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BACKGROUND: Deeper insight is needed on how monoclonal antibodies (mAbs) affect vaccine-mediated immune responses when targeting the same protein. We describe the first prospective randomised trial designed to understand mAb-mediated alterations in vaccine-induced immune responses to SARS-CoV-2 spike protein epitopes. METHODS: This randomised, open-label, parallel-group study assessed the potential interaction of a mAb combination, casirivimab and imdevimab, with a vaccine, Moderna's mRNA-1273, in healthy SARS-CoV-2 immunologically naive, seronegative adults at six centres in the USA. Participants were randomly assigned (per prespecified randomisation ratios within enrolment waves) according to a computer-generated randomisation scheme, stratified by age (<65 years and ≥65 years), to various intravenous or subcutaneous doses of casirivimab and imdevimab before, after, or at the same time as mRNA-1273 or to mRNA-1273 only. The doses of casirivimab and imdevimab were chosen to mimic various time intervals between receipt of 1200 mg of the mAb and the first dose of a primary series with mRNA-1273. The primary endpoint was vaccine-induced 50% inhibitory dilution neutralising antibody titres to SARS-CoV-2 spike protein, 56 days after the first vaccination. Secondary endpoints included vaccine-induced total antibodies to SARS-CoV-2 antigens and incidence of treatment-emergent adverse events. Exploratory endpoints included blood-derived T-cell and B-cell responses. The per-protocol set was used for the analysis of the primary endpoint and included all randomly assigned participants who received both doses of the vaccine and completed the injection or infusion of casirivimab and imdevimab per protocol, had no evidence of SARS-CoV-2 infection in the past or in the 56 days after the first dose of vaccine, and did not receive any intervention outside of the study that could alter the immune response. Safety was assessed in the safety analysis set, which included all randomly assigned participants who had received one or more doses of mRNA-1273 or any study drug, and analysed based on treatment received. The study is registered with ClinicalTrials.gov, NCT04852978, and is complete. FINDINGS: Between April 29, 2021, and Nov 21, 2022, 807 participants were assessed for eligibility and 295 were randomly assigned. 293 participants were included in the safety analysis set and 260 were included in the per-protocol set. All vaccinated participants developed neutralising antibodies to SARS-CoV-2, with median titres above the published protective threshold (100 IU/mL) against the SARS-CoV-2 D614G variant (considered a reference strain at the time the initial COVID-19 vaccines were developed). Titres were decreased up to 4-fold (median titres 280-450 IU/mL for casirivimab and imdevimab vs 1160 IU/mL for vaccine only on day 56) when casirivimab and imdevimab was given 85 days or less before vaccination (150-1200 mg intravenously) or co-administered subcutaneously (600 mg or 1200 mg) with vaccination. Minimal reduction in neutralisation titres was observed in the 48 mg and 12 mg intravenous groups, corresponding to receipt of casirivimab and imdevimab 113 days and 169 days, respectively, before vaccination, and when administering the vaccine 6 days before the mAb. Across all groups, mAbs had a minimal effect on vaccine-induced total antibodies and T-cell responses to the spike protein. Casirivimab and imdevimab plus mRNA-1273 was generally well tolerated; a slight increase in treatment-emergent adverse events was observed in the casirivimab and imdevimab plus vaccine groups versus the vaccine-only group. INTERPRETATION: Casirivimab and imdevimab administration before or at the time of COVID-19 vaccination reduced the elicitation of SARS-CoV-2 neutralising antibodies, but minimal effect was observed when vaccination occurred before mAb administration. Although the clinical significance of this decrease in neutralisation is unclear, this evidence suggests that further investigation of potential interactions could be warranted before concurrent clinical use of mAbs and vaccines targeting the same viral proteins as their main modes of action for the prevention or treatment of infectious diseases. FUNDING: Regeneron Pharmaceuticals and F Hoffmann-La Roche.
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OBJECTIVES: A phase 1, double-blind, placebo-controlled trial was conducted to evaluate the safety, tolerability, and exploratory efficacy of repeat monthly doses of subcutaneous (SC) casirivimab and imdevimab (CAS+IMD) in uninfected adult volunteers. METHODS: Participants were randomized (3:1) to SC CAS+IMD 1200 mg or placebo every 4 weeks for up to six doses. Primary and secondary end points evaluated safety, pharmacokinetics, and immunogenicity. Exploratory efficacy was evaluated by the incidence of COVID-19 or SARS-CoV-2 seroconversion. RESULTS: In total, 969 participants received CAS+IMD. Repeat monthly dosing of SC CAS+IMD led to a 92.4% relative risk reduction in clinically defined COVID-19 compared with placebo (3/729 [0.4%] vs 13/240 [5.4%]; odds ratio 0.07 [95% CI 0.01-0.27]), and a 100% reduction in laboratory-confirmed COVID-19 (0/729 vs 10/240 [4.2%]; odds ratio 0.00). Development of anti-drug antibodies occurred in a small proportion of participants (<5%). No grade ≥3 injection-site reactions (ISRs) or hypersensitivity reactions were reported. Slightly more participants reported treatment-emergent adverse events with CAS+IMD (54.9%) than with placebo (48.3%), a finding that was due to grade 1-2 ISRs. Serious adverse events were rare. No deaths were reported in the 6-month treatment period. CONCLUSION: Repeat monthly administration of 1200 mg SC CAS+IMD was well-tolerated, demonstrated low immunogenicity, and showed a substantial risk reduction in COVID-19 occurrence.
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Tratamiento Farmacológico de COVID-19 , COVID-19 , Adulto , Anticuerpos Monoclonales Humanizados , COVID-19/prevención & control , Método Doble Ciego , Humanos , SARS-CoV-2RESUMEN
Importance: Easy-to-administer anti-SARS-CoV-2 treatments may be used to prevent progression from asymptomatic infection to symptomatic disease and to reduce viral carriage. Objective: To evaluate the effect of combination subcutaneous casirivimab and imdevimab on progression from early asymptomatic SARS-CoV-2 infection to symptomatic COVID-19. Design, Setting, and Participants: Randomized, double-blind, placebo-controlled, phase 3 trial of close household contacts of a SARS-CoV-2-infected index case at 112 sites in the US, Romania, and Moldova enrolled July 13, 2020-January 28, 2021; follow-up ended March 11, 2021. Asymptomatic individuals (aged ≥12 years) were eligible if identified within 96 hours of index case positive test collection. Results from 314 individuals positive on SARS-CoV-2 reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) testing are reported. Interventions: Individuals were randomized 1:1 to receive 1 dose of subcutaneous casirivimab and imdevimab, 1200 mg (600 mg of each; n = 158), or placebo (n = 156). Main Outcomes and Measures: The primary end point was the proportion of seronegative participants who developed symptomatic COVID-19 during the 28-day efficacy assessment period. The key secondary efficacy end points were the number of weeks of symptomatic SARS-CoV-2 infection and the number of weeks of high viral load (>4 log10 copies/mL). Results: Among 314 randomized participants (mean age, 41.0 years; 51.6% women), 310 (99.7%) completed the efficacy assessment period; 204 were asymptomatic and seronegative at baseline and included in the primary efficacy analysis. Subcutaneous casirivimab and imdevimab, 1200 mg, significantly prevented progression to symptomatic disease (29/100 [29.0%] vs 44/104 [42.3%] with placebo; odds ratio, 0.54 [95% CI, 0.30-0.97]; P = .04; absolute risk difference, -13.3% [95% CI, -26.3% to -0.3%]). Casirivimab and imdevimab reduced the number of symptomatic weeks per 1000 participants (895.7 weeks vs 1637.4 weeks with placebo; P = .03), an approximately 5.6-day reduction in symptom duration per symptomatic participant. Treatment with casirivimab and imdevimab also reduced the number of high viral load weeks per 1000 participants (489.8 weeks vs 811.9 weeks with placebo; P = .001). The proportion of participants receiving casirivimab and imdevimab who had 1 or more treatment-emergent adverse event was 33.5% vs 48.1% for placebo, including events related (25.8% vs 39.7%) or not related (11.0% vs 16.0%) to COVID-19. Conclusions and Relevance: Among asymptomatic SARS-CoV-2 RT-qPCR-positive individuals living with an infected household contact, treatment with subcutaneous casirivimab and imdevimab antibody combination vs placebo significantly reduced the incidence of symptomatic COVID-19 over 28 days. Trial Registration: ClinicalTrials.gov Identifier: NCT04452318.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/efectos adversos , Infecciones Asintomáticas , COVID-19/epidemiología , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19 , Niño , Progresión de la Enfermedad , Método Doble Ciego , Combinación de Medicamentos , Femenino , Humanos , Incidencia , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Factores de Riesgo , Carga ViralRESUMEN
BACKGROUND: REGEN-COV (previously known as REGN-COV2), a combination of the monoclonal antibodies casirivimab and imdevimab, has been shown to markedly reduce the risk of hospitalization or death among high-risk persons with coronavirus disease 2019 (Covid-19). Whether subcutaneous REGEN-COV prevents severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and subsequent Covid-19 in persons at high risk for infection because of household exposure to a person with SARS-CoV-2 infection is unknown. METHODS: We randomly assigned, in a 1:1 ratio, participants (≥12 years of age) who were enrolled within 96 hours after a household contact received a diagnosis of SARS-CoV-2 infection to receive a total dose of 1200 mg of REGEN-COV or matching placebo administered by means of subcutaneous injection. At the time of randomization, participants were stratified according to the results of the local diagnostic assay for SARS-CoV-2 and according to age. The primary efficacy end point was the development of symptomatic SARS-CoV-2 infection through day 28 in participants who did not have SARS-CoV-2 infection (as measured by reverse-transcriptase-quantitative polymerase-chain-reaction assay) or previous immunity (seronegativity). RESULTS: Symptomatic SARS-CoV-2 infection developed in 11 of 753 participants in the REGEN-COV group (1.5%) and in 59 of 752 participants in the placebo group (7.8%) (relative risk reduction [1 minus the relative risk], 81.4%; P<0.001). In weeks 2 to 4, a total of 2 of 753 participants in the REGEN-COV group (0.3%) and 27 of 752 participants in the placebo group (3.6%) had symptomatic SARS-CoV-2 infection (relative risk reduction, 92.6%). REGEN-COV also prevented symptomatic and asymptomatic infections overall (relative risk reduction, 66.4%). Among symptomatic infected participants, the median time to resolution of symptoms was 2 weeks shorter with REGEN-COV than with placebo (1.2 weeks and 3.2 weeks, respectively), and the duration of a high viral load (>104 copies per milliliter) was shorter (0.4 weeks and 1.3 weeks, respectively). No dose-limiting toxic effects of REGEN-COV were noted. CONCLUSIONS: Subcutaneous REGEN-COV prevented symptomatic Covid-19 and asymptomatic SARS-CoV-2 infection in previously uninfected household contacts of infected persons. Among the participants who became infected, REGEN-COV reduced the duration of symptomatic disease and the duration of a high viral load. (Funded by Regeneron Pharmaceuticals and others; ClinicalTrials.gov number, NCT04452318.).
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Anticuerpos Monoclonales Humanizados/uso terapéutico , COVID-19/prevención & control , SARS-CoV-2 , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades Asintomáticas , COVID-19/virología , Niño , Método Doble Ciego , Combinación de Medicamentos , Femenino , Humanos , Incidencia , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Gravedad del Paciente , Carga Viral , Adulto Joven , Tratamiento Farmacológico de COVID-19RESUMEN
IMPORTANCE: Easy-to-administer antiviral treatments may be used to prevent progression from asymptomatic infection to COVID-19 and to reduce viral carriage. OBJECTIVE: Evaluate the efficacy and safety of subcutaneous casirivimab and imdevimab antibody combination (REGEN-COV) to prevent progression from early asymptomatic SARS-CoV-2 infection to COVID-19. DESIGN: Randomized, double-blind, placebo-controlled, phase 3 study that enrolled asymptomatic close contacts living with a SARS-CoV-2-infected household member (index case). Participants who were SARS-CoV-2 RT-qPCR-positive at baseline were included in the analysis reported here. SETTING: Multicenter trial conducted at 112 sites in the United States, Romania, and Moldova. PARTICIPANTS: Asymptomatic individuals ≥12 years of age were eligible if identified within 96 hours of collection of the index case's positive SARS-CoV-2 test sample. INTERVENTIONS: A total of 314 asymptomatic, SARS-CoV-2 RT-qPCR-positive individuals living with an infected household contact were randomized 1:1 to receive a single dose of subcutaneous REGEN-COV 1200mg (n=158) or placebo (n=156). MAIN OUTCOMES AND MEASURES: The primary endpoint was the proportion of participants who developed symptomatic COVID-19 during the 28-day efficacy assessment period. The key secondary efficacy endpoints were the number of weeks of symptomatic SARS-CoV-2 infection and the number of weeks of high viral load (>4 log10 copies/mL). Safety was assessed in all treated participants. RESULTS: Subcutaneous REGEN-COV 1200mg significantly prevented progression from asymptomatic to symptomatic disease compared with placebo (31.5% relative risk reduction; 29/100 [29.0%] vs 44/104 [42.3%], respectively; P=.0380). REGEN-COV reduced the overall population burden of high-viral load weeks (39.7% reduction vs placebo; 48 vs 82 total weeks; P=.0010) and of symptomatic weeks (45.3% reduction vs placebo; 89.6 vs 170.3 total weeks; P=.0273), the latter corresponding to an approximately 5.6-day reduction in symptom duration per symptomatic participant. Six placebo-treated participants had a COVID-19-related hospitalization or ER visit versus none for those receiving REGEN-COV. The proportion of participants receiving placebo who had ≥1 treatment-emergent adverse events was 48.1% compared with 33.5% for those receiving REGEN-COV, including events related (39.7% vs 25.8%, respectively) or not related (16.0% vs 11.0%, respectively) to COVID-19. CONCLUSIONS AND RELEVANCE: Subcutaneous REGEN-COV 1200mg prevented progression from asymptomatic SARS-CoV-2 infection to COVID-19, reduced the duration of high viral load and symptoms, and was well tolerated. TRIAL REGISTRATION: ClinicalTrials.gov Identifier, NCT04452318.
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Background: Casirivimab and imdevimab (REGEN-COV™) markedly reduces risk of hospitalization or death in high-risk individuals with Covid-19. Here we explore the possibility that subcutaneous REGEN-COV prevents SARS-CoV-2 infection and subsequent Covid-19 in individuals at high risk of contracting SARS-CoV-2 by close exposure in a household with a documented SARS-CoV-2-infected individual. Methods: Individuals ≥12 years were enrolled within 96 hours of a household contact being diagnosed with SARS-CoV-2 and randomized 1:1 to receive 1200 mg REGEN-COV or placebo via subcutaneous injection. The primary efficacy endpoint was the proportion of participants without evidence of infection (SARS-CoV-2 RT-qPCR-negative) or prior immunity (seronegative) who subsequently developed symptomatic SARS-CoV-2 infection during a 28-day efficacy assessment period. Results: Subcutaneous REGEN-COV significantly prevented symptomatic SARS-CoV-2 infection compared with placebo (81.4% risk reduction; 11/753 [1.5%] vs. 59/752 [7.8%], respectively; P<0.0001), with 92.6% risk reduction after the first week (2/753 [0.3%] vs. 27/752 [3.6%], respectively). REGEN-COV also prevented overall infections, either symptomatic or asymptomatic (66.4% risk reduction). Among infected participants, the median time to resolution of symptoms was 2 weeks shorter with REGEN-COV vs. placebo (1.2 vs. 3.2 weeks, respectively), and the duration of time with high viral load (>104 copies/mL) was lower (0.4 vs. 1.3 weeks, respectively). REGEN-COV was generally well tolerated. Conclusions: Administration of subcutaneous REGEN-COV prevented symptomatic Covid-19 and asymptomatic SARS-CoV-2 infection in uninfected household contacts of infected individuals. Among individuals who became infected, REGEN-COV reduced the duration of symptomatic disease, decreased maximal viral load, and reduced the duration of detectable virus.(ClinicalTrials.gov number, NCT04452318.).
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Mobile health (mHealth) technologies have the potential to capture dense patient data on the background of real-life behavior. Merck & Co., Inc. (Kenilworth, NJ), in collaboration with Koneksa Health, conducted a phase I clinical trial to validate cardiovascular mHealth technologies for concordance with traditional approaches and to establish sensitivity to detect effects of pharmacological intervention. This two-part study enrolled 18 healthy male subjects. Part I, a 5-day study, compared mHealth measures of heart rate (HR) and blood pressure (BP) to those from traditional methods. Hypotheses of similarity, in the clinic and at home, were tested individually for HR, systolic BP, and diastolic BP, at a 2-sided 0.05 alpha level, with a prespecified criterion for similarity being the percentage differences between the 2 measurements within 15%. Part II, a 7-day, 3-period randomized balanced crossover study, evaluated the mHealth technology's ability to detect effects of bisoprolol and salbutamol. Hypotheses that the changes from baseline in HR were greater in the bisoprolol (reduction in HR) and salbutamol (increase in HR) groups compared with no treatment were tested, at a 1-sided 0.05 alpha level. Linear mixed-effects models, Pearson's correlation coefficients, summary statistics, and exploratory plots were applied to analyze the data. The mHealth measures of HR and BP were demonstrated to be similar to those from traditional methods, and sensitive to changes in cardiovascular parameters induced by bisoprolol and salbutamol.
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Albuterol/farmacología , Bisoprolol/farmacología , Presión Sanguínea/efectos de los fármacos , Análisis de Datos , Frecuencia Cardíaca/efectos de los fármacos , Telemedicina/métodos , Adolescente , Adulto , Anciano , Antihipertensivos/farmacología , Presión Sanguínea/fisiología , Estudios Cruzados , Frecuencia Cardíaca/fisiología , Humanos , Masculino , Persona de Mediana Edad , Telemedicina/estadística & datos numéricos , Adulto JovenRESUMEN
RATIONALE: Suvorexant is a first-in-class orexin receptor antagonist for treating insomnia. There is a general concern that hypnotics may impair next-morning driving ability. OBJECTIVE: The objective of this study was to evaluate next-morning driving performance in older adults after single and repeated doses of suvorexant. METHODS: Double-blind, randomized, placebo-controlled, 4-period crossover study in 24 healthy volunteers (10 females), aged 65-80 years. Subjects were treated with suvorexant (15 and 30 mg) for eight consecutive nights, zopiclone 7.5 mg nightly on days 1 and 8, and placebo. Driving performance was assessed on days 2 and 9 (9 h after dosing) using a 1-h standardized highway driving test in normal traffic, measuring standard deviation of lateral position (SDLP). Drug-placebo differences in SDLP >2.4 cm were considered to reflect clinically meaningful driving impairment. RESULTS: Driving performance as measured by SDLP was not impaired following suvorexant. Mean drug-placebo differences in SDLP following suvorexant 15 and 30 mg on day 2 and 9 were 0.6 cm or less. Their 90 % CIs were all below the threshold of 2.4 cm for clinical relevance and included zero, indicating effects were not clinically meaningful or statistically significant. Symmetry analysis showed no significant differences between the number of participants who had SDLP differences >2.4 cm and those who had SDLP differences <-2.4 cm following suvorexant. CONCLUSIONS: There was no clinically meaningful residual effect of suvorexant 15 and 30 mg on next-morning driving (9 h after bedtime dosing) in healthy older adults, as assessed by mean changes in SDLP and by the number of participants on drug versus placebo that exceeded a predetermined threshold for clinically meaningful impairment.
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Conducción de Automóvil , Compuestos de Azabiciclo/farmacología , Azepinas/farmacología , Hipnóticos y Sedantes/farmacología , Piperazinas/farmacología , Desempeño Psicomotor/efectos de los fármacos , Fármacos Inductores del Sueño/farmacología , Triazoles/farmacología , Anciano , Anciano de 80 o más Años , Azepinas/administración & dosificación , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Voluntarios Sanos , Humanos , Masculino , Fármacos Inductores del Sueño/administración & dosificación , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Factores de Tiempo , Triazoles/administración & dosificaciónRESUMEN
OBJECTIVES: We sought to validate Cognitive Research Corporation's Driving Simulator (CRCDS Mini-Sim) for studies of drug safety with respect to driving ability. METHODS: A total of 30 healthy subjects were randomized to receive placebo or 7.5 mg zopiclone, a hypnotic known to impair driving, in random order during the 2 treatment periods of a 2 period crossover design. RESULTS: Evening administration of 7.5 mg zopiclone increased next-day standard deviation of lateral lane position (SDLP) by 2.62 cm on average compared with evening administration of placebo, and caused significant effects on symmetry analysis. The magnitude of the change in SDLP is highly similar to changes previously observed using on-the-road driving methods. CONCLUSIONS: Further validation of the CRCDS Mini-Sim is warranted to develop this platform for drug safety studies.
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STUDY OBJECTIVE: To evaluate next-morning driving performance in adults younger than 65 years, after single and repeated doses of suvorexant 20 and 40 mg. DESIGN: Double-blind, placebo-controlled, 4-period crossover study. SETTING: Maastricht University, The Netherlands. PARTICIPANTS: 28 healthy volunteers (15 females), aged 23 to 64 years. INTERVENTIONS: Suvorexant (20 and 40 mg) for 8 consecutive nights; zopiclone 7.5 mg nightly on day 1 and 8; placebo. MEASUREMENTS: Performance on day 2 and 9 (9 h after dosing) using a one-hour standardized highway driving test in normal traffic, measuring standard deviation of lateral position (SDLP). Drug-placebo changes in SDLP > 2.4 cm were considered to reflect meaningful driving impairment. RESULTS: Mean drug-placebo changes in SDLP following suvorexant 20 and 40 mg were 1.01 and 1.66 cm on day 2, and 0.48 and 1.31 cm on Day 9, respectively. The 90% CIs of these changes were all below 2.4 cm. Symmetry analysis showed that more subjects had SDLP changes > 2.4 cm than < -2.4 cm following suvorexant 20 and 40 mg on day 2, and following suvorexant 40 mg on day 9. Four female subjects requested that a total of 5 driving tests--all following suvorexant--stop prematurely due to self-reported somnolence. CONCLUSIONS: As assessed by mean changes in standard deviation of lateral position (SDLP), there was no clinically meaningful residual effect of suvorexant in doses of 20 and 40 mg on next-morning driving (9 h after bedtime dosing) in healthy subjects < 65 years old. There may be some individuals who experience next-day effects, as suggested by individual changes in SDLP and prematurely stopped tests. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov NCT01311882.
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Conducción de Automóvil/psicología , Azepinas/administración & dosificación , Azepinas/farmacología , Voluntarios Sanos , Fármacos Inductores del Sueño/administración & dosificación , Fármacos Inductores del Sueño/farmacología , Triazoles/administración & dosificación , Triazoles/farmacología , Adulto , Compuestos de Azabiciclo/administración & dosificación , Compuestos de Azabiciclo/farmacología , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Individualidad , Masculino , Persona de Mediana Edad , Países Bajos , Piperazinas/administración & dosificación , Piperazinas/farmacología , Desempeño Psicomotor/efectos de los fármacos , Autoinforme , Fases del Sueño/efectos de los fármacos , Fases del Sueño/fisiología , Adulto JovenRESUMEN
Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H(2)O(2) in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.
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Autofagia/efectos de los fármacos , Mitocondrias/metabolismo , Orgánulos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Antracenos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitofagia/efectos de los fármacos , Orgánulos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Perileno/análogos & derivados , Perileno/farmacología , Factores de TiempoRESUMEN
Both the commitment event and the modality of cell death in photodynamic therapy (PDT) remain poorly defined. We report that PDT with endoplasmic reticulum (ER)-associating hypericin leads to an immediate loss of SERCA2 protein levels, causing disruption of Ca2+ homeostasis and cell death. Protection of SERCA2 protein rescues ER-Ca2+ levels and prevents cell death, suggesting that SERCA2 photodestruction with consequent incapability of the ER to maintain intracellular Ca2+ homeostasis is causal to cell killing. Apoptosis is rapidly initiated after ER-Ca2+ depletion and strictly requires the BAX/BAK gateway at the mitochondria. Bax-/-Bak-/- double-knockout (DKO) cells are protected from apoptosis but undergo autophagy-associated cell death as revealed by electron microscopy and biochemical analysis. Autophagy inhibitors, but not caspase antagonists, significantly reduce death of DKO cells, suggesting that sustained autophagy is lethal. Thus, following ER photodamage and consequent disruption of Ca2+ homeostasis, BAX and BAK proteins model PDT-mediated cell killing, which is executed through apoptosis in their presence or via an autophagic pathway in their absence.
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Apoptosis/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Perileno/análogos & derivados , Fotoquimioterapia , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Antracenos , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de la radiación , Fibroblastos/metabolismo , Eliminación de Gen , Humanos , Ratones , Perileno/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Using tumor cell-restricted overexpression of glutathione peroxidase 4 (GP x 4), we investigated the contribution of tumor cell eicosanoids to solid tumor growth and malignant progression in two tumor models differing in tumorigenic potential. By lowering cellular lipid hydroperoxide levels, GP x 4 inhibits cyclooxygenase (COX) and lipoxygenase (LOX) activities. GP x 4 overexpression drastically impeded solid tumor growth of weakly tumorigenic L929 fibrosarcoma cells, whereas B16BL6 melanoma solid tumor growth was unaffected. Yet, GP x 4 overexpression did markedly increase the sensitivity of B16BL6 tumors to angio-destructive TNF-alpha therapy and abolished the metastatic lung colonizing capacity of B16BL6 cells. Furthermore, the GP x 4-mediated suppression of tumor cell prostaglandin E(2) (PGE(2)) production impeded the induction of COX-2 expression by the tumor stress conditions hypoxia and inflammation. Thus, our results reflect a PGE(2)-driven positive feedback loop for COX-2 expression in tumor cells. This was further supported by the restoration of COX-2 induction capacity of GP x 4-overexpressing L929 tumor cells when cultured in the presence of exogenous PGE(2). Thus, although COX-2 expression and eicosanoid production may be enabled by PGE(2) from the tumor microenvironment, our results demonstrate the predominant tumor cell origin of protumoral eicosanoids, promoting solid tumor growth of weakly tumorigenic tumors and malignant progression of strongly tumorigenic tumors.
Asunto(s)
Eicosanoides/biosíntesis , Fibrosarcoma/fisiopatología , Glutatión Peroxidasa/metabolismo , Melanoma/fisiopatología , Metástasis de la Neoplasia/prevención & control , Animales , Hipoxia de la Célula/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 2/efectos de los fármacos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Modelos Animales de Enfermedad , Eicosanoides/antagonistas & inhibidores , Femenino , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/genética , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Transferencia de Gen , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/farmacología , Lipooxigenasa/efectos de los fármacos , Lipooxigenasa/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Porcinos , Factores de Tiempo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/uso terapéuticoRESUMEN
BACKGROUND/AIMS: Mycophenolate mofetil (MMF) is known to attenuate glomerulosclerosis in experimental models of renal failure. We investigated whether this is mediated by reduction of oxidative stress. METHODS: Effects of MMF on oxidative stress are studied in an experimental rat model (NA model) involving unilateral nephrectomy and two intravenous injections with adriamycin (2 mg/kg). Rats are sacrificed after 2 and 6 weeks. Glomerulosclerosis and tubulointerstitial lesions are demonstrated by histological techniques. Presence of macrophages/monocytes (ED1) and myofibroblasts (alpha-SMA) is demonstrated by immunohistochemistry. Oxidative stress is evaluated by enzymatic measurements (AOE), spectrofluorometry (TBARS), immunohistochemistry (MDA and HNE) and histology (ferric iron deposition). RESULTS: The NA model shows proteinuria, hypercholesterolemia, beginning glomerulosclerosis, tubulointerstitial sclerosis and tubular dilatation, glomerular, periglomerular and interstitial presence of alpha-SMA and increased presence of macrophages/monocytes after 6 weeks. Oxidative stress in renal cortex is apparent (increased cortex TBARS concentration, increased glomerular presence of MDA and HNE, decreased activity of antioxidant enzymes, ferric iron deposition in proximal tubules) after 6 weeks. MMF administration results in a decrease of glomerulosclerosis, interstitial sclerosis, glomerular and periglomerular expression of alpha-SMA and the number of ED1-positive cells in tubulointerstitium and glomeruli. Proteinuria and cholesterolemia are not decreased. TBARS level, and activities of catalase, Mn and Cu/Zn superoxide dismutase as well as the presence of ferric iron in the proximal tubules are not changed by MMF treatment. Cortex activity of glutathione peroxidase returns to normal. CONCLUSION: MMF has a favorable effect on glomerular and interstitial fibrosis in the NA model of kidney disease, but not on proteinuria and cholesterolemia. Improvement of fibrosis cannot be explained by major changes in oxidative stress or antioxidant defense.