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Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the ß-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction. Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one ß-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected ß-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage. Significance: These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.
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BACKGROUND: Capsule endoscopy (CE) provides a novel approach to evaluate obscure gastrointestinal bleeding. Yet CE is not routinely utilized in the inpatient setting for a variety of reasons. We sought to identify factors that predict complete CE and diagnostically meaningful CE, as well as assess the impact of inpatient CE on further hospital management.1 na d2 METHODS: We conducted a retrospective review of patients undergoing inpatient CE at a tertiary referral, academic center over a 3 year period. We analyzed data on patient demographics, medical history, endoscopic procedures, hospital course, and results of CE. The primary outcome was complete CE and the secondary outcome was positive findings of pathology on CE. RESULTS: 131 patients were included (56.5% were men 43.5% women, median age of 71.0 years). Overall, CE was complete in 77.1% of patients. Complete CE was not related to motility risk factors, gender, or administration modality. Patients with incomplete CE tended to be older, have lower BMI, and Caucasian, however results did not reach statistical significance (p = 0.06; p = 0.06; p = 0.08 respectively). Positive CE was noted in 73.3% of patients, with 35.1% of all patients having active bleeding. Positive CE was not associated with AVM risk factors or medication use. 28.0% of patients underwent subsequent hospital procedures, among which 67.6% identified the same pathology seen on CE. CONCLUSIONS: Contrary to previous studies, we found the majority of inpatient CEs were complete and positive for pathology. We found high rates of correlation between CE and subsequent procedures. The use of CE in the inpatient setting helps to guide the diagnosis and treatment of hospitalized patients with obscure gastrointestinal bleeding.
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Endoscopia por Cápsula , Idoso , Endoscopia por Cápsula/efeitos adversos , Endoscopia Gastrointestinal/métodos , Feminino , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/etiologia , Hemorragia Gastrointestinal/patologia , Humanos , Pacientes Internados , Masculino , Encaminhamento e Consulta , Estudos RetrospectivosRESUMO
CRISPR-Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR.
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Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endodesoxirribonucleases/metabolismo , Edição de Genes , Reparo de DNA por Recombinação , Linhagem Celular , Humanos , Mutação , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.
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Acidaminococcus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes/métodos , Acidaminococcus/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Células Cultivadas , Endonucleases/genética , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Jurkat , Células Matadoras Naturais/metabolismo , Reprodutibilidade dos Testes , Linfócitos T/metabolismoRESUMO
Controlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable in medical practice. Current algorithms for analyzing off-target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. Here we present CRISPECTOR, a software tool that supports the detection and quantification of on- and off-target genome-editing activity from NGS data using paired treatment/control CRISPR experiments. In particular, CRISPECTOR facilitates the statistical analysis of NGS data from multiplex-PCR comparative experiments to detect and quantify adverse translocation events. We validate the observed results and show independent evidence of the occurrence of translocations in human cell lines, after genome editing. Our methodology is based on a statistical model comparison approach leading to better false-negative rates in sites with weak yet significant off-target activity.
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Sistemas CRISPR-Cas , Biologia Computacional/métodos , Edição de Genes/métodos , Algoritmos , Proteínas de Ligação a DNA/genética , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Proteínas Nucleares/genética , Software , Fatores de Transcrição/genéticaRESUMO
CRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair fingerprints that result from non-homologous end joining (NHEJ) after double-stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building biological understanding of the repair into a novel analysis tool (CRISPAltRations) improved the quality of the results. We validated our software using simulated, targeted amplicon sequencing data (11 guide RNAs [gRNAs] and 603 on- and off-target locations) and demonstrated that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology-directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a user interface (UI), we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics.
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LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.
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Proteases Dependentes de ATP/antagonistas & inibidores , Desenho de Fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Inibidores de Proteases/química , Proteases Dependentes de ATP/metabolismo , Sítios de Ligação , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Ácidos Borônicos/farmacologia , Bortezomib/química , Bortezomib/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-AtividadeRESUMO
Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.
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Sangue Fetal/citologia , Imunoterapia Adotiva , Interleucina-15/genética , Células Matadoras Naturais/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Aerobiose , Animais , Antígenos CD19/imunologia , Linfoma de Burkitt/patologia , Linfoma de Burkitt/terapia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Glicólise , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/transplante , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores de Antígenos Quiméricos , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)-grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.
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Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Humanos , Receptores de Glucocorticoides/genética , Linfócitos TRESUMO
Genome editing of human cluster of differentiation 34+ (CD34+) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, ex vivo genome editing using the CRISPR-Cas9 system in CD34+ HSPCs and to create a clear workflow for precise identification of off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered to CD34+ HSPCs as part of ribonucleoprotein (RNP) complexes, targeting therapeutically relevant genes. The addition of an Alt-R electroporation enhancer (EE), a short, single-stranded oligodeoxynucleotide (ssODN), significantly increased editing efficiency in CD34+ HSPCs. Notably, similar editing improvement was observed when excess gRNA over Cas9 protein was used, providing a DNA-free alternative suitable for therapeutic applications. Furthermore, we demonstrated that sgRNA may be preferable over 2-part gRNA in a locus-specific manner. Finally, we present a clear experimental framework suitable for the unbiased identification of bona fide off-target sites by Genome-Wide, Unbiased Identification of Double-Strand Breaks (DSBs) Enabled by Sequencing (GUIDE-seq), as well as subsequent editing quantification in CD34+ HSPCs using rhAmpSeq. These findings may facilitate the implementation of genome editing in CD34+ HSPCs for research and therapy and can be adapted for other hematopoietic cells.
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Acne Vulgar/imunologia , Doença de Crohn/complicações , Hidradenite/imunologia , Pioderma Gangrenoso/imunologia , Acne Vulgar/diagnóstico , Acne Vulgar/patologia , Biópsia , Doença de Crohn/imunologia , Face/diagnóstico por imagem , Hidradenite/diagnóstico , Hidradenite/patologia , Humanos , Masculino , Pioderma Gangrenoso/diagnóstico , Pioderma Gangrenoso/patologia , Pele/imunologia , Pele/patologia , Síndrome , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.
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Proteína 9 Associada à CRISPR/genética , Edição de Genes , Células-Tronco Hematopoéticas/metabolismo , Mutação/genética , Ribonucleoproteínas/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/terapia , Antígenos CD34/metabolismo , Sequência de Bases , Escherichia coli , Células HEK293 , HumanosRESUMO
BACKGROUND: Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. RESULTS: Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. CONCLUSIONS: For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.
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Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normasRESUMO
Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.
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Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Nicotina/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Benzimidazóis/síntese química , Benzimidazóis/química , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Conformação Molecular , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Nicotina/síntese química , Nicotina/química , Nicotina/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.
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Antineoplásicos/farmacologia , Benzenoacetamidas/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Proteínas Mutantes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab/farmacologia , Modelos Animais de Doenças , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacosRESUMO
GOAL: The purpose of this study was to assess the effect of decreased colonoscopy reimbursement on gastroenterologist practice behavior, including time to retirement and procedure volume. BACKGROUND: In 2015, the Centers for Medicare and Medicaid Services proposed reductions in colonoscopy reimbursements. With new initiatives for increased colorectal cancer screening, it is crucial to understand how reimbursement changes could affect these efforts. STUDY: Randomly selected respondents from the American College of Gastroenterology membership database were surveyed on incremental changes in practice behavior if colonoscopy reimbursement were to decrease by 10, 20, 30, or 40 %. Data were analyzed using both Pearson's Chi-square and analysis of variance. RESULTS: Two thousand and nine gastroenterologists received the survey with a 16.3 % response rate. Procedure volume significantly decreased with degree of reimbursement reductions (p < 0.001). With a 10 % decrease, 72 % of respondents reported no change in the number of colonoscopies performed. With a 20 % decrease, 39 % would decrease their procedure volume, while 21 % of respondents would increase their procedure volume. With a 30 and 40 % decrease, procedure volume decreased by 48 and 50 %, respectively. In terms of retirement, current plans predict a cumulative retirement rate of 29.4 % at 10 years. More than 42 % of respondents plan to retire after 2030. In the 2014-2023 retirement subgroup (N = 74 responses), there was a significant hastening of retirement year at 20 % (p = 0.016), 30 % (p < 0.001), and 40 % (p < 0.001) reimbursement reductions as compared to baseline responses. CONCLUSION: Decreasing colonoscopy reimbursements may have a significant effect on the effective gastroenterology work force.
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Colonoscopia/economia , Gastroenterologistas/economia , Necessidades e Demandas de Serviços de Saúde/tendências , Padrões de Prática Médica , Mecanismo de Reembolso , Adulto , Idoso , Coleta de Dados , Humanos , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
Non-small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M.
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Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Mutação/efeitos dos fármacos , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Systematic SAR optimization of the GPR119 agonist lead 1, derived from an internal HTS campaign, led to compound 29. Compound 29 displays significantly improved in vitro activity and oral exposure, leading to GLP1 elevation in acutely dosed mice and reduced glucose excursion in an OGTT study in rats at doses ⩾10 mg/kg.