled trial [MSC-derived EVs with cargo consisting of proteins, mitochondria, miRs, ions, and lipids are also being evaluated preclinically [In summary, there are several technologies to aid in the selection or engineering of basally fit autologous or allogeneic MSC therapeutic products or engineering MSC-derived EVs that can be specifically curated for OA therapeutics. Ultimately, MSCs with multimodal anti-inflammatory, immunomodulatory, chondroprotective and antifibrotic functionalities will likely be of greatest benefit. Dosing remains an open question, although meta-analysis results seem to suggest that cartilage structural changes are feasible with >40–50 × 10 | PMC10229578 |
Considerations for successful clinical trial design for MSC products for OA | osteoarthritis, OA | OSTEOARTHRITIS, EMA | Clinical trials investigating MSC treatments in OA should incorporate additional readouts on putative mechanisms of action to facilitate future regulatory approvals of MSC products in OA and to tailor next-generation MSC therapies that have specific therapeutic properties and CQAs that are tuned for relevance in OA (Fig. Levers for enabling successful mesenchymal stromal cell trials in osteoarthritis - Four aspects that need to be addressed to enable successful patient outcomes. PROMs patient-reported outcome measures, CPPs critical process parameters, CQAs critical quality attributes, MSCs mesenchymal stromal cells, OA osteoarthritisThe episodic nature of OA, termed “flare-ups”, is poorly understood etiologically [There are existing guidelines for clinical trial design considerations, including the selection of OA patient populations, as put out by the European Medicines Agency (EMA) [Cost is another consideration for the commercialization of successful MSC therapeutic products. The extent of the effect size and whether there are symptom-modifying or disease-modifying effects (as evaluated by a combination of PROMs, imaging, and biomarker and biomechanic assays) will determine the willingness to pay of the health care system and market. Using patient survey-based methods, Piuzzi et al. obtained pricing information for same-day minimally manipulated autologous cell preparations from 65 centers in the USA and found that the mean price was US$ 5156 ± 2446 for a single treatment [ | PMC10229578 |
Summary | P., OA, pain | DISEASE PROGRESSION, DISEASE | Even with 15 completed RCTs, the sample size of 610 treated patients (with MSCs and control treatments) is relatively small to reach definitive conclusions on optimal dosing, baseline patient endotype and phenotype, delivery strategies, MSC sourcing, manufacturing, and overall treatment efficacy. Nonetheless, the findings of meta-analyses and our own analyses revealed positive effects on pain, function and symptoms at final follow-up endpoints in MSC-treated patients relative to baseline (12/15). In 11/15 RCTs, there were improvements in these metrics relative to the control group (HA, PRP, or surgical intervention) at 6 months or longer, depending on the dose of MSC, MSC basal fitness, the baseline clinical phenotype of the patient and the choice of the control comparator. MSCs have also demonstrated chondroprotective or regenerative effects on cartilage in 18/21 clinical studies, including 11/15 RCTs. MSCs have multimodal mechanisms of action, including chondroprotective effects, effects on cartilage repair and ECM anabolism, and pro-angiogenic, antifibrotic, anti-inflammatory and immunomodulatory effects, but there is a dearth of clinical data to confirm these mechanisms in OA. Defining baseline patient disease endotype along with clinical phenotyping will help identify and stratify patients who are receptive to receiving MSC therapies. Patient endotyping will require combinatorial tools, including different types of omics data, levels of systemic and local inflammatory markers, and immune cell subpopulations, which will need to be correlated with patient clinical outcomes and structural changes to validate them as sensitive and reliable biomarkers of patient endotypes. The episodic nature of OA further confounds the categorization of patients by clinical phenotype and/or disease endotyping but should be factored in to better understand the highly heterogeneous, personalized and temporally variable nature of disease progression. The selection of basally fit MSC products involves selecting MSCs from a host of donors that display CQAs that are relevant in the context of OA, including immunomodulatory properties, angiogenic properties and tissue remodeling properties. CQAs are modifiable by culture expansion and cryopreservation, and hence, care needs to be taken to optimize these parameters to maintain the CQAs of MSC products for clinical use. Ultimately, basally fit MSC products may be genetically engineered or nongenetically primed to be “fit-for-OA" with enhanced immunomodulatory and/or pro-angiogenic and/or cartilage-reparative properties to override reliance on donor heterogeneity and culture parameters. A combination of basally fit MSCs that are applied to a subset of knee OA patients who are receptive to receiving MSC treatments (based on disease endotyping and clinical phenotyping) will result in successful outcomes. Statistical modeling will need to be used to collect data from different trials to show the correlation between patient basal status, MSC basal fitness status and ultimately responder/nonresponder status. To enable this, larger sample sizes (for example, a 1020 patient Phase III trial to investigate the safety and efficacy of INVOSSA™, a gene-engineered cell therapy product for OA [These authors contributed equally: Griffin Copp, Kevin P. Robb. | PMC10229578 |
Acknowledgements | Arthritis | ARTHRITIS | This research was funded by the Canadian Institutes of Health Research (CIHR) (PJT-166089) and the Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN-2018-05737). This work is in part supported by the Schroeder Arthritis Institute via the Toronto General and Western Hospital Foundation (University Health Network). We gratefully acknowledge editing support from Anu Thomas. | PMC10229578 |
Competing interests | SV has 60% ownership of Regulatory Cell Therapy Consultants Inc., which does not conflict with this paper in any way. KR and GC declare no conflicts of interest. | PMC10229578 | ||
References | PMC10229578 | |||
1. Introduction | cancer [Behaviors, Dietary behaviors, food-seeking behaviors, pancreatic and gastrointestinal hormones, satiety, dietary behaviors | SECONDARY, TYPE 2 DIABETES MELLITUS, CHRONIC DISEASES, HEART DISEASE | Hunger and satiety are controlled by several physiological mechanisms, including pancreatic and gastrointestinal hormones. While the influence of exercise and fasting have been described individually, in relation to these hormones, there is a paucity of work showing the effects of the two modalities (fasting and exercise) combined. Twenty healthy adults (11 males, 9 females) completed both conditions of this study, each consisting of a 36-h water-only fast. One of the fasts began with treadmill exercise, and the differences between the conditions on various appetite hormones were measured every 12 h. The difference in the area under the curve between conditions for ghrelin was 211.8 ± 73.1 pg/mL (F = 8.40, Dietary behaviors are linked to metabolic health and have been shown to predict non-communicable chronic diseases such as type 2 diabetes mellitus, heart disease, and cancer [Behaviors surrounding food consumption are influenced by a number of environmental, social, psychological, and physiological factors. This interplay between drivers of food consumption makes the control of dietary behaviors very complex and creates a challenge when attempting to regulate food intake. Physiologically, the drive to consume food is largely regulated by a balance of hormones that signal both satiety and hunger [During a period of fasting, circulating levels of GLP-1, PYY, PP, GIP, leptin, and insulin are reduced, and levels of antagonistic hormones such as ghrelin and glucagon increase. These changes in hormones drive food-seeking behaviors [While previous works have assessed the independent effects of fasting and exercise on hunger, to date there are only a few that have evaluated the two in combination [We previously published the primary outcomes of this study. These results show that initiating a fast with a bout of exercise accelerated some of the metabolic changes that occur during fasting [The aim of this study was to evaluate the impact of initiating a 36-h fast with or without a bout of exercise on plasma concentrations of ghrelin, GLP-1, GIP, PYY, PP, leptin, and insulin. We also describe the overall change in hormone concentrations over the course of a 36-h fast. These results represent secondary outcomes to data that has been previously reported [ | PMC10142171 |
2. Materials and Methods | The study was designed to compare the impact of fasting with and without an initiating bout of exercise on appetite hormones (PYY, PP, GLP-1, GIP, insulin, ghrelin, and leptin). To accomplish this purpose, we used a randomized crossover design with conditions counterbalanced. All participants were exposed to two treatment conditions. Both conditions took place on identical days of the week, and the washout between conditions was at least six days (the longest washout was 13 days). Both intervention days were identical, with the exception of the addition of an exercise bout that took place 30 min into the fast and after the standardized meal [Prior to beginning the study, randomizer.org was used to randomly assign the condition order to participant number [ | PMC10142171 | ||
2.1. Participants | word of mouth | A total of 11 men and 9 women were recruited to participate in the study. Participants were recruited using advertisements, fliers, and word of mouth. Baseline physical activity was self-reported. Based on this self-report, 5% of participants participated in less than 30 min of exercise per week, 10% participated in 30 to 90 min of exercise per week, 35% participated in 90 to 150 min of exercise per week, 20% participated in 150 to 225 min of exercise per week, and 30% participated in more than 225 min of exercise per week.The following are the exclusion criteria used for the study:Diagnosed with a metabolic disease;Diagnosed with an orthopedic impairment;Diagnosed with an eating disorder;Taking metabolism-altering medications [Consuming more than 60 mg of caffeine daily [Pregnant or lactating;Postmenopausal [underweight (BMI < 18.5 kg/mPracticing calorie or carbohydrate diets. | PMC10142171 | |
2.2. Measurements | PMC10142171 | |||
2.2.1. Anthropometric Measurements | Body mass and height were assessed at the beginning of each condition. Participants were assessed with their shoes removed and wearing a t-shirt and athletic shorts. Body mass was assessed using a digital scale accurate to ±0.1 kg (Seca, Hamburg, Germany). Height was assessed using a stadiometer (Seca, Hamburg, Germany) accurate to ±0.1 cm. Body composition was assessed on the initial visit using a GE iDXA (GE, Fairfield, CT, USA) [ | PMC10142171 | ||
2.2.2. Plasma Hormone Levels | BLOOD, EMD | Blood draws were taken every 12 h of each fast (0, 12, 24, and 36 h). Samples were centrifuged for 15 min within 10 min of collection and then stored at −80 °F. Ghrelin, GLP-1, PYY, PP, leptin, insulin, and GIP were quantified using a standard 96-well multiplex human metabolic hormone magnetic bead panel according to the manufacturer’s instructions (EMD Millipore Corporation, Billerica, MA, USA, Catalog # HMHEMAG-34K). | PMC10142171 | |
2.3. Procedures | PMC10142171 | |||
2.3.1. Screening | FOOD ALLERGIES | Potential participants were emailed a link to an online survey, which was used to ensure all inclusion criteria for the study were met. In the survey, participants were asked to complete the PAR-Q, a food preference questionnaire, and report any food allergies. Candidates who qualified were invited to participate in the study. They were instructed to arrive at the lab prepared to exercise, and to refrain from vigorous activity for 24 h prior to testing, along with avoiding caffeine and other stimulant consumption. Adherence to these protocols was assessed through verbal questioning at the beginning of each testing session. Adherence was later reaffirmed through the assessment of baseline insulin, which was found not to differ between conditions, suggesting that participants began each condition of the study in a similar metabolic state. If participants were non-compliant with pretest protocols, they were rescheduled. | PMC10142171 | |
2.3.2. Orientation | Dietary and sleep | Participants gave informed consent prior to participation in the study. Each participant was familiarized with the study procedures and informed of the purpose of the study. During the testing period, participants were asked to go about their normal daily activities while avoiding strenuous activity, including cardiovascular or strength training, hiking, yard work, or other forms of vigorous exercise or activity. Participants were also asked to maintain their regular sleeping patterns during the testing period. Dietary and sleep adherence were verified using an activity and sleep log and were also verbally verified at each visit. | PMC10142171 | |
2.3.3. Standardized Meals | To initiate each fast, a standardized meal was given to each participant. Equations validated for reliability and accuracy by Hall et al. [ | PMC10142171 | ||
2.3.4. Treatment Sessions | Participants were asked to maintain normal eating habits leading up to each fast and to abstain from eating for at least four hours before consuming the standardized meal at the initiation of their fast in an effort to normalize blood markers and prevent pre-fast caloric loading [ | PMC10142171 | ||
2.3.5. Exercise Protocol | Exercise protocols were completed on a treadmill at a speed and grade designed to bring the estimated heart rate reserve (HRR) of each participant to 70%. This type of exercise is considered intense [The following formula was used to estimate maximal HR [A strap-on heart rate monitor (Garmin, Olathe, KS, USA) was fitted to the participant, and they were then instructed to sit for 5 min to establish their resting HR (HRrest). Once HR calculations were complete, subjects began the exercise. On the treadmill, the speed and grade were adjusted and set to cause the participant to meet their target HR within 5 min after beginning exercise. Once set, the speed and grade were not adjusted. Participants were expected to maintain this intensity for the entire duration of the exercise; however, if a participant was unable to maintain the exercise intensity, they were permitted a 60-s break before resuming.The exercise duration was individualized in order for each participant to expend approximately the same number of calories as they consumed in the standardized meal. The energy expenditure calculation was based on the ACSM metabolic equation, which converts oxygen to calories by multiplying liters of oxygen by 5.Standard ACSM metabolic equation:Equations to estimate exercise duration:E = energy from a meal (kilocalories);kg = participant’s weight (kilograms);S = treadmill speed (meters per minute);G = grade of the treadmill (percent incline);min = time on the treadmill (minutes).All calculations were performed in a protected spreadsheet using the above preset equations to ensure accuracy. Energy expenditure was verified using indirect calorimetry throughout the entire duration of the exercise (COSMED, Rome, Italy). | PMC10142171 | ||
2.4. Statistical Analysis | Means and standard deviations were used to describe participant data. The primary factors in the statistical models were condition and sex. The two levels of the condition variable were fasting alone and fasting with exercise. The sample size was calculated a priori. Using an alpha of 0.05, a beta of 0.20, and an effect size of 0.66 (a ten percent difference between conditions) resulted in a sample of 20 participants.A mixed-model repeated-measures ANOVA was used to evaluate the time course differences for each hormone over the 36-h fast for each condition, controlling for baseline values. Where there were significant interactions between time and condition, a post hoc least significant difference pairwise comparison was used to examine the differences. Gender was included in the model as a covariate, and the interactive affect between gender and condition was evaluated. The area under the treatment curve was calculated using the trapezoidal rule for the plasma concentration of each hormone. The area under the curve was used to better represent the total response of each variable. A mixed-model repeated-measures ANOVA was used to compare areas under the curve for each condition. The cut point for statistical significance was | PMC10142171 | ||
4. Discussion | nutritional ketosis, depressed, appetite | INSULIN SENSITIVITY | Recent research indicates that fasting may lead to weight loss but may also increase concentrations of ghrelin while reducing concentrations of GLP-1, PYY, and PP throughout the fast [We found that completing a bout of vigorous exercise at the beginning of a fast depressed concentrations of ghrelin by 17% while raising concentrations of GLP-1 by 13%, an effect independent of biological sex. In contrast, PYY, PP, leptin, and insulin were not altered with exercise. These results help describe the impact of acute exercise on appetite and generally agree with the notion that exercise may be a mild to moderate anorexigenic stimulus [Previous studies have examined how different exercise intensities and durations can affect GLP-1 by examining post-exercise GLP-1 concentrations. For example, Martins et al. reported that total plasma GLP-1 concentration increased between 16–70% an hour after exercising at 60% of max heart rate [Because blood was taken every 12 h, our study was not designed to capture the acute effects of exercise. In contrast to the acute post-exercise studies described previously, the current study demonstrated that GLP-1 was suppressed in both conditions 12 to 24 h into the fast. However, the findings from this study agree with previous research in that exercise elevated GLP-1 by comparison to the control condition. One of the unique contributions of this study is that we observed the elevation of GLP-1 beyond the 2 h post-exercise seen in other research, demonstrating that the impact of exercise on GLP-1 lasted the duration of the fast. Interestingly, between 24 and 36 h of fasting, GLP-1 rose above baseline in both conditions, suggesting that the impact of fasting on GLP-1 is not linear, though the mechanism describing the rise in GLP-1 after 24 h of fasting remains uncertain [Similar to GLP-1, multiple studies have evaluated how moderate to intense exercise influences ghrelin. However, the findings from these studies are less consistent [We demonstrated that exercise mitigates the rise in ghrelin typically observed with fasting, as there was no change in baseline ghrelin concentrations for the first 24 h of the fast in the exercise condition. Our study agrees with most of the current research because, relative to the control, adding exercise to the fast reduced ghrelin concentrations. Though there are significant differences between the experimental conditions during the first 24 h of the fast, our results indicate that the impact of exercise on ghrelin during a fast disappears by 36 h.While additional work is needed to describe the mechanisms responsible for changes in ghrelin in response to acute exercise [Several studies have also shown that aerobic exercise can alter plasma concentrations of PYY [The results from this study show that exercise also did not alter insulin concentrations following 12 h of fasting. Physical activity is considered a means of improving insulin sensitivity, which stems from increased membrane permeability and glucose transport at the skeletal muscle independent of insulin [A host of evidence suggests that hunger increases over the course of a fast. This is based on subjective ratings of hunger as well as alterations in hormones such as ghrelin, GLP-1, PYY, PP, GIP, and leptin [After 24 h, GLP-1 rebounded to concentrations higher than baseline, and ghrelin concentrations dropped below baseline. The exact impact of these two hormones on hunger and the drive to consume food is complicated since other appetite-suppressing hormone concentrations remained low throughout the duration of the fast. However, the changes in GLP-1 and ghrelin may partially mitigate the drive to consume food and could at least partially explain why hunger is highest during the first 24 h of a fast [Results from our lab recently found that a state of nutritional ketosis could be reached approximately 3.5 h sooner when a fast was initiated with exercise [ | PMC10142171 |
Limitations and Strengths | This was an acute study evaluating the change in appetite hormones over the course of a single 36-h fast. While the results are informative, we are unable to describe how these results may change with repeated fasts over time. In addition, while each participant abstained from food for four hours prior to presenting at the lab and was fed a standardized meal at the beginning of each fast, the food intake prior to that was not controlled. The food and drink consumed prior to that point may have had a small residual impact on the participant’s metabolic state. However, participants were instructed to follow normal eating patterns and not overconsume at these meals in anticipation of the fast. There were no differences between appetite hormones at baseline, indicating that participants started each fast in a similar metabolic state. The fast was performed under free-living conditions, which lack the control of a lab environment. However, while this introduces some variability between fasts, the results are more generalizable since behaviors were not constrained. In addition, condition order was randomized, and any random differences in behavior between conditions should be controlled. Finally, blood draws were taken every 12 h, occurring at baseline, 12, 24, and 36 h. More frequent blood samples would better describe the time course of metabolic changes during the fast, especially within the first few hours after exercise. Additionally, we did not control the phases of the menstrual cycle of the women in the study. This could allow for more variability in appetite hormones since the hormone concentrations change with the phase of the menstrual cycle [Despite these limitations, this study adds to the current literature regarding the effects of combined fasting and exercise. While most research has evaluated the impact of fasting or exercise on appetite-regulating hormones separately, this study is one of the first to evaluate the behaviors in combination. In addition, most studies only evaluate a few hormones at a time or each hormone individually. The current study described the impact of exercise and fasting on seven hormones, which allows a broader perspective on how fasting and exercise influence the biological drive to consume food. While much of the existing literature captures the acute impact (30 min to 3 h) of exercise or fasting on appetite hormones, this study evaluated the impact up to 36 h. This enabled us to describe the prolonged effects of exercise on these hormones and to observe the non-linear response of both GLP-1 and ghrelin. Finally, the practice of intermittent fasting and time-restricted eating is very popular because of their potential to improve body composition and metabolic health. This study adds to the current body of literature by describing how exercise could be used in conjunction with fasting to potentially enhance the benefits of this behavior while mitigating the negative effects of hunger. | PMC10142171 | ||
5. Conclusions | This study found that initiating a fast with a bout of vigorous treadmill exercise lowers ghrelin and elevates GLP-1 concentrations compared to fasting alone. However, the relationship between hormone concentration and hunger seems to be complicated and the changes that we observed in GLP-1 and ghrelin did not seem to be enough to alter the subjective rating of hunger. These results, coupled with our previously reported findings [ | PMC10142171 | ||
Author Contributions | L.E.D. | Conceptualization, L.S.D., B.T.B., L.E.D., L.A.T. and B.W.B.; methodology, L.S.D., B.T.B., L.E.D., L.A.T. and B.W.B.; software, L.S.D.; validation, L.S.D., B.T.B., L.E.D., L.A.T. and B.W.B.; formal analysis, B.T.B., L.E.D., L.A.T. and B.W.B.; investigation, L.S.D., C.J.C., G.R.L. and J.L.C.; data curation, L.S.D. and B.W.B.; writing—original draft preparation, L.S.D., C.J.C., G.R.L., J.L.C. and B.W.B.; writing—review and editing, L.S.D., E.Z.G., B.T.B., L.E.D., L.A.T. and B.W.B.; visualization, L.S.D., G.R.L. and B.W.B.; supervision, B.W.B.; project administration, L.S.D., C.J.C., J.L.C. and B.W.B.; funding acquisition, L.S.D., L.A.T. and B.W.B. All authors have read and agreed to the published version of the manuscript. | PMC10142171 | |
Institutional Review Board Statement | The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Brigham Young University (IRB2019-319, approved 14 October 2019). | PMC10142171 | ||
Informed Consent Statement | Informed consent was obtained from all subjects involved in the study. | PMC10142171 | ||
Data Availability Statement | The data supporting the reported results are available on the Open Science Framework at | PMC10142171 | ||
Conflicts of Interest | The authors declare no conflict of interest. | PMC10142171 | ||
References | Participant flow diagram.Concentrations of ghrelin (Demographic characteristics of participants.Hormone concentrations (pg/mL) at four time points during a 36 h fast with or without exercise.Mean ± standard deviation. F and Differences in area under the curve for fasting with and without exercise over 36 h. | PMC10142171 | ||
Objective | weight gain | PRETERM BIRTH, COMPLICATIONS | Preterm birth is one of the most important health problems in the world. Feeding intolerance is one of the most common and serious complications of premature infant. The purpose of this study was to explore the effect of Chinese pediatric Tuina on the prevention of feeding intolerance in favour of weight gain in premature infants. | PMC10782327 |
Methods | weight gain | PREMATURE | A prospective randomized controlled study was conducted in the Department of Neonatology in our hospital. Premature infants were recruited and randomly assigned to an intervention group or a control group. Premature infants in the intervention group received a Chinese pediatric Tuina intervention by professional chiropractors, while premature infants in the control group received standard care. The incidence of feeding intolerance and weight gain situation were compared between the two groups. | PMC10782327 |
Result | gastrointestinal bleeding, liver insufficiency | GASTROINTESTINAL BLEEDING, NECROTIZING ENTEROCOLITIS | After 1 week of intervention, the body weight (2.5±0.5 vs 2.0±0.4, p=0.038), head circumference (32.8±1.7 vs 29.9±1.4, p=0.041), albumin (34.6±5.8 vs 28.4±6.1, p-0.026) and prealbumin (155.8±35.2 vs 113.6±36.8, p=0.021) of preterm infants in the intervention group were significantly better than those in the control group. The incidence of feeding intolerance (7 vs 15, p=0.032) in the intervention group was significantly lower than that in the control group. Although there were no statistically significant differences (P>0.05), the incidences of gastrointestinal bleeding, necrotizing enterocolitis, and liver insufficiency were lower in the intervention group than in the control group. | PMC10782327 |
Conclusion | weight gain | Chinese pediatric Tuina can effectively prevent the occurrence of feeding intolerance in premature infants and be conducive to the weight gain and improving nutritional status of premature infants. | PMC10782327 | |
Introduction | PRETERM BIRTH, COMPLICATIONS | Preterm birth is one of the most important health problems in the world.Due to their low gestational age and low weight at birth, the immature development of various systems and organs, poor sucking and swallowing function, and insufficient secretion of digestive enzymes, preterm infants are prone to various complications, and feeding intolerance is one of the most common and serious complications. | PMC10782327 |
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