RESUMEN
Antisense oligonucleotides (ASO) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2'MOE) ASO have shown dose- and sequence-specific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count. Phenotype 2 is rare, severe thrombocytopenia. This article focuses on the underlying cause of the more common phenotype 1, investigating the effects of ASO on platelet production and platelet function. Five phosphorothioate ASO were studied: three 2'MOE sequences; 487660 (no effects on platelet count), 104838 (associated with phenotype 1), and 501861 (effects unknown) and two CpG sequences; 120704 and ODN 2395 (known to activate platelets). Human cord bloodderived megakaryocytes were treated with these ASO to study their effects on proplatelet production. Platelet activation (determined by surface Pselectin) and platelet-leukocyte aggregates were analyzed in ASO-treated blood from healthy human volunteers. None of the ASO inhibited proplatelet production by human megakaryocytes. All the ASO were shown to bind to the platelet receptor glycoprotein VI (KD ~0.2-1.5 mM). CpG ASO had the highest affinity to glycoprotein VI, the most potent platelet-activating effects and led to the greatest formation of platelet-leukocyte aggregates. 2'MOE ASO 487660 had no detectable platelet effects, while 2'MOE ASOs 104838 and 501861 triggered moderate platelet activation and SYKdependent formation of platelet-leukocyte aggregates. Donors with higher platelet glycoprotein VI levels had greater ASO-induced platelet activation. Sequence-dependent ASO-induced platelet activation and platelet-leukocyte aggregates may explain phenotype 1 (moderate drops in platelet count). Platelet glycoprotein VI levels could be useful as a screening tool to identify patients at higher risk of ASO-induced platelet side effects.
Asunto(s)
Plaquetas , Oligonucleótidos Antisentido , Humanos , Leucocitos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Activación Plaquetaria , Recuento de PlaquetasRESUMEN
2'-methoxyethyl (MOE) antisense oligonucleotides (ASOs) tested in multidose intrathecal nonhuman primate (NHP) toxicity studies have consistently revealed the presence of single large vacuoles in pyramidal neurons of the hippocampus in the absence of any cellular response. Termed "macrovesicular," these vacuoles were characterized by immunohistochemistry and transmission electron microscopy which showed that these vacuoles are dilated lysosomes in neurons containing accumulated ASO. Additionally, two NHP studies were conducted to investigate the role of tissue fixation on their histogenesis. In Fixation Study 1, 6 doses of 5 mg 2'-MOE ASO with a full phosphorothioate backbone were administered by lumbar puncture over 5 weeks; in Fixation Study 2, 5 doses of 35 mg 2'-MOE ASO with a mixed phosphorothioate/phosphodiester backbone were administered over 12 weeks. At necropsy in each study, brain slices were either immersion fixed in neutral buffered 10% formalin or Carnoy's fixative; frozen at -80 °C; or perfusion fixed with modified Karnovsky's fixative. Fixed samples were processed to paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and compared with H&E cryosections prepared from the frozen tissue of the same brain. The presence of vacuoles in fixed brain tissue but never in fresh frozen tissue showed that they arose during postmortem tissue fixation, and as such represent a processing artifact that is not relevant to human safety assessment of intrathecally administered 2'-MOE ASOs.
Asunto(s)
Neuronas , Oligonucleótidos Antisentido , Animales , Fijadores , Hipocampo , PrimatesRESUMEN
Cellular uptake of antisense oligonucleotides (ASOs) is one of the main determinants of in vivo activity and potency. A significant advancement in improving uptake into cells has come through the conjugation of ASOs to triantenarry N-acetyl-galactosamine (GalNAc3), a ligand for the asialoglycoprotein receptor on hepatocytes. The impact for antisense oligonucleotides, which are already taken up into hepatocytes, is a 10-fold improvement in potency in mice and up to a 30-fold potency improvement in humans, resulting in overall lower effective dose and exposure levels. 2'-Methoxyethyl-modified antisense oligonucleotide conjugated to GalNAc3 (ISIS 702843) is specific for human transmembrane protease serine 6 and is currently in clinical trials for the treatment of ß-thalassemia. This report summarizes a chronic toxicity study of ISIS 702843 in nonhuman primates (NHPs), including pharmacokinetic and pharmacology assessments. Suprapharmacologic doses of ISIS 702843 were well tolerated in NHPs after chronic dosing, and the data indicate that the overall safety profile is very similar to that of the unconjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) ASOs. Notably, the GalNAc3 moiety did not cause any new toxicities nor exacerbate the known nonspecific class effects of the 2'-MOE ASOs. This observation was confirmed with multiple GalNAc3-MOE conjugates by querying a data base of monkey studies containing both GalNAc3-conjugated and unconjugated 2'-MOE ASOs. SIGNIFICANCE STATEMENT: This report documents the potency, pharmacology, and overall tolerability profile of a triantenarry N-acetyl-galactosamine (GalNAc3)-conjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) antisense oligonucleotide (ASO) specific to transmembrane protease serine 6 after chronic treatment in the cynomolgus monkey. Collective analysis of 15 independent GalNAc3-conjugated and unconjugated 2'-MOE ASOs shows the consistency in the dose response and character of hepatic and platelet tolerability across sequences that will result in much larger safety margins for the GalNAc3-conjugated 2'-MOE ASOs when compared with the unconjugated 2'-MOE ASOs given the increased potency.
Asunto(s)
Calicreínas/metabolismo , Nanoconjugados/toxicidad , Oligonucleótidos Antisentido/toxicidad , Acetilglucosamina/química , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Macaca fascicularis , Masculino , Nanoconjugados/química , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinéticaRESUMEN
The 6-month Tg.rasH2 mouse carcinogenicity model provides an acceptable alternative to the 2-year carcinogenicity study in CD-1 mice. However, key questions related to the use of this model for testing antisense oligonucleotides (ASOs) include the similarity in the biologic response between mouse strains and the feasibility of using data from the CD-1 mouse to set doses and dose schedules for a Tg.rasH2 carcinogenicity study. To evaluate the potential strain differences, four distinct 2'- O-(2-methoxyethyl) ASOs were administered to CByB6F1 (wild type), Tg.rasH2 (hemizygous), and CD-1 mice. There were no meaningful differences in clinical signs, body weight, food consumption, or serum chemistry and hematology parameters. Histopathology evaluation indicated little to no difference in the spectrum or magnitude of changes present. The cytokine/chemokine response was also not appreciably different between the strains. This was consistent with the similarity in ASO concentration in the liver between the mouse strains tested. As the class effects of the ASOs were not meaningfully different between CD-1, CByB6F1, or Tg.rasH2 mice, data from nonclinical studies in CD-1 mice can be used for dose selection and expectation of effect in the Tg.rasH2 mouse.
Asunto(s)
Carcinógenos/toxicidad , Genes ras , Oligonucleótidos Antisentido/toxicidad , Oligorribonucleótidos/toxicidad , Pruebas de Toxicidad , Animales , Secuencia de Bases , Carcinógenos/clasificación , Carcinógenos/farmacocinética , Citocinas/sangre , Femenino , Hemicigoto , Masculino , Ratones Endogámicos ICR , Ratones Transgénicos , Oligonucleótidos Antisentido/clasificación , Oligonucleótidos Antisentido/farmacocinética , Oligorribonucleótidos/clasificación , Oligorribonucleótidos/farmacocinética , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos , Especificidad de la Especie , Factores de Tiempo , Distribución Tisular , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad/normasRESUMEN
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.
Asunto(s)
Hígado/efectos de los fármacos , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos/toxicidad , ARN Mensajero/genética , Ribonucleasa H/genética , Alanina Transaminasa/sangre , Alanina Transaminasa/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Análisis por Micromatrices , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Precursores del ARN/antagonistas & inhibidores , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleasa H/antagonistas & inhibidores , Ribonucleasa H/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
PURPOSE: Age-related macular degeneration (AMD) is the leading cause of permanent vision loss among the elderly in many industrialized countries, and the complement system plays an important role in the pathogenesis of AMD. Inhibition of complement factor B, a key regulator of the alternative pathway, is implicated as a potential therapeutic intervention for AMD. Here we investigated the effect of liver factor B reduction on systemic and ocular factor B levels. METHODS: Second-generation antisense oligonucleotides (ASOs) targeting mouse and monkey factor B mRNA were administered by subcutaneous injection to healthy mice or monkeys, and the level of factor B mRNA was assessed in the liver and the eye. In addition, the factor B protein level was determined in plasma and whole eyes from the treated animals. RESULTS: Mice and monkeys treated with factor B ASOs demonstrated a robust reduction in liver factor B mRNA levels with no change in ocular factor B mRNA levels. Plasma factor B protein levels were significantly reduced in mice and monkeys treated with factor B ASOs, leading to a dramatic reduction in ocular factor B protein, below the assay detection levels. CONCLUSIONS: The results add to the increasing evidence that the liver is the main source of plasma and ocular factor B protein, and demonstrate that reduction of liver factor B mRNA by an ASO results in a significant reduction in plasma and ocular factor B protein levels. The results suggest that inhibition of liver factor B mRNA by factor B ASOs would reduce systemic alternative complement pathway activation and has potential to be used as a novel therapy for AMD.
Asunto(s)
Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Ojo/metabolismo , Hígado/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , ARN Mensajero/metabolismo , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Éteres Metílicos/química , Oligonucleótidos Antisentido/efectos adversos , Oligonucleótidos Antisentido/química , Tionucleótidos/efectos adversos , Tionucleótidos/química , Adulto , Anciano , Animales , Plaquetas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Voluntarios Sanos , Humanos , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Macaca fascicularis , Masculino , Persona de Mediana Edad , Oligonucleótidos Antisentido/administración & dosificación , Tionucleótidos/administración & dosificación , Adulto JovenRESUMEN
Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. Although DIVI in laboratory animal species has been well characterized for vasoactive small molecules, there is little available information regarding DIVI associated with biotherapeutics such as peptides/proteins or antibodies. Because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans and the lack of robust biomarkers of DIVI, preclinical DIVI findings can cause considerable delays in or even halt development of promising new drugs. This review discusses standard terminology, characteristics, and mechanisms of DIVI associated with biotherapeutics. Guidance and points to consider for the toxicologist and pathologist facing preclinical cases of biotherapeutic-related DIVI are outlined, and examples of regulatory feedback for each of the mechanistic types of DIVI are included to provide insight into risk assessment.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Lesiones del Sistema Vascular/inducido químicamente , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Oligonucleótidos Antisentido/efectos adversos , Lesiones del Sistema Vascular/inducido químicamente , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
Differences in sensitivity of monkeys and humans to antisense oligonucleotide (ASO)-induced complement alternative pathway (AP) activation were evaluated in monkeys, humans, and in serum using biochemical assays. Transient AP activation was evident in monkeys at higher doses of two 2'-O-methoxyethyl (2'-MOE) ASOs (ISIS 426115 and ISIS 183750). No evidence of AP activation was observed in humans for either ASO, even with plasma ASO concentrations that reached the threshold for activation in monkeys. The absence of complement activation in humans is consistent with a query of the Isis Clinical Safety Database containing 767 subjects. The in vivo difference in sensitivity was confirmed in vitro, as monkey and human serum exposed to increasing concentrations of ASO indicated that monkeys were more sensitive to AP activation with this class of compounds. The mechanistic basis for the greater sensitivity of monkeys to AP activation by 2'-MOE ASO was evaluated using purified human or monkey factor H protein. The binding affinities between a representative 2'-MOE ASO and either purified protein are similar. However, the IC50 of fluid-phase complement inhibition for monkey factor H is about 3-fold greater than that for human protein using either monkey serum or factor H-depleted human serum. Interestingly, there is a sequence variant in the monkey complement factor H gene similar to a single nucleotide polymorphism in humans that is correlated with decreased factor H protein function. These findings show that monkeys are more sensitive to 2'-MOE ASO-mediated complement activation than humans likely because of differences in factor H inhibitory capacity.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Activación de Complemento/fisiología , Factor H de Complemento/genética , Comprensión , Oligonucleótidos Antisentido/farmacología , Oligorribonucleótidos/farmacología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Humanos , Macaca fascicularis , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oligonucleótidos , Oligonucleótidos Antisentido/genética , Oligorribonucleótidos/genética , Adulto JovenRESUMEN
Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously, we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here, we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared with several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and nonhuman primates (NHPs). Intracerebroventricular bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared with i.c.v. infusion. For both methods of delivery, the duration of ISIS 396443-mediated SMN2 splicing correction is long lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development.
Asunto(s)
Encéfalo/efectos de los fármacos , Atrofia Muscular Espinal/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , Empalme del ARN/efectos de los fármacos , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Encéfalo/metabolismo , Femenino , Infusiones Intraventriculares , Inyecciones Intraventriculares , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Atrofia Muscular Espinal/tratamiento farmacológico , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Oligodesoxirribonucleótidos Antisentido/farmacocinética , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Oligonucleótidos/administración & dosificación , Oligonucleótidos/farmacocinética , Oligonucleótidos/uso terapéuticoRESUMEN
A strategy to produce sufficient anticoagulant properties with reduced risk of bleeding may be possible through inhibition of factor XI (FXI), a component of the intrinsic coagulation cascade. The objective of this work was to determine the safety profile of ISIS 416858, a 2'-methoxyethoxy (2'-MOE) antisense oligonucleotide inhibitor of FXI, with focus on assessment of bleeding risk. Cynomolgus monkeys administered ISIS 416858 (4, 8, 12, and 40 mg/kg/wk, subcutaneous) for up to 13 weeks produced a dose-dependent reduction in FXI (mRNA in liver and plasma activity) and a concomitant increase in activated partial thromboplastin time (APTT). ISIS 416858 (20 or 40 mg/kg/wk) reduced plasma FXI activity by 80% at 4 weeks of treatment that resulted in a 33% increase in APTT by 13 weeks with no effects on PT, platelets, or increased bleeding following partial tail amputation or gum and skin laceration. The dose-dependent presence of basophilic granules in multiple tissues in ISIS 416858-treated animals was an expected histologic change for a 2'-MOE antisense oligonucleotide, and no toxicity was attributed to hepatic FXI reduction. Basophilic granules reflect cellular drug uptake and subsequent visualization on hematoxylin staining. These results suggest that ISIS 416858 has an acceptable preclinical safety profile and is a promising clinical candidate to treat thrombotic disease.
Asunto(s)
Factor XI/antagonistas & inhibidores , Hemorragia/prevención & control , Oligonucleótidos Antisentido/farmacología , Tiempo de Tromboplastina Parcial , Animales , Coagulación Sanguínea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Factor XI/genética , Factor XI/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hemorragia/sangre , Infusiones Intravenosas , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Oligonucleótidos/administración & dosificación , Oligonucleótidos/genética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Pulmón/efectos de los fármacos , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos/toxicidad , Receptores de Superficie Celular/genética , Animales , Femenino , Pulmón/metabolismo , Pulmón/patología , Pulmón/fisiología , Macaca , Masculino , Ratones , Oligonucleótidos/sangre , Oligonucleótidos/farmacocinética , Oligonucleótidos Antisentido/sangre , Oligonucleótidos Antisentido/farmacocinética , ARN Mensajero/metabolismoRESUMEN
Raised blood C-reactive protein (CRP) level is a predictor of cardiovascular events, but whether blood CRP is causal in the disease process is unknown. The latter would best be defined by pharmacological inhibition of the protein in the context of a randomized case-control study. However, no CRP specific drug is currently available so such a prospective study cannot be performed. Blood CRP is synthesized primarily in the liver and the liver is an organ where antisense oligonucleotide (ASO) drugs accumulate. Taking advantage of this we evaluated the efficacy of CRP specific ASOs in rodents with experimentally induced cardiovascular damage. Treating rats for 4 weeks with a rat CRP-specific ASO achieved >60% reduction of blood CRP. Notably, this effect was associated with improved heart function and pathology following myocardial infarction (induced by ligation of the left anterior descending artery). Likewise in human CRP transgenic mice treated for 2 weeks with a human CRP-specific ASO, blood human CRP was reduced by >70% and carotid artery patency was improved (2 weeks after surgical ligation). CRP specific ASOs might pave the way towards a placebo-controlled trial that could clarify the role of CRP in cardiovascular disease.
Asunto(s)
Proteína C-Reactiva/metabolismo , Enfermedades Cardiovasculares/tratamiento farmacológico , Animales , Proteína C-Reactiva/antagonistas & inhibidores , Enfermedades Cardiovasculares/sangre , Ecocardiografía , Femenino , Masculino , Ratones , Infarto del Miocardio/sangre , Infarto del Miocardio/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Ratas , Ratas Sprague-DawleyRESUMEN
This analysis sought to assess the clinical predictivity of an in vitro assay which utilized the human B-lymphoma BJAB cell line, for identification of antisense oligonucleotides (ASOs) with the potential to elicit innate immune activation in humans. Adverse events (AEs) from clinical trial data were analyzed based on prior clinical knowledge and network analysis of the clinical data to identify correlations with the BJAB assay. Clinically evaluated ASOs were ranked by the BJAB assay's mean log-fold increase in TNF expression levels. Flu-like reactions (FLRs) and injection site reactions (ISRs), were chosen as AEs of interest, along with those Medical Dictionary for Regulatory Activities preferred terms identified using AE network analysis. Fifteen different 2'-O-methoxyethyl (2'MOE) modified ASOs were ranked by the incidence of each AE group in the integrated safety data from 35 clinical trials. ISRs are considered to be local to the injection site, whereas FLRs are reflected by systemic constitutional symptoms. The correlations identified in this analysis of integrated clinical data provide evidence that the ASO sequences selected by the BJAB assay have a lower likelihood of causing systemic inflammatory AEs associated with FLRs, but not ISRs.
Asunto(s)
Oligonucleótidos Antisentido , Humanos , Oligonucleótidos Antisentido/efectos adversos , Línea CelularRESUMEN
2'-O-Methoxyethyl antisense oligonucleotide (2'-MOE ASO)-induced severe thrombocytopenia (TCP) [platelet (PLT) count <50 K/µL] was observed in the Asian-sourced cynomolgus monkeys with low incidence (2%-4% at doses >5 mg/kg/week). The potential mechanisms for TCP were studied using the Mauritian-sourced cynomolgus monkeys, which were shown to be more susceptible to ASO-induced TCP, along with the Asian-sourced animals. ISIS 405879, a 2'-MOE ASO, induced severe TCP (PLT <50 K/µL) in seven of nine Mauritian-sourced monkeys but not in the Asian-sourced monkeys after 16 weeks of treatment at 40 mg/kg/week. Marked increases in PLT-bound C3d/C4d were detected in all thrombocytopenic Mauritian-sourced monkeys but not in the unaffected Mauritian- or Asian-sourced monkeys, suggesting increased PLT clearance due to complement deposition on the PLTs. However, this effect was independent of the ASO-mediated fluid-phase alternative complement activation. A correlation was also observed between serum antiglycoprotein (GP) IIb/IIIa immunoglobulin G (IgG) and PLT reduction. In addition, increases in total serum IgM, anti-PLT IgM, and anti-PLT factor 4 IgM levels were observed in monkeys from both sources but were more evident in the Mauritian-sourced monkeys. These data suggest an enhanced innate immune cell activation to ISIS 405879, leading to increased PLT destruction through complement fixation on the PLTs or PLT crossreacting polyclonal antibody production.
Asunto(s)
Plaquetas , Trombocitopenia , Animales , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Complemento C3d , Macaca fascicularis , Oligonucleótidos , Trombocitopenia/inducido químicamente , Trombocitopenia/genética , Inmunoglobulina MRESUMEN
ISIS 388626, a 2'-methoxyethyl (MOE)-modified antisense oligonucleotide (ASO) that targets human sodium glucose cotransporter 2 (SGLT2) mRNA, is in clinical trials for the management of diabetes. SGLT2 plays a pivotal role in renal glucose reabsorption, and inhibition of SGLT2 is anticipated to reduce hyperglycemia in diabetic subjects by increasing urinary glucose elimination. To selectively inhibit SGLT2 in the kidney, ISIS 388626 was designed as a "shortmer" ASO, consisting of only 12 nucleotides with two 2'-MOE-modified nucleotides at the termini. Mice and monkeys received up to 30 mg/kg/week ISIS 388626 via subcutaneous injection for 6 or 13 weeks. Dose-dependent decreases in renal SGLT2 mRNA expression were observed, which correlated with dose-related increases in glucosuria without concomitant hypoglycemia. There were no histologic changes in the kidney attributed to SGLT2 inhibition after 6 or 13 weeks of treatment. The remaining changes observed in these studies were typical of those produced in these species by the administration of oligonucleotides, correlated with high doses of ISIS 388626, and were unrelated to the inhibition of SGLT2 expression. The kidney contained the highest concentration of ISIS 388626, and dose-dependent basophilic granule accumulation in tubular epithelial cells of the kidney, which is evidence of oligonucleotide accumulation in these cells, was the only histologic change identified. No changes in kidney function were observed. These results revealed only readily reversible changes after the administration of ISIS 388626 and support the continued investigation of the safety and efficacy of ISIS 388626 in human trials.
Asunto(s)
Oligodesoxirribonucleótidos/farmacología , Oligonucleótidos Antisentido/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Área Bajo la Curva , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Electrocardiografía/efectos de los fármacos , Femenino , Semivida , Hemodinámica/efectos de los fármacos , Inyecciones Subcutáneas , Riñón/efectos de los fármacos , Riñón/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos ICR , Oligodesoxirribonucleótidos/farmacocinética , Oligodesoxirribonucleótidos/toxicidad , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/toxicidad , Farmacocinética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transportador 2 de Sodio-GlucosaRESUMEN
Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , ADN/inmunología , Inmunidad Innata/inmunología , Interferón Tipo I/inmunología , Oligonucleótidos Antisentido/inmunología , Ribosa/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Alanina Transaminasa/genética , Alanina Transaminasa/inmunología , Alanina Transaminasa/metabolismo , Animales , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN/genética , ADN/metabolismo , Hepatocitos/inmunología , Hepatocitos/metabolismo , Inmunidad Innata/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1 , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Oligonucleótidos Antisentido/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Receptores de Interleucina-1/metabolismo , Ribosa/genética , Ribosa/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability. This compelled us to develop a more convenient high-throughput assay. In this study, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPBMC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs through comparison to known benchmark PS-ASO controls that were previously shown to be safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO TLR9-dependent innate immune activation.
Asunto(s)
Linfoma de Burkitt , Oligonucleótidos Antisentido , Humanos , Oligonucleótidos Antisentido/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/terapia , Leucocitos Mononucleares , Receptor Toll-Like 9/genéticaRESUMEN
A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.