RESUMO
Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.
Assuntos
Endossomos , Oligonucleotídeos , Animais , Camundongos , Oligonucleotídeos/metabolismo , Endossomos/genética , Endocitose/fisiologia , Transporte Biológico , Oligonucleotídeos Antissenso/genética , RNA Interferente Pequeno/genéticaRESUMO
The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.
Assuntos
Oligonucleotídeos Antissenso/síntese química , Índice Terapêutico do Medicamento , Animais , Células HEK293 , Células HeLa , Humanos , Fígado/metabolismo , Masculino , Mesilatos/química , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/toxicidade , Fosforamidas/química , Ligação Proteica , Distribuição TecidualRESUMO
We recently showed that site-specific incorporation of 2'-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5'-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5'-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2'-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5'-Me and R-5'-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5'-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.
Assuntos
DNA/química , Oligonucleotídeos Antissenso/química , Animais , Glucose/análogos & derivados , Glucose/química , Células HeLa , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/toxicidade , Compostos Organofosforados/síntese química , Ribonuclease HRESUMO
Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2'-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2'-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.
Assuntos
DNA/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Fosforotioatos/genética , Ribonuclease H/genética , Animais , DNA/química , Camundongos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Ligação Proteica/genética , Ribonuclease H/químicaRESUMO
Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.
Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleotídeos Antissenso/química , Organofosfonatos/química , Oligonucleotídeos Fosforotioatos/química , Células 3T3-L1 , Animais , Caspases/metabolismo , Linhagem Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Proteínas de Ligação a DNA , Células HeLa , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Fosforotioatos/administração & dosagem , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismoRESUMO
A convenient method for the synthesis of several triantennary GalNAc clusters based on a nitromethanetrispropionic acid core was developed. The synthetic approach involves pentafluorophenolic ester intermediates which can be used in a one-pot, seven reaction procedure to quickly prepare a variety of triantennary GalNAc conjugated ASOs. The GalNAc clusters were conjugated to the 5'-end of an antisense oligonucleotide and evaluated for activity in primary mouse hepatocytes where they showed â¼10-fold improvement in activity.
Assuntos
Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/síntese química , Nitrocompostos/síntese química , Oligonucleotídeos Antissenso/síntese química , Propionatos/síntese química , Acetilgalactosamina/farmacologia , Animais , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Indicadores e Reagentes , Camundongos , Nitrocompostos/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Propionatos/farmacologia , Receptores Depuradores Classe B/metabolismoRESUMO
Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.
Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Animais , Células Cultivadas , Proteínas Fúngicas/metabolismo , Camundongos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ribonuclease H/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Estereoisomerismo , TemperaturaRESUMO
Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. â¼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. â¼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.
Assuntos
Acetilgalactosamina/química , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/farmacologia , Animais , Células Cultivadas , Hepatócitos/citologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
To increase understanding of the interactions and effects of the diastereoisomeric character of phosphorothioate (PS) oligonucleotides on chromatographic retention, three chromatographic methods [in-series reversed phase-strong anion exchange (RP-SAX), ion pair-reversed phase and metal ion complexation chromatography (MICC)] were applied to the characterization of stereo-enriched compounds. Chromatographic systems are widely available, amenable to routine applications, and simple to deploy in comparison to more advanced instrumentation (e.g., 31P NMR) and procedures (e.g., enzymatic digestion). Analogous diastereoisomeric distribution profiles were obtained by RP-SAX and IP-RP based on their common mechanism of separation involving the combination of hydrophobic and electrostatic interactions. Similar linear relationships between retention time (tR) and the numbers of stereo random, Rp, and Sp PS linkages were obtained with both methods. Sp-enriched diastereoisomers were retained longer than stereo random and Rp-enriched diastereoisomers. MICC produced much broader diastereoisomeric peak distributions than the other two methods due to its more complicated nature of interaction. Average mass spectra showed a smaller number of Ag ions (1-7) complex with early eluting diastereoisomers than with later eluting diastereoisomers (which complex between 6-12 Ag ions). A higher late-to-early peak UV area ratio was obtained for a sample containing 10 Sp linkages vs one containing 10 Rp linkages pointing to the tendency of the Sp diastereoisomers for increased interactions which could be explained by structures with more open or stretched configurations. Consideration of the peak shapes of the MICC distributions led to comparable hierarchical cluster analysis (HCA) classification to that produced by the IP-RP method, indicating a good orthogonality between the two methods. Preliminary analysis of the data using partial least squares showed that it should be possible to determine the diastereoisomeric composition of PS oligonucleotides from chromatographic data following appropriate data training.
Assuntos
Oligonucleotídeos Fosforotioatos , Estereoisomerismo , Oligonucleotídeos Fosforotioatos/química , Oligonucleotídeos Fosforotioatos/análise , Cromatografia de Fase Reversa/métodos , Cromatografia por Troca Iônica/métodos , Interações Hidrofóbicas e Hidrofílicas , Cromatografia Líquida/métodosRESUMO
The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.
Assuntos
Oligonucleotídeos Antissenso , Oligonucleotídeos Fosforotioatos , Animais , Endossomos/metabolismo , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Fosforotioatos/genética , Fósforo , Índice TerapêuticoRESUMO
We utilized BALB/c mice infected with murine CMV (MCMV) or severe combined immunodeficient (SCID) mice implanted with human fetal tissue and infected with HCMV to determine the efficacy of (S)-9-[3-hydroxy-2-(phophonomethoxy)propyl]adenine ((S)-HPMPA), hexadecyloxypropyl-(S)-HPMPA (HDP-(S)-HPMPA) or octadecyloxyethyl-(S)-HPMPA (ODE-(S)-HPMPA). In MCMV-infected BALB/c mice, oral HDP-(S)-HPMPA at 30 mg/kg significantly reduced mortality when started 24-48 h post inoculation. In the experimental HCMV infection, oral administration of vehicle or 10mg/kg of (S)-HPMPA, HDP-(S)-HPMPA or ODE-(S)-HPMPA was initiated 24h after infection and continued for 28 consecutive days. Cidofovir (CDV), at 20mg/kg given i.p., was used as a positive control. HDP-(S)-HPMPA or ODE-(S)-HPMPA significantly reduced viral replication compared to vehicle-treated mice, while oral (S)-HPMPA was ineffective.
Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Herpesviridae/tratamento farmacológico , Organofosfonatos/uso terapêutico , Adenina/administração & dosagem , Adenina/farmacologia , Adenina/uso terapêutico , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , Cidofovir , Citomegalovirus/efeitos dos fármacos , Citosina/administração & dosagem , Citosina/análogos & derivados , Citosina/uso terapêutico , Fígado/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Muromegalovirus/efeitos dos fármacos , Organofosfonatos/administração & dosagem , Organofosfonatos/farmacologia , Análise de SobrevidaRESUMO
Second-generation (Gen 2) Antisense oligonucleotides (ASOs) show increased nuclease stability and affinity for their RNA targets, which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using the phosphorothioate (PS) backbone modification, which enhances ASO interactions with plasma, cell surface, and intracellular proteins. This facilitates ASO distribution to peripheral tissues and also promotes cellular uptake after injection into animals. Previous work identified that Stabilin receptors specifically internalize PS-ASOs in the sinusoidal endothelial cells of the liver and the spleen. By modulating expression of specific proteins involved in the trafficking and maturation of the endolysosomal compartments, we show that Rab5C and EEA1 in the early endosomal pathway, and Rab7A and lysobisphosphatidic acid in the late endosomal pathway, are important for trafficking of PS-ASOs and facilitate their escape from endolysosomal compartments after Stabilin-mediated internalization. In conclusion, this work identifies key rate-limiting proteins in the pathway for PS-ASO translocation and escape from the endosome.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Ácidos Docosa-Hexaenoicos/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Células HEK293 , Humanos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Receptores de Retorno de Linfócitos/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Oligonucleotide-based therapeutics have made rapid progress in the clinic for treatment of a variety of disease indications. Unmodified oligonucleotides are polyanionic macromolecules with poor drug-like properties. Over the past two decades, medicinal chemists have identified a number of chemical modification and conjugation strategies which can improve the nuclease stability, RNA-binding affinity, and pharmacokinetic properties of oligonucleotides for therapeutic applications. In this perspective, we present a summary of the most commonly used nucleobase, sugar and backbone modification, and conjugation strategies used in oligonucleotide medicinal chemistry.
Assuntos
Oligonucleotídeos/uso terapêutico , Química Farmacêutica , Humanos , Modelos Moleculares , Conformação Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/químicaRESUMO
The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.
Assuntos
Acetilgalactosamina/síntese química , Acetilgalactosamina/farmacologia , Hepatócitos/efeitos dos fármacos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/farmacologia , Animais , Apolipoproteína C-III/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Fator XI/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Depuradores Classe B/biossíntese , Receptores Depuradores Classe B/genética , Relação Estrutura-AtividadeRESUMO
Smallpox was eradicated by vaccination in the 1970s. However, concerns have arisen about the potential use of variola virus as a biological weapon. Most of the world's population has little residual immunity because systematic vaccination against smallpox ceased in the early 1970s. Vaccination of key elements of the population against smallpox is again being considered. However, there are now large numbers of persons who cannot be safely vaccinated with the current vaccine because of AIDS, immunosuppressive drugs, and certain common skin disorders. It would be useful to have a potent orally active drug as an alternative for these persons in case of an outbreak of smallpox. Alkoxyalkyl esters of cidofovir (CDV) have been shown to be highly active and selective against poxviruses in vitro with activities several logs greater than the activity of unmodified CDV. This is due in large part to increased cellular penetration and conversion to CDV-diphosphate, the active antiviral. In this paper, the oral pharmacokinetics of 14C-labeled hexadecyloxypropyl-cidofir (HDP-CDV), octadecyloxyethyl-cidofir (ODP-CDV), and oleyloxypropyl-cidofir (OLP-CDV) are examined and oral bioavailability and tissue distribution assessed and compared with parenteral CDV. The alkoxyalkyl CDVs are highly orally bioavailable and do not concentrate in kidney, the site of the dose-limiting toxicity of CDV. Plasma and tissue drug levels are many times greater than the in vitro EC(50s) for variola, cowpox, and vaccinia viruses. Thus, the compounds are good candidates for further development for prevention and treatment of smallpox infection and the complications of vaccination.
Assuntos
Antivirais/química , Antivirais/farmacocinética , Disponibilidade Biológica , Citosina/análogos & derivados , Citosina/química , Citosina/farmacocinética , Rim/química , Organofosfonatos , Compostos Organofosforados/química , Compostos Organofosforados/farmacocinética , Administração Oral , Animais , Antivirais/administração & dosagem , Radioisótopos de Carbono/farmacocinética , Cidofovir , Citosina/administração & dosagem , Esterificação , Ésteres/química , Feminino , Camundongos , Compostos Organofosforados/administração & dosagem , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Cidofovir (CDV) is an effective therapy for certain human cytomegalovirus (HCMV) infections in immunocompromised patients that are resistant to other antiviral drugs, but the compound is not active orally. To improve oral bioavailability, a series of lipid analogs of CDV and cyclic CDV (cCDV), including hexadecyloxypropyl-CDV and -cCDV and octadecyloxyethyl-CDV and -cCDV, were synthesized and found to have multiple-log-unit enhanced activity against HCMV in vitro. On the basis of the activity observed with these analogs, additional lipid esters were synthesized and evaluated for their activity against herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus, murine cytomegalovirus, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and HHV-8. Using several different in vitro assays, concentrations of drug as low as 0.001 microM reduced herpesvirus replication by 50% (EC50) with the CDV analogs, whereas the cCDV compounds were generally less active. In most of the assays performed, the EC50 values of the lipid esters were at least 100-fold lower than the EC50 values for unmodified CDV or cCDV. The lipid analogs were also active against isolates that were resistant to CDV, ganciclovir, or foscarnet. These results indicate that the lipid ester analogs are considerably more active than CDV itself against HSV, VZV, CMV, EBV, HHV-6, and HHV-8 in vitro, suggesting that they may have potential for the treatment of infections caused by a variety of herpesviruses.
Assuntos
Antivirais/farmacologia , Citosina/análogos & derivados , Herpesviridae/efeitos dos fármacos , Organofosfonatos/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/química , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cidofovir , Ciclização , Citomegalovirus/efeitos dos fármacos , Citosina/química , Citosina/farmacologia , Ensaio de Imunoadsorção Enzimática , Ésteres/farmacologia , Fibroblastos/virologia , Citometria de Fluxo , Humanos , Hibridização In Situ , Organofosfonatos/química , Relação Estrutura-Atividade , Sais de Tetrazólio , Tiazóis , Ensaio de Placa ViralRESUMO
Four newly synthesized ether lipid esters of cidofovir (CDV), hexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxypropyl-CDV (OLP-CDV), and oleyloxyethyl-CDV (OLE-CDV), were found to have enhanced activities against vaccinia virus (VV) and cowpox virus (CV) in vitro compared to those of CDV. The compounds were administered orally and were evaluated for their efficacies against lethal CV or VV infections in mice. HDP-CDV, ODE-CDV, and OLE-CDV were effective at preventing mortality from CV infection when treatments were initiated 24 h after viral inoculation, but only HDP-CDV and ODE-CDV maintained efficacy when treatments were initiated as late as 72 h postinfection. Oral pretreatment with HDP-CDV and ODE-CDV were also effective when they were given 5, 3, or 1 day prior to inoculation with CV, even when each compound was administered as a single dose. Both HDP-CDV and ODE-CDV were also effective against VV infections when they were administered orally 24 or 48 h after infection. In animals treated with HDP-CDV or ODE-CDV, the titers of both CV and VV in the liver, spleen, and kidney were reduced 3 to 7 log(10). In contrast, virus replication in the lungs was not significantly reduced. These data indicate that HDP-CDV or ODE-CDV given orally is as effective as CDV given parenterally for the treatment of experimental CV and VV infections and suggest that these compounds may be useful for the treatment of orthopoxvirus infections in humans.
Assuntos
Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Vírus da Varíola Bovina/efeitos dos fármacos , Varíola Bovina/tratamento farmacológico , Citosina/análogos & derivados , Citosina/farmacologia , Citosina/uso terapêutico , Organofosfonatos , Compostos Organofosforados/farmacologia , Compostos Organofosforados/uso terapêutico , Vaccinia virus/efeitos dos fármacos , Vacínia/tratamento farmacológico , Administração Oral , Animais , Cidofovir , Varíola Bovina/virologia , Citosina/toxicidade , Éteres/química , Feminino , Lipídeos/química , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organofosforados/toxicidade , Vacínia/virologia , Carga Viral , Replicação Viral/efeitos dos fármacosRESUMO
Infection with human cytomegalovirus (HCMV) can cause serious complications in bone-marrow and solid-organ transplant recipients, and current therapies are not optimal. We evaluated 2 orally active ether lipid ester analogues of cidofovir (CDV)--hexadecyloxypropyl-CDV (HDP-CDV) and octadecyloxyethyl-CVD (ODE-CDV)--in severe combined immunodeficient mice in which either human fetal retinal tissue or human fetal thymus and liver tissue had been implanted and was later infected with HCMV. Our results indicate that orally administered treatment with either HDP-CDV or ODE-CDV is 4-8-fold more active, on a molar basis, than is intraperitoneally administered CDV. These data suggest that HDP-CDV and ODE-CDV should be further evaluated as potential antiviral agents for treatment of HCMV infection.
Assuntos
Antivirais , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Citosina , Citosina/análogos & derivados , Organofosfonatos , Compostos Organofosforados , Pró-Fármacos , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/química , Antivirais/uso terapêutico , Cidofovir , Infecções por Citomegalovirus/virologia , Citosina/administração & dosagem , Citosina/química , Citosina/uso terapêutico , Modelos Animais de Doenças , Ésteres/química , Éter/química , Humanos , Lipídeos/química , Masculino , Camundongos , Camundongos SCID , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/química , Compostos Organofosforados/uso terapêutico , Pró-Fármacos/administração & dosagem , Pró-Fármacos/uso terapêutico , Resultado do TratamentoRESUMO
To improve the oral bioavailability of cidofovir (CDV), a series of ether lipid ester prodrugs were synthesized and evaluated for activity against murine cytomegalovirus (MCMV) infection. Four of these analogs, hexadecyloxypropyl (HDP)-CDV, octadecyloxyethyl (ODE)-CDV, oleyloxyethyl (OLE)-CDV, and oleyloxypropyl (OLP)-CDV, were found to have greater activity than CDV against human CMV and MCMV in vitro. The efficacy of oral treatment with these compounds against MCMV infections in BALB/c mice was then determined. Treatment with HDP-CDV, ODE-CDV, OLE-CDV, or OLP-CDV at 2.0 to 6.7 mg/kg of body weight provided significant protection when daily treatments were initiated 24 to 48 h after viral inoculation. Additionally, HDP-CDV or ODE-CDV administered twice weekly or as a single dose of 1.25 to 10 mg/kg was effective in reducing mortality when treatment was initiated at 24 h, 48 h, or, in some cases, 72 h after viral inoculation. In animals treated daily with HDP-CDV or ODE-CDV, virus titers in lung, liver, spleen, kidney, pancreas, salivary gland, and blood were reduced 3 to 5 log(10)-fold, which was comparable to CDV given intraperitoneally. These results indicated that HDP-CDV or ODE-CDV given orally was as effective as parenteral CDV for the treatment of experimental MCMV infection and suggest that further evaluation for use in CMV infections in humans is warranted.