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Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an â¼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.
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Tratamiento Basado en Trasplante de Células y Tejidos , Ejercicio Físico , Humanos , Biblioteca de Genes , Inmunoterapia , InvestigaciónRESUMEN
Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.
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Exones , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Empalme del ARN , Análisis de Secuencia de ADN/métodos , Separación Celular , Biología Computacional , Citometría de Flujo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Intrones , Células K562 , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los ResultadosRESUMEN
Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.
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Sistemas CRISPR-Cas/genética , ADN de Cadena Simple/genética , Farmacorresistencia Microbiana/genética , Ingeniería Genética , Genoma Bacteriano/genética , Alelos , ADN de Cadena Simple/biosíntesis , Escherichia coli/genética , Biblioteca de Genes , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento , Saccharomyces cerevisiae/genética , Biología SintéticaRESUMEN
The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 "recalcitrant" AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional "safe replacement zone" (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.
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Arginina/genética , Escherichia coli/genética , ARN Mensajero/genética , Aminoácidos/genética , Codón/genética , Genes Esenciales/genética , Código Genético , Genoma Bacteriano , Biosíntesis de Proteínas/genética , ARN Mensajero/biosíntesisRESUMEN
Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by â¼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is â¼10-fold more efficient at achieving allelic diversity than previous best practices.
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Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Ingeniería Genética/métodos , Proteínas de Transporte de Membrana/genética , Alelos , Biomarcadores , Escherichia coli/genética , Eliminación de Gen , Duplicación de Gen , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , FenotipoRESUMEN
The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.
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Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Biblioteca de Genes , Ingeniería Genética/métodos , Modelos Genéticos , ARN Mensajero/genética , Biología de Sistemas/métodos , Clonación Molecular , Cartilla de ADN/genética , Escherichia coli/genética , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Regiones Promotoras Genéticas/genética , Elementos Reguladores de la Transcripción/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosomas/genéticaRESUMEN
Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.
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Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.
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Euterios , Evolución Molecular , Animales , Femenino , Humanos , Secuencia Conservada/genética , Euterios/genética , Genoma HumanoRESUMEN
PURPOSE: This study evaluated the pharmacokinetics (PK), pharmacodynamics (PD), safety/tolerability, and cardiac safety of liposomal muramyl tripeptide phosphatidyl-ethanolamine [mifamurtide (L-MTP-PE)] in healthy adults. METHODS: L-MTP-PE 4 mg was administered intravenously over 30 min. Study participants were monitored from 24 h preinfusion until 72 h postinfusion. Blood samples were drawn over 0-72 h postdose to determine serum MTP-PE, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and C-reactive protein (CRP) concentrations. Electrocardiograpic (ECG) data were collected via continuous Holter monitoring beginning 24 h predose through 24 h postdose. Changes from time-matched pretreatment baseline QTc and associated two-sided 90 % confidence intervals were calculated. RESULTS: Twenty-one participants received L-MTP-PE. Total serum MTP-PE declined rapidly with a terminal half-life of 2.05 ± 0.40 h. PK variability was low, with <30 % coefficient of variation in systemic exposure. Serum concentrations of IL-6, TNF-α, and CRP increased following L-MTP-PE infusion. Maximum observed increases in IL-6 and TNF-α occurred at 4 and 2 h, respectively, returning toward baseline by 8 h postdose. L-MTP-PE was generally well tolerated, with no adverse events greater than grade 3. Headache, chills, tachycardia, nausea, and pyrexia were the most frequent adverse events. L-MTP-PE infusion resulted in an increased heart rate without readily apparent QTc prolongation. CONCLUSIONS: MTP-PE PK following L-MTP-PE administration were characterized by a short serum half-life and low variability. Increases in IL-6, TNF-α, and CRP and the safety profile were consistent with the immunomodulatory mechanism of action. No clinically significant effect of L-MTP-PE on cardiovascular repolarization was observed based on analysis of ECG QTc intervals.
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Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Corazón/efectos de los fármacos , Fosfatidiletanolaminas/administración & dosificación , Fosfatidiletanolaminas/farmacocinética , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/efectos adversos , Acetilmuramil-Alanil-Isoglutamina/farmacocinética , Adulto , Área Bajo la Curva , Proteína C-Reactiva/metabolismo , Electrocardiografía/métodos , Femenino , Corazón/fisiología , Humanos , Interleucina-6/sangre , Interleucina-6/metabolismo , Liposomas/administración & dosificación , Masculino , Persona de Mediana Edad , Fosfatidiletanolaminas/efectos adversos , Estudios Prospectivos , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismo , Adulto JovenRESUMEN
Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.
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Recurrencia Local de Neoplasia , Receptores Quiméricos de Antígenos , Humanos , Recurrencia Local de Neoplasia/metabolismo , Antígeno de Maduración de Linfocitos B , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Linfocitos T , Inmunoterapia , Transducción de SeñalRESUMEN
Rezafungin is a new echinocandin in development for treatment of candidemia and invasive candidiasis, and for prophylaxis of invasive fungal infections. Rezafungin is the first echinocandin to undergo definitive QT/QTc study. This phase 1, single-center, randomized, double-blind trial was conducted to assess effects of intravenous rezafungin vs intravenous placebo (with moxifloxacin as positive control) on the QT interval of the electrocardiogram, corrected for heart rate by Fridericia's formula (QTcF), in healthy adults. Therapeutic (600 mg) and supratherapeutic (1400 mg) rezafungin doses were selected to achieve exposures 2.5-fold higher than produced by the highest dose used in a phase 2 trial (400 mg once weekly). The primary end point was change in QTcF from baseline (ΔQTcF) as a function of plasma concentration, assessed by comparing upper bounds of the 2-sided 90% confidence interval. The estimated mean ΔΔQTcF at the mean plasma concentrations for the rezafungin doses had upper bounds <10 milliseconds, within the upper bound of the 2-sided 90% confidence interval. Intravenous rezafungin up to 1400 mg in a single dose did not prolong QT interval and had no apparent effect on repolarization or QRS duration. Electrocardiogram results showed no clinically significant effects of concern. These findings support the continued development of rezafungin.
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Antifúngicos/efectos adversos , Equinocandinas/efectos adversos , Electrocardiografía , Adulto , Antifúngicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Equinocandinas/administración & dosificación , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Síndrome de QT Prolongado/etiología , Masculino , Persona de Mediana Edad , Moxifloxacino/administración & dosificación , Moxifloxacino/efectos adversos , Adulto JovenRESUMEN
The amplification of millions of single molecules in parallel can be performed on microscopic magnetic beads that are contained in aqueous compartments of an oil-buffer emulsion. These bead-emulsion amplification (BEA) reactions result in beads that are covered by almost-identical copies derived from a single template. The post-amplification analysis is performed using different fluorophore-labeled probes. We have identified BEA reaction conditions that efficiently produce longer amplicons of up to 450 base pairs. These conditions include the use of a Titanium Taq amplification system. Second, we explored alternate fluorophores coupled to probes for post-PCR DNA analysis. We demonstrate that four different Alexa fluorophores can be used simultaneously with extremely low crosstalk. Finally, we developed an allele-specific extension chemistry that is based on Alexa dyes to query individual nucleotides of the amplified material that is both highly efficient and specific.
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ADN/análisis , ADN/genética , Colorantes Fluorescentes/química , Microesferas , Técnicas de Amplificación de Ácido Nucleico/métodos , Absorción , Alelos , Animales , Composición de Base , Bovinos , Color , ADN/química , Emulsiones , HumanosRESUMEN
We describe a method that enables the multiplex screening of a pool of many different donor cell lines. Our method accurately predicts each donor proportion from the pool without requiring the use of unique DNA barcodes as markers of donor identity. Instead, we take advantage of common single nucleotide polymorphisms, whole-genome sequencing, and an algorithm to calculate the proportions from the sequencing data. By testing using simulated and real data, we showed that our method robustly predicts the individual proportions from a mixed-pool of numerous donors, thus enabling the multiplexed testing of diverse donor cells en masse.More information is available at https://pgpresearch.med.harvard.edu/poolseq/.
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Linfocitos B/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Donantes de Tejidos , Secuenciación Completa del Genoma , Algoritmos , Simulación por Computador , Genoma Humano , Humanos , Polimorfismo de Nucleótido Simple/genética , Tamaño de la MuestraRESUMEN
BACKGROUND: Reference ranges for electrocardiogram (ECG) intervals, heart rate, and QRS axis in general use by medical personnel and ECG readers are unrepresentative of true age- and sex-related values in large populations and are not based on modern electrocardiographic and ECG reading technology. METHODS AND RESULTS: The results of ECG interpretation by cardiologists using digital technology for viewing and interpreting ECGs were compiled from single, baseline ECGs of 79,743 individuals included in pharmaceutical company-sponsored clinical trials. Women comprised 48% of the total population. Ages ranged from 3 months to 99 years, and the bulk of the population (56%) was aged 40 to 70 years. Striking differences in numerical ECG values based on age and sex were observed. A subgroup of 46,129 individuals with a very low probability of cardiovascular disease was identified. The following were the reference ranges for this subgroup, determined using the 2nd and 98th percentiles: heart rate, 48 to 98 beats/min; PR interval, 113 to 212 milliseconds; QRS interval, 69 to 109 milliseconds; frontal plane QRS axis, -40 degrees to 91 degrees ; QT interval, 325 to 452 milliseconds; QTc-Bazett, 361 to 457 milliseconds; and QTc-Fridericia, 359 to 445 milliseconds. There were marked age- and sex-related variations in the reference ranges of this subgroup, and they differ substantially from previously reported norms. Small differences were observed in ECG values obtained using our digital methods as compared with readings done using paper tracings and values computed by 2 commercial computer algorithms. CONCLUSIONS: We observed large differences in electrocardiographic heart rate, interval, and axis reference ranges in this study compared with those reported previously and with reference ranges in general use. We also observed a large influence of age and sex upon normal values. Very large cohorts are required to fully assess age- and sex-related variation of reference ranges. Electrocardiographic reference ranges should be modernized.
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Electrocardiografía Ambulatoria/estadística & datos numéricos , Electrocardiografía Ambulatoria/normas , Frecuencia Cardíaca/fisiología , Adolescente , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Nevada/epidemiología , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución por SexoRESUMEN
DNAplotlib ( www.dnaplotlib.org ) is a computational toolkit for the programmable visualization of highly customizable, standards-compliant genetic designs. Functions are provided to aid with both visualization tasks and to extract and overlay associated experimental data. High-quality output is produced in the form of vector-based PDFs, rasterized images, and animated movies. All aspects of the rendering process can be easily customized or extended by the user to cover new forms of genetic part or regulation. DNAplotlib supports improved communication of genetic design information and offers new avenues for static, interactive and dynamic visualizations that map and explore the links between the structure and function of genetic parts, devices and systems; including metabolic pathways and genetic circuits. DNAplotlib is cross-platform software developed using Python.
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Biología Computacional/métodos , Programas Informáticos , Biología Sintética/métodos , Redes y Vías Metabólicas/genética , Interfaz Usuario-ComputadorRESUMEN
We present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.∆A. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.
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Escherichia coli/genética , Ingeniería Genética , Genoma Bacteriano/genética , Genómica , Variación Genética , Genotipo , MutaciónRESUMEN
Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.
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Biología Computacional/instrumentación , Genoma Microbiano/genética , Genómica , Programas Informáticos , Genoma , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Internet , Oligonucleótidos/genéticaRESUMEN
Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.
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Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Sintéticos , Código Genético/fisiología , Genoma Bacteriano , Genes Esenciales , Genes Letales , Código Genético/genética , Ingeniería Genética , Fenotipo , Biosíntesis de Proteínas/genéticaRESUMEN
OBJECTIVE: A clinical trial was conducted in healthy adult volunteers to assess the effect of levofloxacin, moxifloxacin, and ciprofloxacin on the QT and QTc interval. METHODS: Electrocardiograms were recorded 24 hours before and after subjects took placebo, 1000 mg levofloxacin, 800 mg moxifloxacin, and 1500 mg ciprofloxacin in a double-blind, randomized, 4-period, 4-treatment, 4-sequence crossover trial. Changes in QT and QTc interval from baseline were assessed by several different methods. RESULTS: Increases in QT and QTc interval compared with placebo were consistently greater after moxifloxacin compared with either levofloxacin or ciprofloxacin. The mean postdose change from baseline QTc (Bazett) intervals for the 24-hour period after treatment with moxifloxacin ranged from 16.34 to 17.83 ms (P < .001, compared with placebo). For levofloxacin, this change ranged from 3.53 to 4.88 ms (P < .05, compared with placebo), and for ciprofloxacin, this change ranged from 2.27 to 4.93 ms (P < .05, compared with placebo, with the use of 3 of 5 baseline methods). CONCLUSIONS: A change in QTc (Bazett) interval from baseline can be demonstrated safely in healthy volunteers after single high doses of fluoroquinolones that achieve approximately 1.5 times the maximum plasma drug concentration that occurs after recommended doses. There is substantial daily variation in both QT and QTc interval, and the magnitude and frequency of changes in QTc interval can depend on the methods used. These factors need to be considered because clinical trials measuring the effects of drugs on QT intervals are used to estimate the risk of using these drugs. Greater changes in QT and QTc intervals after treatment with moxifloxacin compared with levofloxacin or ciprofloxacin are consistent with in vitro observations related to the effect of these drugs on rapid potassium (IK(r)) channels. The clinical relevance of these differences is not known.
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Antiinfecciosos/farmacología , Compuestos Aza , Electrocardiografía/efectos de los fármacos , Fluoroquinolonas , Quinolinas , Adulto , Anciano , Antiinfecciosos/administración & dosificación , Antiinfecciosos/sangre , Ciprofloxacina/administración & dosificación , Ciprofloxacina/sangre , Ciprofloxacina/farmacología , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Levofloxacino , Masculino , Persona de Mediana Edad , Moxifloxacino , Ofloxacino/administración & dosificación , Ofloxacino/sangre , Ofloxacino/farmacologíaRESUMEN
A clinical trial was conducted in healthy volunteers using both periodic and continuous ECG recordings to assess the effect of increasing doses of levofloxacin on the QT and QTc interval. Periodic and continuous ECGs were recorded before and after subjects were dosed with placebo and increasing doses of levofloxacin (500 mg, 1000 mg, 1500 mg) that included doses twice the maximum recommended dose of 750 mg in a double-blind, randomized, four-period, four-sequence crossover trial. Mean heart rate (HR) and the QT and QTc interval after dosing with levofloxacin and placebo were compared, and HR-QT interval relationships defined by linear regression analysis were calculated. After single doses of 1000 and 1500 mg of levofloxacin, HR increased significantly, as measured by periodic and continuous ECG recordings. This transient increase occurred at times of peak plasma concentration and was without symptoms. Mean QT intervals after placebo and mean intervals after levofloxacin were indistinguishable. Using periodic ECG recordings, single doses of 1500 mg were associated with small increases in QTc that were statistically significant. In contrast, an effect on QTc was shown only using the Bazett formula with data obtained from continuous ECG recordings. Together with the finding that levofloxacin does not influence HR-QT relationships, these findings suggest that levofloxacin has little effect on prolonging ventricular repolarization and that small increases in HR associated with high doses of levofloxacin contribute to the drug's apparent effect on QTc. Single doses of 1000 or 1500 mg of levofloxacin transiently increase HR without affecting the uncorrected QT interval. Differences in mean QTc after levofloxacin compared to placebo vary depending on the correction formula used and whether the data analyzed are from periodic or continuous ECG recordings. This work suggests that using continuous ECG recordings in assessing QT/QTc effects of drugs may be of value, particularly with drugs that might influence HR.