RESUMO
Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.
Assuntos
Desenvolvimento Embrionário , Camadas Germinativas , Células-Tronco Pluripotentes , Humanos , Diferenciação Celular , Implantação do Embrião , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Células-Tronco Pluripotentes/citologia , Interleucina-6/metabolismo , Gástrula/citologia , Gástrula/embriologia , Âmnio/citologia , Âmnio/embriologia , Âmnio/metabolismo , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismoRESUMO
Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1-3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4-6 and to advance stem-cell-based regenerative approaches7. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
Assuntos
Callithrix , Gastrulação , Útero , Animais , Callithrix/embriologia , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Endoderma/citologia , Endoderma/embriologia , Feminino , Perfilação da Expressão Gênica , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic approaches, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E6.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Taken together, our multiple analyses evaluated the efficacy of the method for the derivation of marmoset EPSCs.
Assuntos
Callithrix , Células-Tronco Embrionárias , Animais , Humanos , Camundongos , Células-Tronco Embrionárias/metabolismo , Diferenciação Celular , Perfilação da Expressão Gênica , TranscriptomaRESUMO
Mutations in C8orf37 cause Bardet-Biedl syndrome (BBS), retinitis pigmentosa (RP), and cone-rod dystrophy (CRD), all manifest in photoreceptor degeneration. Little is known about which proteins C8orf37 interacts with to contribute to photoreceptor survival. To determine the proteins that potentially interact with C8orf37, we carried out a yeast two-hybrid (Y2H) screen using C8orf37 as a bait. FAM161A, a microtubule-binding protein localized at the photoreceptor cilium required for photoreceptor survival, was identified as one of the preys. Double immunofluorescence staining and proximity ligation assay (PLA) of marmoset retinal sections showed that C8orf37 was enriched and was co-localized with FAM161A at the ciliary base of photoreceptors. Epitope-tagged C8orf37 and FAM161A, expressed in HEK293 cells, were also found to be co-localized by double immunofluorescence staining and PLA. Furthermore, interaction domain mapping assays identified that the N-terminal region of C8orf37 and amino acid residues 341-517 within the PFAM UPF0564 domain of FAM161A were critical for C8orf37-FAM161A interaction. These data suggest that the two photoreceptor survival proteins, C8orf37 and FAM161A, interact with each other which may contribute to photoreceptor health.
Assuntos
Proteínas do Olho , Proteínas , Retinose Pigmentar , Aminoácidos/metabolismo , Epitopos/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Células HEK293 , Humanos , Células Fotorreceptoras/metabolismo , Proteínas/genética , Proteínas/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismoRESUMO
The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis.
Assuntos
Callithrix/genética , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Análise de Célula Única , Transcriptoma/genética , Animais , Sequência Conservada/genética , Endoderma/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Camadas Germinativas/metabolismo , Humanos , Camundongos , Fatores de Transcrição Otx , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Transcrição GênicaRESUMO
A seven-year-old female common marmoset (Callithrix jacchus) presented with weight loss. Imaging revealed a left thoracic mass, confirmed at necropsy. Histology and immunohistochemistry suggested a well-differentiated pulmonary adenocarcinoma. No evidence of local lymphovascular invasion or distant metastasis was observed. This is the first report of pulmonary adenocarcinoma in marmosets.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Doenças dos Macacos , Animais , Callithrix , Feminino , Imuno-Histoquímica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/veterinária , Doenças dos Macacos/diagnósticoRESUMO
BACKGROUND: The common marmoset (Callithrix jacchus) is one of the most studied primate model organisms. However, the marmoset genomes available in the public databases are highly fragmented and filled with sequence gaps, hindering research advances related to marmoset genomics and transcriptomics. RESULTS: Here we utilize single-molecule, long-read sequence data to improve and update the existing genome assembly and report a near-complete genome of the common marmoset. The assembly is of 2.79 Gb size, with a contig N50 length of 6.37 Mb and a chromosomal scaffold N50 length of 143.91 Mb, representing the most contiguous and high-quality marmoset genome up to date. Approximately 90% of the assembled genome was represented in contigs longer than 1 Mb, with approximately 104-fold improvement in contiguity over the previously published marmoset genome. More than 98% of the gaps from the previously published genomes were filled successfully, which improved the mapping rates of genomic and transcriptomic data on to the assembled genome. CONCLUSIONS: Altogether the updated, high-quality common marmoset genome assembly provide improvements at various levels over the previous versions of the marmoset genome assemblies. This will allow researchers working on primate genomics to apply the genome more efficiently for their genomic and transcriptomic sequence data.
Assuntos
Callithrix/genética , Mapeamento Cromossômico/métodos , Genoma/genética , Animais , Biologia Computacional/métodos , Mapeamento de Sequências Contíguas/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Alinhamento de SequênciaRESUMO
Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.
Assuntos
Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Preparações Farmacêuticas/administração & dosagem , RNA/administração & dosagem , Transfecção/métodos , Idoso , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , PlatirrinosRESUMO
BACKGROUND: For the diagnosis and treatment of adult T-cell leukemia/lymphoma (ATLL) caused by human T-lymphotropic virus type 1 (HTLV-1) are required therapeutic modalities urgently. Non-human primate models for ATLL would provide a valuable information for clinical studies. We did a pilot study to establish an ATLL non-human primate model using common marmosets (Callithrix jacchus). METHODS: We inoculated HTLV-1-producing MT-2 cells into 9-month-old marmosets, either intraperitoneally or intravenously. We next administrated MT-2 cells into 13-month-old marmosets under cyclosporine A (CsA) treatment to promote infection. HTLV-1 infection was determined by measuring HTLV-1 antibody titer in the common marmosets. RESULTS: The HTLV-1 antibody titer increased in the intraperitoneally inoculated marmoset with or without CsA treatment, and it kept over five 5 years though proviral copy number (proviral load, PVL) remained low throughout the study. CONCLUSION: We obtained HTLV-1 asymptomatic carriers of common marmosets by inoculating MT-2 cells.
Assuntos
Callithrix , Modelos Animais de Doenças , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Leucemia-Linfoma de Células T do Adulto/virologia , Animais , Projetos PilotoRESUMO
The common marmoset (marmoset; Callithrix jacchus) shows anatomical and physiological features that are in common with humans. Establishing induced pluripotent stem cells (iPSCs) from marmosets holds promise for enhancing the utility of the animal model for biomedical and preclinical studies. However, in spite of the presence of some previous reports on marmoset iPSCs, the reprogramming technology in marmosets is still under development. In particular, the efficacy of RNA-based reprogramming has not been thoroughly investigated. In this study, we attempted RNA-based reprogramming for deriving iPSCs from marmoset fibroblasts. Although we failed to derive iPSC colonies from marmoset fibroblasts by using a conventional RNA-based reprogramming method previously validated in human fibroblasts, we succeeded in deriving colony-forming cells with a modified induction medium supplemented with a novel set of small molecules. Importantly, following one-week culture of the colony-forming cells in conventional embryonic stem cell (ESC) medium, we obtained iPSCs which express endogenous pluripotent markers and show a differentiation potential into all three germ layers. Taken together, our results indicate that RNA-based reprogramming, which is valuable for deriving transgene-free iPSCs, is applicable to marmosets.
Assuntos
Técnicas de Cultura de Células , Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , RNA/química , Animais , Callithrix , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica , Humanos , Transcriptoma , TransgenesRESUMO
The common marmoset is a small nonhuman primate in which the application of transgenesis and genetic knockout techniques allows the generation of gene-modified models of human diseases. However, its longer generation time than that of rodents is a major obstacle to the widespread use of gene-modified marmosets for biomedical research. In this study, we examined the feasibility of shortening the generation time by using prepubertal marmoset males as gamete donors. We collected late round stage spermatids (Steps 5-7), elongated spermatids, and testicular spermatozoa from the testis of a prepubertal 11-month-old male marmoset and injected them into in vitro-matured oocytes. After 7 days in culture, two embryos from elongated spermatid injection and two embryos from sperm injection were transferred into two separate recipient females. The recipient female that received elongated spermatid injection-derived embryos became pregnant and gave birth to one female infant. This is the first demonstration that a spermatid from a prepubertal male primate can support full-term development. Using this method, we can expect to obtain offspring of gene-modified males 6 months to a year earlier than with natural mating.
Assuntos
Nascido Vivo , Injeções de Esperma Intracitoplásmicas , Espermátides , Animais , Callithrix , Feminino , Masculino , GravidezRESUMO
The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low Km value was relatively comparable to that for marmoset livers. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.
Assuntos
Callithrix/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Fígado/enzimologia , Propofol/metabolismo , Animais , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Oxazinas/metabolismo , Oxirredução , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência , Testosterona/metabolismoRESUMO
Eight years prior to her present admission, a 61-year-old Japanese woman was diagnosed with autoimmune hepatitis, slowly progressive insulin-dependent diabetes mellitus, and chronic thyroiditis; she had been treated with oral prednisolone (PSL). After she suddenly discontinued PSL, she newly developed systemic lupus erythematosus. A combination therapy of oral PSL and intravenous cyclophosphamide resulted in remission. She was finally diagnosed with autoimmune polyglandular syndrome (APS) type 3 (3A ,3B, 3D), complicated with four different autoimmune diseases. Since patients with type 3 APS may present many manifestations over a long period of time, they should be carefully monitored.
Assuntos
Hepatite Autoimune/complicações , Lúpus Eritematoso Sistêmico/complicações , Poliendocrinopatias Autoimunes/diagnóstico , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Feminino , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/tratamento farmacológico , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Pessoa de Meia-Idade , Poliendocrinopatias Autoimunes/complicações , Poliendocrinopatias Autoimunes/tratamento farmacológico , Prednisolona/administração & dosagem , Prednisolona/uso terapêuticoRESUMO
Nonhuman primate (NHP) experimental models have contributed greatly to human health research by assessing the safety and efficacy of newly developed drugs, due to their physiological and anatomical similarities to humans. To generate NHP disease models, drug-inducible methods, and surgical treatment methods have been employed. Recent developments in genetic and developmental engineering in NHPs offer new options for producing genetically modified disease models. Moreover, in recent years, genome-editing technology has emerged to further promote this trend and the generation of disease model NHPs has entered a new era. In this review, we summarize the generation of conventional disease model NHPs and discuss new solutions to the problem of mosaicism in genome-editing technology.
Assuntos
Animais Geneticamente Modificados/genética , Modelos Animais de Doenças , Edição de Genes/métodos , Haplorrinos/genética , Animais , HumanosRESUMO
The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The spermatids acquired oocyte-activating capacity at the late round spermatid stage, as confirmed by the resumption of meiosis and Ca2+ oscillations upon injection into mouse oocytes. The spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation.
Assuntos
Sinalização do Cálcio , Núcleo Celular/metabolismo , Criopreservação , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Espermátides/metabolismo , Animais , Callithrix , Feminino , Masculino , Camundongos , Microinjeções , Oócitos/citologia , Espermátides/citologiaRESUMO
1. Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined. 2. The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6ß-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1'-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2 C and 3 A antibodies suppressed progesterone 21/17α- and 16α/6ß-hydroxylation, respectively, in marmoset liver microsomes. 3. Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/Km values of 0.53 and 0.089 mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6ß-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients). 4. These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6ß-hydroxylation by marmoset P450 2 C and 3 A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Progesterona/farmacocinética , Animais , Callithrix , Hidroxilação , Progesterona/farmacologiaRESUMO
1. Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine-ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys. 2. Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects. 3. Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84-144 µM (under 26-76 µM of Km values), in similar manners to liver and intestine microsomes. 4. These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP3A/metabolismo , Intestino Delgado/enzimologia , Fígado/enzimologia , Terfenadina/farmacocinética , Animais , Humanos , Hidroxilação , Macaca fascicularis , Microssomos Hepáticos/enzimologiaRESUMO
1. A potentially useful animal model for preclinical studies is the common marmoset (Callithrix jacchus). In this study, using reverse-transcription polymerase chain reaction from marmoset livers, we identified a novel cytochrome P450 (P450) 2F1 cDNA with an open reading frame of 1473 bp. 2. High sequence identities of 92-94% with primate P450 2 F amino acid sequences were indicated by deduced amino acid sequences of P450 2F1 cDNA. Phylogenetic analysis indicates that marmoset P450 2F1 is more congruent with primate P450 2 F forms than those of other species such as rodents. 3. Among five tissue types examined, abundant expression of marmoset P450 2F1 mRNA and P450 2F1 protein in lungs was shown. Cynomolgus monkey P450 2F1 mRNA was abundantly expressed in lungs as well as testes and ovaries in 10 tissue types. 4. Similar to those of humans and cynomolgus monkeys, marmoset P450 2F1 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation and biphenyl hydroxylation, however unlike human P450 2F1, marmoset P450 2F1 exhibited hydroxylation activity toward coumarin and chlorzoxazone. 5. These findings indicated that P450 2F1 enzyme expressed in marmoset lungs and also catalyzed metabolism of xenobiotics, suggesting the importance of P450 2 F-dependent drug metabolism in marmoset lungs.
Assuntos
Compostos de Bifenilo/metabolismo , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Pulmão/enzimologia , Sequência de Aminoácidos , Animais , Callithrix , Sistema Enzimático do Citocromo P-450/química , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Cinética , Macaca fascicularis , Masculino , Oxirredução , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato , Xenobióticos/metabolismoRESUMO
1. Simulated clearances of R-warfarin and efavirenz were recently reported for individual cynomolgus monkeys genotyped for cytochrome P450 2C19 and 2C9, respectively. To expand and verify this modeling procedure, simulations of R/S-omeprazole and R/S-warfarin clearances after oral administrations in individual marmosets were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling consisting of gut, liver and central compartments. 2. Pharmacokinetics of R/S-omeprazole were chirally determined using the previously reported plasma microsamples in this study. The areas under the plasma concentration/time curves (AUC) of R-omeprazole and S-warfarin, but not S-omeprazole and R-warfarin, after oral administrations in the P450 2C19 homozygous mutant group were significantly higher than those in the wild-type group. These modeled hepatic intrinsic clearances were also significantly associated with the marmoset P450 2C19 genotypes. Other parameter values, e.g. absorption rate constants or systemic circulation volumes, were not likely determining factors. 3. The reported individual AUC values measured in 4-6 marmosets after oral R-omeprazole and S-warfarin administrations were significantly correlated with the AUC values predicted using the PBPK models after virtual administrations. 4. This study indicates that clearances of R-omeprazole, S-warfarin and related medicines associated with polymorphic P450 2C19 in individual marmosets can be simulated using simplified individual PBPK models.