RESUMO
Antisense oligonucleotides (ASOs) are a promising therapeutic modality. However, failure to predict acute kidney injury induced by SPC5001 ASO observed in a clinical trial suggests the need for additional preclinical models to complement the preceding animal toxicity studies. To explore the utility of in vitro systems in this space, we evaluated the induction of nephrotoxicity and kidney injury biomarkers by SPC5001 in human renal proximal tubule epithelial cells (HRPTEC), cultured in 2D, and in a recently developed kidney proximal tubule-on-a-chip. 2D HRPTEC cultures were exposed to the nephrotoxic ASO SPC5001 or the safe control ASO 556089 (0.16-40 µM) for up to 72 h, targeting PCSK9 and MALAT1, respectively. Both ASOs induced a concentration-dependent downregulation of their respective mRNA targets but cytotoxicity (determined by LDH activity) was not observed at any concentration. Next, chip-cultured HRPTEC were exposed to SPC5001 (0.5 and 5 µM) and 556089 (1 and 10 µM) for 48 h to confirm downregulation of their respective target transcripts, with 74.1 ± 5.2% for SPC5001 (5 µM) and 79.4 ± 0.8% for 556089 (10 µM). During extended exposure for up to 20 consecutive days, only SPC5001 induced cytotoxicity (at the higher concentration; 5 µM), as evaluated by LDH in the perfusate medium. Moreover, perfusate levels of biomarkers KIM-1, NGAL, clusterin, osteopontin and VEGF increased 2.5 ± 0.2-fold, 3.9 ± 0.9-fold, 2.3 ± 0.6-fold, 3.9 ± 1.7-fold and 1.9 ± 0.4-fold respectively, in response to SPC5001, generating distinct time-dependent profiles. In conclusion, target downregulation, cytotoxicity and kidney injury biomarkers were induced by the clinically nephrotoxic ASO SPC5001, demonstrating the translational potential of this kidney on-a-chip.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Túbulos Renais Proximais/efeitos dos fármacos , Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , Injúria Renal Aguda/patologia , Biomarcadores/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Túbulos Renais Proximais/patologia , L-Lactato Desidrogenase/metabolismo , Dispositivos Lab-On-A-Chip , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Drug discovery with phosphorothioate oligonucleotides is an area of intensive research. In this study we have controlled the stereochemistry of the phosphorothioate backbone of LNA oligonucleotides to investigate the differences in safety profile, target mRNA knock down, and cellular uptake in vitro. The study reveals that controlling only four stereocenters in an isomeric phosphorothioate mixture can improve the therapeutic index significantly by improving safety without compromising activity.
Assuntos
Oligonucleotídeos/química , Animais , Sobrevivência Celular , Células Cultivadas , Química Farmacêutica , Células Epiteliais/metabolismo , Hepatócitos/metabolismo , Humanos , Túbulos Renais/metabolismo , Camundongos , Estrutura Molecular , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/toxicidade , Oligonucleotídeos Fosforotioatos/química , RNA Mensageiro/antagonistas & inibidoresRESUMO
The human telomerase reverse transcriptase catalytic subunit (hTERT) is the rate-limiting subunit of the telomerase holoenzyme. Down-regulating the expression of hTERT mRNA by antisense oligonucleotides would reduce the expression of hTERT, inhibit telomerase activity, and impair the growth of cancer cells in vitro. In this work, we propose a locked nucleic acid-functionalized gold nanoparticle flare probe (AuNP-probe). After transferring these probes into cells by endocytosis of the gold nanoparticles, the binding process of the antisense locked nucleic acid with hTERT mRNA along with gene regulation can be visualized by fluorescence recovery of flare-sequences. A significant decline in hTERT mRNA levels and the hTERT content occurred in cancer cells after treatment with the AuNP-probes, and only approximately 25% of the original level of hTERT mRNA remained after 72 h. AuNP-probe treated cancer cells were arrested in the G1 phase of the cell cycle and underwent apoptosis; cell viability decreased obviously compared with that of telomerase-negative normal cells.
Assuntos
DNA/química , Corantes Fluorescentes/química , Ouro/química , Nanopartículas Metálicas/química , RNA Mensageiro/metabolismo , Telomerase/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Carbocianinas/química , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/toxicidade , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Fluorescência , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/toxicidade , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/toxicidade , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/toxicidade , RNA Mensageiro/genética , Telomerase/antagonistas & inibidores , Telomerase/genética , Fatores de TempoRESUMO
TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP-43 exists as a full-length protein and as two shorter forms of 25 and 35 kDa. Full-length mutant TDP-43s found in amyotrophic lateral sclerosis patients re-localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP-43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kDa truncated form of TDP-43 is restricted to the intermembrane space, while the full-length forms also localize in the mitochondrial matrix in cultured neuronal NSC-34 cells. Interestingly, the full-length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial-transcribed mRNAs, while the 35 kDa form does not. In the light of the known differential contribution of the full-length and short isoforms to generate toxic aggregates, we propose that the presence of full-length TDP-43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP-43 forms play a major role.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Neurônios , Oligonucleotídeos/toxicidade , Isoformas de Proteínas/metabolismo , Linhagem Celular Transformada , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Citosol/ultraestrutura , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Imunoprecipitação , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mutação/efeitos dos fármacos , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Consumo de Oxigênio/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.
Assuntos
Oligonucleotídeos/uso terapêutico , Animais , Ativação do Complemento , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Excipientes/toxicidade , Humanos , Dose Máxima Tolerável , Testes de Mutagenicidade , Oligonucleotídeos/farmacocinética , Oligonucleotídeos/toxicidade , Medição de RiscoRESUMO
Over the last decade, single stranded oligonucleotides (ON) have gained increased attention as a new drug modality. Because the assessment of genotoxicity risk during early development of pharmaceuticals is essential, we evaluated the potential of locked nucleic acids (LNA)-ONs to induce DNA damage in L5178Y tk+/- cells both with the mouse lymphoma assay (MLA) and the micronucleus test (MNT). Further, the MLA was performed to assess gene and chromosome mutation over 3 and 24h (± metabolic activation). In addition, the MNT was performed to assess, in addition, a potential aneugenic liability. None of the experiments demonstrated a genotoxic effect for the five tested LNA-ONs. We further show data from four proprietary LNA-ONs tested in standard genotoxicity assays in vitro and partially in vivo, which were all negative. In addition, cellular and nuclear uptake of LNA-ONs in L5178Y tk+/- cells was demonstrated. Based on the results presented here as well as in the literature about other representatives of this class, we consider LNA-ONs as generally not DNA reactive and question whether genotoxicity testing of this class of ONs should be required. This is in line with recent recommendation made by the OSWG that extensively assessed the genotoxicity of oligonucleotides. Environ. Mol. Mutagen. 58:112-121, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Oligonucleotídeos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Biofarmácia/métodos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Camundongos , Testes de Mutagenicidade , Oligonucleotídeos/genética , RNA Longo não Codificante/genéticaRESUMO
Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.
Assuntos
Modelos Animais , Oligonucleotídeos/toxicidade , Porco Miniatura , Animais , Área Sob a Curva , Relação Dose-Resposta a Droga , Feminino , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/farmacocinética , Suínos , Distribuição Tecidual , ToxicocinéticaRESUMO
Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.
Assuntos
Bioensaio/métodos , Fígado/patologia , Oligonucleotídeos/toxicidade , Preparações Farmacêuticas/química , Testes de Toxicidade , Animais , Apoptose/efeitos dos fármacos , Técnicas de Cocultura , Criopreservação , Citocinas/biossíntese , DNA de Cadeia Simples/toxicidade , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Camundongos , MicroRNAs/metabolismo , Reprodutibilidade dos TestesRESUMO
Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.
Assuntos
Inativação Gênica , Fígado/metabolismo , Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , Ribonuclease H/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Ribonuclease H/metabolismoRESUMO
The Oligonucleotide Safety Working Group subcommittee on genotoxicity testing considers therapeutic oligonucleotides (ONs) unlikely to be genotoxic based on their properties and on the negative results for ONs tested to date. Nonetheless, the subcommittee believes that genotoxicity testing of new ONs is warranted because modified monomers could be liberated from a metabolized ON and incorporated into DNA and could hypothetically cause chain termination, miscoding, and/or faulty replication or repair. The standard test battery as described in Option 1 of International Conference on Harmonisation S2(R1) is generally adequate to assess such potential. However, for the in vitro assay for gene mutations, mammalian cells are considered more relevant than bacteria for most ONs due to their known responsiveness to nucleosides and their greater potential for ON uptake; on the other hand, bacterial assays may be more appropriate for ONs containing non-ON components. Testing is not recommended for ONs with only naturally occurring chemistries or for ONs with chemistries for which there is documented lack of genotoxicity in systems with demonstrated cellular uptake. Testing is recommended for ONs that contain non-natural chemical modifications and use of the complete drug product (including linkers, conjugates, and liposomes) is suggested to provide the most clinically relevant assessment. Documentation of uptake into cells comparable to those used for genotoxicity testing is proposed because intracellular exposure cannot be assumed for these large molecules. ONs could also hypothetically cause mutations through triple helix formation with genomic DNA and no tests are available for detection of such sequence-specific mutations across the entire genome. However, because the potential for triplex formation by therapeutic ONs is extremely low, this potential can be assessed adequately by sequence analysis.
Assuntos
Oligonucleotídeos/toxicidade , Animais , Células Cultivadas , Dano ao DNA , Avaliação Pré-Clínica de Medicamentos , Humanos , Testes de Mutagenicidade , Oligonucleotídeos/uso terapêuticoRESUMO
High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.
Assuntos
Fígado/efeitos dos fármacos , Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , RNA Mensageiro/genética , Ribonuclease H/genética , Alanina Transaminase/sangue , Alanina Transaminase/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise em Microsséries , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/antagonistas & inibidores , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of efficient miRNA inhibitor delivery systems. As a cationic polymer transfection material for nucleic acids, the poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer combines the high transfection efficiency of polyethylenimine (PEI) and the good biodegradability of polyllysine (PLL). In this work, PEI-PLL was successfully synthesized and confirmed to transfect plasmid and oligonucleotide more effectively than PEI in MCF-7 cells (human breast cancer cells). In this regard, two kinds of miR-21 inhibitors, miR-21 sponge plasmid DNA (Sponge) and anti-miR-21 oligonucleotide (AMO), were transported into MCF-7 cells by PEI-PLL respectively. The miR-21 expression and the cellular physiology were determined post transfection. Compared with the negative control, PEI-PLL/Sponge or PEI-PLL/AMO groups exhibited lower miR-21 expression and cell viability. The anti-tumor mechanism of PEI-PLL/miR-21 inhibitors was further studied by cell cycle and western blot analyses. The results indicated that the miR-21 inhibition could induce the cell cycle arrest in G1 phase, upregulate the expression of Programmed Cell Death Protein 4 (PDCD4) and thus active the caspase-3 apoptosis pathway. Interestingly, the PEI-PLL/Sponge and PEI-PLL/AMO also sensitized the MCF-7 cells to anti-tumor drugs, doxorubicin (DOX) and cisplatin (CDDP). These results demonstrated that PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. STATEMENT OF SIGNIFICANCE: This work was a combination of the high transfection efficiency of polyethylenimine (PEI), the good biodegradability of polyllysine (PLL) and the breast cancer-killing effect of miR-21 inhibitors. The poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer was employed as the vector of miR-21 sponge plasmid DNA (Sponge) or anti-miR-21 oligonucleotide (AMO). PEI-PLL showed more transfection efficiency and lower cytotoxicity in human breast cancer cells than PEI. Moreover, the breast cancer cells exhibited significantly lower miR-21 expression and cell viability post transfection with sponge or AMO. Interestingly, the PEI-PLL/miR-21 inhibitor complexes also sensitized the cancer cells to anti-cancer chemotherapy drugs, doxorubicin (DOX) and cisplatin (CDDP). This synergistic effect provides a good application prospect of co-delivery miR-21 inhibitors and chemical drugs in breast cancer therapy.
Assuntos
Neoplasias da Mama/terapia , MicroRNAs/antagonistas & inibidores , Oligonucleotídeos/genética , Polietilenoimina/química , Polilisina/química , Poríferos/química , Animais , Western Blotting , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética , Endocitose/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Células MCF-7 , MicroRNAs/genética , Oligonucleotídeos/toxicidade , Tamanho da Partícula , Polietilenoimina/toxicidade , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , TransfecçãoRESUMO
This white paper summarizes the current consensus of the Reproductive Subcommittee of the Oligonucleotide Safety Working Group on strategies to assess potential reproductive and/or developmental toxicities of therapeutic oligonucleotides (ONs). The unique product characteristics of ONs require considerations when planning developmental and reproductive toxicology studies, including (a) chemical characteristics, (b) assessment of intended and unintended mechanism of action, and (c) the optimal exposure, including dosing regimen. Because experience across the various classes of ONs as defined by their chemical backbone is relatively limited, best practices cannot be defined. Rather, points to consider are provided to help in the design of science-based reproductive safety evaluation programs based upon product attributes.
Assuntos
Aptidão Genética/efeitos dos fármacos , Guias como Assunto , Oligonucleotídeos/farmacocinética , Reprodução/efeitos dos fármacos , Animais , Biomarcadores Farmacológicos/análise , Vias de Administração de Medicamentos , Esquema de Medicação , Cálculos da Dosagem de Medicamento , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Animais , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/toxicidadeRESUMO
This document was prepared by the Safety Pharmacology Subcommittee of the Oligonucleotide Safety Working Group (OSWG), a group of industry and regulatory scientists involved in the development and regulation of therapeutic oligonucleotides. The mission of the Subcommittee was to develop scientific recommendations for the industry regarding the appropriate scope and strategies for safety pharmacology evaluations of oligonucleotides (ONs). These recommendations are the consensus opinion of the Subcommittee and do not necessarily reflect the current expectations of regulatory authorities. 1) Safety pharmacology testing, as described in the International Conference on Harmonisation (ICH) S7 guidance, is as applicable to ONs as it is to small molecule drugs and biotherapeutics. 2) Study design considerations for ONs are similar to those for other classes of drugs. In general, as with other therapeutics, studies should evaluate the drug product administered via the clinical route. Species selection should ideally consider relevance of the model with regard to the endpoints of interest, pharmacological responsiveness, and continuity with the nonclinical development program. 3) Evaluation of potential effects in the core battery (cardiovascular, central nervous, and respiratory systems) is recommended. In general: a. In vitro human ether-a-go-go-related gene (hERG) testing does not provide any specific value and is not warranted. b. Emphasis should be placed on in vivo evaluation of cardiovascular function, typically in nonhuman primates (NHPs). c. Due to the low level of concern, neurologic and respiratory function can be assessed concurrently with cardiovascular safety pharmacology evaluation in NHPs, within repeat-dose toxicity studies, or as stand-alone studies. In the latter case, rodents are most commonly used. 4) Other dedicated safety pharmacology studies, beyond the core battery, may have limited value for ONs. Although ONs can accumulate in the kidney and liver, evaluation of functional changes in these organs, as well as gastrointestinal (GI) and unintended "pro-inflammatory" effects, may be best evaluated during repeat-dose toxicity studies. Broad receptor- or ligand-binding profiling has not historically been informative for most ON subclasses, but may have value for investigative purposes.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Oligonucleotídeos/toxicidade , Segurança do Paciente , Animais , Doenças Cardiovasculares/prevenção & controle , Consenso , Gastroenteropatias/prevenção & controle , Humanos , Doenças Neurodegenerativas/prevenção & controle , Oligonucleotídeos/farmacocinética , Guias de Prática Clínica como Assunto , Projetos de PesquisaRESUMO
Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.
Assuntos
Pulmão/efeitos dos fármacos , Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , Receptores de Superfície Celular/genética , Animais , Feminino , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiologia , Macaca , Masculino , Camundongos , Oligonucleotídeos/sangue , Oligonucleotídeos/farmacocinética , Oligonucleotídeos Antissenso/sangue , Oligonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/metabolismoRESUMO
Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure-toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure-toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.
Assuntos
Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , Animais , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Motivos de Nucleotídeos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Hyperactivation of signal transducer and activator of transcription 3 (STAT3) has been linked to tumorigenesis in most malignancies, including head and neck squamous cell carcinoma. Intravenous delivery of a chemically modified cyclic STAT3 decoy oligonucleotide with improved serum and thermal stability demonstrated antitumor efficacy in conjunction with downmodulation of STAT3 target gene expression such as cyclin D1 and Bcl-X(L) in a mouse model of head and neck squamous cell carcinoma. The purpose of the present study was to determine the toxicity and dose-dependent antitumor efficacy of the cyclic STAT3 decoy after multiple intravenous doses in Foxn1 nu mice in anticipation of clinical translation. The two doses (5 and 10 mg/kg) of cyclic STAT3 decoy demonstrated a significant decrease in tumor volume compared with the control groups (mutant cyclic STAT3 decoy or saline) in conjunction with downmodulation of STAT3 target gene expression. There was no dose-dependent effect of cyclic STAT3 decoy on tumor volume or STAT3 target gene expression. There were no significant changes in body weights between the groups during the dosing period, after the dosing interval or on the day of euthanasia. No hematology or clinical chemistry parameters suggested toxicity of the cyclic STAT3 decoy compared with saline control. No gross or histological pathological abnormalities were noted at necropsy in any of the animals. These findings suggest a lack of toxicity of intravenous administration of a cyclic STAT3 decoy oligonucleotide. In addition, comparable antitumor effects indicate a lack of dose response at the two dose levels investigated.
Assuntos
Antineoplásicos/administração & dosagem , Oligonucleotídeos/administração & dosagem , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Administração Intravenosa , Animais , Antineoplásicos/toxicidade , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mutação , Nível de Efeito Adverso não Observado , Oligonucleotídeos/toxicidade , Ratos Sprague-DawleyRESUMO
Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Endocitose/efeitos dos fármacos , Fígado/efeitos dos fármacos , Oligonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , Transcriptoma/efeitos dos fármacos , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Clatrina/metabolismo , Endocitose/genética , Perfilação da Expressão Gênica , Injeções Subcutâneas , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismoRESUMO
Prostate cancer represents approximately 10 percent of all cancer cases in men and accounts for more than a quarter of all cancer types. Advances in understanding the molecular mechanisms of prostate cancer progression, however, have not translated well to the clinic. Patients with metastatic and hormone-refractory disease have only palliative options for treatment, as chemotherapy seldom produces durable or complete responses, highlighting the need for novel therapeutic approaches. T-oligo, a single-stranded deoxyribonucleic acid with partial sequence homology to human telomeric DNA, has elicited cytostatic and/or cytotoxic effects in multiple cancer cell types. In contrast, normal primary cells of varying tissue types are resistant to cytotoxic actions of T-oligo, underscoring its potential utility as a novel targeted cancer therapeutic. Mechanistically, T-oligo is hypothesized to interfere with normal telomeric structure and form G-quadruplex structures, thereby inducing genomic stress in addition to aberrant upregulation of DNA damageresponse pathways. Here, we present data demonstrating the enhanced effectiveness of a deoxyguanosine-enriched sequence of T-oligo, termed (GGTT)4, which elicits robust cytotoxic effects in prostate cancer cells at lower concentrations than the most recent T-oligo sequence (5'-pGGT TAG GTG TAG GTT T 3') described to date and used for comparison in this study, while exerting no cytotoxic actions on nontransformed human prostate epithelial cells. Additionally, we provide evidence supporting the T-oligo induced activation of cJun N-terminal kinase (JNK) signaling in prostate cancer cells consistent with G-quadruplex formation, thereby significantly advancing the understanding of the T-oligo mechanism of action.
Assuntos
Citotoxinas/toxicidade , Desoxiguanosina/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Oligonucleotídeos/toxicidade , Próstata/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , DNA de Cadeia Simples , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Quadruplex G , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Especificidade de Órgãos , Próstata/metabolismo , Próstata/patologia , Transdução de Sinais , Telômero/químicaRESUMO
Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.