RESUMO
The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.
Assuntos
Oligorribonucleotídeos/administração & dosagem , Oligorribonucleotídeos/química , Receptor 7 Toll-Like/efeitos dos fármacos , Receptor 8 Toll-Like/efeitos dos fármacos , Receptor Toll-Like 9/efeitos dos fármacos , Animais , Células Cultivadas , Quimiocinas/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Feminino , Células HEK293/efeitos dos fármacos , Células HEK293/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Oligonucleotídeos Fosforotioatos/administração & dosagem , Oligonucleotídeos Fosforotioatos/química , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/imunologiaRESUMO
A manufacturing and purification process for duplex oligonucleotides was established, which shortens and simplifies currently used procedures, yielding a product of higher purity. The reported procedure is based on nondenaturing anion-exchange (AEX) chromatography, which is performed on the annealed duplex rather than the individual single strands. The duplex is formed early in the process by annealing of the crude single strands directly after solid-phase synthesis. Two 30 µmol manufacturing runs using duplex purification were performed on 2 different AEX resins and compared with a manufacturing run of the same scale using conventional single-strand chromatography. The same pooling strategy was employed for all purifications. Content of optimal duplex (duplex exclusively comprising full-length single strands) was 90.5% and 90.2% for the batches obtained by duplex purification and 86.1% for the batch obtained by single-strand purification. Maximum chromatographic recoveries were 67% for the duplex purification and 68% for the single-strand purification. Hence, the manufacture of small interfering RNA (siRNA) using duplex purification was simpler and faster than conventional single-strand purification and provided better purity and similar yield of final siRNA.
Assuntos
Cromatografia por Troca Iônica/métodos , Oligonucleotídeos/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/isolamento & purificação , Resinas de Troca Aniônica/química , Espectrometria de Massas/métodos , Oligonucleotídeos/síntese química , RNA Interferente Pequeno/análiseRESUMO
Small interfering RNAs (siRNA) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. siRNA are typically formed by annealing of two complementary single stranded oligoribonucleotides. Compared to purity determination of non-hybridized single strands by denaturing chromatographic methods, characterization of the hybridized duplex is challenging. Here we are reporting a non-denaturing ion pairing-reversed phase (IP-RP) chromatography method capable of separating optimal duplex (full-length single strands only) from non-optimal duplex variants (containing shortmers, longmers and 2',5'-isomers) using ultraviolet- and mass spectrometric detection. The impact of different annealing conditions on siRNA composition was investigated. Optimized annealing conditions lead to a significant increase in optimal duplex, while total duplex content remained constant. The non-denaturing method reported herein showed high mass spectrometric sensitivity and superior separation efficiencies compared to other IP-RP buffer systems. The method is useful for in-process control and release testing of therapeutic double stranded nucleic acids such as siRNA.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , RNA Interferente Pequeno/química , Conformação de Ácido Nucleico , Sensibilidade e EspecificidadeRESUMO
Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions-such as 2',3'-isomerization, strand scission, and HF elimination-depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.
Assuntos
RNA Interferente Pequeno/química , Ribose/análogos & derivados , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Temperatura Alta , Espectrometria de Massas , Conformação de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
Unmethylated deoxycytidyl-deoxyguanosin dinucleotide (CpG)-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists for Toll-like receptor 9. We here describe a new class of CpG ODNs, the so-called P-Class, which combines preferred properties of known CpG ODN classes. This P-Class contains two palindromic sequences, enabling it to form concatamers, multimeric units, where each molecule is bound via Watson-Crick basepairing to a second and a third palindrome. The type I interferon-inducing potency and efficacy of the double-palindromic P-Class ODN is substantially higher than that of previously described C-Class ODNs, and they stimulate superior cytokine production upon in vivo application. The multimeric structures of the P-Class can be resolved to monomers and dimers by formulation in low-salt buffer, retaining the strong and potent immune effects. Taken together, we have discovered a novel class of CpG ODNs, the P-Class, with promising superior activity for disease application.
Assuntos
Ilhas de CpG , Sistema Imunitário/efeitos dos fármacos , Interferon Tipo I/biossíntese , Oligodesoxirribonucleotídeos/farmacologia , Animais , Sequência de Bases , Linhagem Celular , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
INTRODUCTION: Radiolabelled particles are an attractive tool in the therapy of malignancies of the liver. We consider particles manufactured from denatured human serum albumin (HSA) as useful carriers of therapeutic radionuclides. Covalent attachment of suitable chelators onto the surface of the spheres promises an easy access to radiolabelled HSA microspheres. METHODS: We synthesized 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) bearing smooth, medium-rough and rough surfaced HSA microspheres (mean diameter: 25 microm). In vitro stability of 86 Y-labelled particles was determined after incubation in human plasma and in a DTPA challenge experiment. In vivo stability of 86 Y DOTA-HSA microspheres was determined after single intravenous application in rats. Subsequently, the particles were completely trapped in the lung microvasculature. Thus, the lung serves in our experiments as target organ. RESULTS: DOTA-HSA microspheres were 86 Y labelled in reproducible high yields (>95%). No differences between smooth and rough surfaced spheres were found for both DOTA coupling and 86 Y labelling. Labelled microspheres showed high in vitro stability in human plasma and in DTPA solution with only 8+/-1% and 2+/-0% loss of radioactivity from the surface, respectively, 48 h postinjection (pi). The three batches (smooth, medium-rough and rough surfaced microspheres) differed considerably in their radioactivity recovery in the lungs of rats 48 h pi. Smooth particles showed the highest in vivo stability of the radiolabel on the surface of the spheres, presumably because of slower proteolytic degradation. CONCLUSION: We found that for the preparation of HSA-derived microspheres for radiotherapeutic application, smooth surfaced spheres are superior to rough spheres due to their higher in vivo stability of the radionuclide fixation.
Assuntos
Albuminas/farmacocinética , Compostos Heterocíclicos/farmacocinética , Microesferas , Compostos Organometálicos/farmacocinética , Albuminas/química , Animais , Quelantes/química , Estabilidade de Medicamentos , Compostos Heterocíclicos/química , Humanos , Marcação por Isótopo/métodos , Pulmão/irrigação sanguínea , Pulmão/diagnóstico por imagem , Pulmão/metabolismo , Taxa de Depuração Metabólica , Compostos Organometálicos/química , Ácido Pentético , Plasma , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Propriedades de Superfície , Distribuição Tecidual , Radioisótopos de Ítrio/química , Radioisótopos de Ítrio/farmacocinéticaRESUMO
A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.
Assuntos
Compostos Heterocíclicos/química , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Compostos Organometálicos/química , Animais , Autorradiografia , Benzeno/química , Compostos Heterocíclicos/metabolismo , Compostos Heterocíclicos/farmacocinética , Isomerismo , Espectroscopia de Ressonância Magnética , Masculino , Oligonucleotídeos/metabolismo , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacocinética , RNA/química , RNA/metabolismo , RNA/farmacocinética , Ratos , Ratos Wistar , Distribuição Tecidual , Radioisótopos de ÍtrioRESUMO
The molecular structures of the two mononuclear title complexes, namely (4-methoxybenzenethiolato-kappaS)oxido[2,2'-(3-phenylpropylimino)bis(ethanethiolato)-kappa(3)S,N,S']technetium(V), [Tc(C14H21NS2)(C7H7OS)O], (I), and (4-methoxybenzenethiolato-kappaS)oxido[2,2'-(propylimino)bis(ethanethiolato)-kappa(3)S,N,S']technetium(V), [Tc(C7H15NS2)(C7H7OS)O], (II), exhibit the same coordination environment for the central Tc atoms. The atoms are five-coordinated (TcNOS3) with a square-pyramidal geometry comprising a tridentate 2,2'-(3-phenylpropylimino)bis(ethanethiolate) or 2,2'-(propylimino)bis(ethanethiolate) ligand, a 4-methoxybenzenethiolate ligand and an additional oxide O atom. Intermolecular C-H...O and C-H...S hydrogen bonds between the monomeric units result in two-dimensional layers with a parallel arrangement.
Assuntos
Compostos Organometálicos/química , Compostos Organometálicos/síntese química , Compostos de Organotecnécio/química , Tecnécio/química , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Estrutura Molecular , Compostos de Organotecnécio/síntese químicaRESUMO
Capillary gel electrophoresis (CGE) is a widely used method for quantification of oligonucleotide-based drugs, such as CpG oligodeoxynucleotides (CpG ODN), aptamers and small interfering ribonucleic acids (siRNAs) that allows accurate quantification of parent compound as well as metabolites. Stable secondary structure formation of these molecules frequently prevents analysis by conventional CGE methods and impedes pharmacokinetic assessment. Herein, we describe development of a CGE method for identification and quantification of complex mixtures of secondary structure forming GC-rich ODN in biological samples at dose levels of 0.5mg/kg and above. Samples containing GC-rich CpG ODN and metabolite markers were treated by solid-phase-extraction (SPE) and subsequently analyzed by CGE using a 50cm neutrally coated capillary at 60 degrees C together with a 7M urea buffer system containing 30% dimethylsulfoxide (DMSO). Peak resolutions >or=1 were typically achieved, enabling pharmacokinetic assessment of secondary structure forming oligonucleotides in biological samples that hitherto were unsusceptible to quantitative analysis.
Assuntos
Eletroforese Capilar/métodos , Sequência Rica em GC/genética , Fígado/química , Oligodesoxirribonucleotídeos/análise , Animais , Sequência de Bases , Humanos , Camundongos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/sangue , Oligodesoxirribonucleotídeos/química , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Deep brain stimulation (DBS) is widely accepted in the treatment of advanced Parkinson's disease (PD) and other movement disorders. The standard implantation procedure is performed under local anesthesia (LA). Certain groups of patients may not be eligible for surgery under LA because of clinical reasons, such as massive fear, reduced cooperativity, or coughing attacks. Microrecording (MER) has been shown to be helpful in DBS surgery. The purpose of this study was to evaluate the feasibility of MER for DBS surgery under general anesthesia (GA) and to compare the data of intraoperative MER as well as the clinical data with that of the current literature of patients undergoing operation under LA. CLINICAL PRESENTATION: The data of nine patients with advanced PD (mean Hoehn and Yahr status, 4.2) who were operated with subthalamic nucleus (STN) DBS under GA, owing to certain clinical circumstances ruling out DBS under LA, were retrospectively analyzed. All operations were performed under analgosedation with propofol or remifentanil and intraoperative MER. For MER, remifentanil was ceased completely and propofol was lowered as far as possible. INTERVENTION: The STN could be identified intraoperatively in all patients with MER. The typical bursting pattern was identified, whereas a widening of the baseline noise could not be as adequately detected as in patients under LA. The daily off phases of the patients were reduced from 50 to 17%, whereas the Unified Parkinson's Disease Rating Scale III score was reduced from 43 (preoperative, medication off) to 19 (stimulation on, medication off) and 12 (stimulation on, medication on). Two patients showed a transient neuropsychological deterioration after surgery, but both also had preexisting episodes of disorientation. One implantable pulse generator infection was noticed. No further significant clinical complications were observed. CONCLUSION: STN surgery for advanced PD with MER guidance is possible with good clinical results under GA. Intraoperative MER of the STN region can be performed under GA with a special anesthesiological protocol. In this setting, the typical STN bursting pattern can be identified, whereas the typical widening of the background noise baseline while entering the STN region is obviously absent. This technique may enlarge the group of patients eligible for STN surgery. Although the clinical improvements and parameter settings in this study were within the range of the current literature, further randomized controlled studies are necessary to compare the results of STN DBS under GA and LA, respectively.
Assuntos
Estimulação Encefálica Profunda/métodos , Eletrodos Implantados , Eletroencefalografia/métodos , Cuidados Intraoperatórios/métodos , Doença de Parkinson/terapia , Implantação de Prótese/métodos , Núcleo Subtalâmico/cirurgia , Idoso , Anestesia Geral , Estimulação Encefálica Profunda/instrumentação , Estudos de Viabilidade , Humanos , Masculino , Resultado do TratamentoRESUMO
To evaluate pharmacokinetics (PK) and biodistribution, CPG 7909, a 24-mer immunostimulatory fully phosphorothioated oligodeoxynucleotide (PS-ODN), was administered by subcutaneous injection at 2, 5 and 12.5mg/kg to mice and at 9mg/kg to rats. Parent compound and metabolites were isolated from plasma and tissues and quantified by capillary gel electrophoresis with UV detection (CGE-UV) and molecular masses were determined by matrix-assisted-laser-desorption-ionization time of flight detection (MALDI-TOF). An established method for PS-ODN isolation from plasma and tissue was modified to prevent oxidation of the phosphorothioate bonds during the extraction process, significantly increasing sensitivity in the subsequent MALDI-TOF analysis. Concentrations of CPG 7909 and metabolites were highest at the injection site (>600mg/kg at 4h). Maximal concentrations in local (draining) lymph nodes (LLN), kidney and liver were 10-15% of that at the injection site. The highest total amount of PS-ODN (percentage of administered dose) was found in the liver (32% at 4h), followed closely by the injection site (23% at 4h). Only very low levels of CPG 7909 and metabolites were found in plasma and only during the first hours. Metabolites identified by MALDI-TOF were similar for both species and all analyzed tissues, although the relative amounts of the different metabolites varied with tissue and over time. Degradation of CPG 7909 in vivo occurred predominantly by 3'exonucleases with additional cleavage by endonucleases.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/metabolismo , Ilhas de CpG/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/metabolismo , Adjuvantes Imunológicos/farmacocinética , Animais , Relação Dose-Resposta a Droga , Feminino , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/farmacocinética , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
UNLABELLED: PET of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Previously, 2 approaches based on the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) have been successfully applied to the heart. Wild-type HSV1-tk was imaged with (124)I-labeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU), and a mutant HSV1-tk (HSV1-sr39tk) was imaged with (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG). The aim of this study was to compare these 2 combinations with regard to specificity, imaging contrast, and reporter probe kinetics using dynamic PET in small and large animals. METHODS: Similar titers of adenovirus-expressing wild-type HSV1-tk (Ad(tk)), mutant HSV1-sr39tk (Ad(sr39tk)), or control genes were directly injected into the myocardium of 24 rats and 8 pigs. Two days later, dynamic PET was performed with a clinical scanner during the 120 min after injection of (124)I-FIAU (Ad(tk) animals and controls) or (18)F-FHBG (Ad(sr39tk) animals and controls). Imaging with (13)N-ammonia was performed to identify cardiac regions of interest. RESULTS: In rats, significant cardiac (124)I-FIAU accumulation occurred in images obtained early (10-30 min) after Ad(tk) injection. Because of tracer washout, however, no difference between Ad(tk)-injected animals and controls was seen in the images obtained later. For (18)F-FHBG, specific myocardial accumulation greater than background levels was detected in Ad(sr39tk)-injected animals at early imaging and, in contrast to (124)I-FIAU accumulation, increased over time until the latest imaging (105-120 min). At maximum, cardiac (18)F-FHBG concentration showed a 4.15 +/- 1.65-fold increase compared with controls (105-120 min), and cardiac (124)I-FIAU concentration reached a maximal increase of 1.34 +/- 0.38-fold compared with controls (10-30 min, P = 0.0014). Global cardiac reporter probe kinetics in rats were confirmed by regional myocardial analysis in pig hearts. Transgene expression was specifically visualized by both approaches. The highest target-to-background ratio of (124)I-FIAU in Ad(tk)-infected pig myocardium was 1.50 +/- 0.20, versus 2.64 +/- 0.49 for (18)F-FHBG in Ad(sr39tk)-infected areas (P = 0.01). In vivo results were confirmed by ex vivo counting and autoradiography. CONCLUSION: Both reporter gene/probe combinations were feasible for noninvasive imaging of cardiac transgene expression in different species. Specific probe kinetics suggest different myocardial handling of pyrimidine (FIAU) and acycloguanosine (FHBG) derivatives. The results favor (18)F-FHBG with mutant HSV1-sr39tk because of continuous accumulation over time and higher imaging contrast.
Assuntos
Arabinofuranosiluracila/análogos & derivados , Perfilação da Expressão Gênica/métodos , Guanina/análogos & derivados , Coração/diagnóstico por imagem , Miocárdio/enzimologia , Timidina Quinase/genética , Timidina Quinase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Arabinofuranosiluracila/farmacocinética , Técnicas de Transferência de Genes , Genes Reporter/genética , Terapia Genética/métodos , Guanina/farmacocinética , Radioisótopos do Iodo/farmacocinética , Masculino , Tomografia por Emissão de Pósitrons/métodos , Ratos , Ratos Wistar , Especificidade da Espécie , Suínos , Distribuição Tecidual , Transgenes/genéticaRESUMO
Synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytosine-deoxyguanosine (CpG) motifs are very potent inducers of the innate immune system, mimicking the effects of bacterial DNA. CpG ODN are recognized by Toll-like receptor 9 (TLR9). Three classes of TLR9 agonists have been described: B-Class CpG ODN that induce strong B- and NK-cell activation and A-Class ODN that induce very high levels of IFN-alpha by plasmacytoid dendritic cells. The recently described C-Class ODN combine most efficiently properties of A- and B-Class ODN in that they induce strong B-cell activation comparable to B-Class ODN together with IFN-alpha secretion comparable to A-Class ODN. Here, we investigate sequence requirements of C-Class ODN regarding optimal IFN-alpha secretion. Sequence as well as backbone modifications like 2'-O-methyl modifications especially in the 5' part of the ODN influence IFN-alpha-producing capacity. Kinetic studies on mRNA level for CD69, IFN-gamma, IP-10 and IL-18 by semi-quantitative PCR demonstrated differences in mRNA transcription for some cytokines suggesting different regulatory mechanisms for different ODN classes. High amounts of IP-10 mRNA and protein as well as up-regulation of IL-18 mRNA were observed especially for the A- and C-Classes. According to these data, C-Class ODN can be described as strong Th1 inducers with the stimulation of type I and II interferon as well as IP-10 production and strong NK activation. These characteristics can be availed to induce potent anti-tumor or anti-viral effects. Consequently, C-Class CpG ODN represent ideal drug candidates for anti-viral and/or anti-tumor therapy.