Pub Date : 2001-04-01DOI: 10.1089/108729001750171335
F. Roque, G. Mon, J. Belardi, A. Rodríguez, L. Grinfeld, R. Long, S. Grossman, A. Malcolm, G. Zon, M. Ormont, D. Fischman, Y. Shi, A. Zalewski
We wished to assess the clinical safety and pharmacokinetics of ascending doses of a synthetic oligodeoxynucleotide (LR-3280) administered after coronary angioplasty. Antisense oligodeoxynucleotides designed to hybridize with target messenger ribonucleic acid (mRNA) in a complementary fashion to inhibit the expression of corresponding protein also have the ability to bind to extracellular growth factors. LR-3280 has been shown to reduce c-myc expression, inhibit growth and collagen biosynthesis in human vascular cells, and reduce neointimal formation in animal models of vascular injury. After successful percutaneous transluminal coronary angioplasty (PTCA), 78 patients were randomized to receive either standard care (n = 26) or standard care and escalating doses of LR-3280 (n = 52) (doses from 1 to 24 mg), administered into target vessel through a guiding catheter. Overall safety was evaluated by clinical adverse events, laboratory tests, and electrocardiograms. Patency was evaluated by quantitative coronary angiography. There were no clinically significant differences between treated and control patients. No adverse effects of LR-3280 on the patency of dilated coronary arteries were observed. Pharmacokinetic data revealed that peak plasma concentrations of LR-3280 occurred at 1 minute over the studied dose range and rapidly decreased after approximately1 hour, with little LR-3280 detected in the urine between 0-6 hours and 12-24 hours. The intracoronary administration of LR-3280 is well tolerated at doses up to 24 mg and produces no adverse effects in dilated coronary arteries. These results provide the basis for the evaluation of local delivery of this phosphorothioate oligodeoxynucleotide for the prevention of human vasculoproliferative disease.
{"title":"Safety of intracoronary administration of c-myc antisense oligomers after percutaneous transluminal coronary angioplasty (PTCA).","authors":"F. Roque, G. Mon, J. Belardi, A. Rodríguez, L. Grinfeld, R. Long, S. Grossman, A. Malcolm, G. Zon, M. Ormont, D. Fischman, Y. Shi, A. Zalewski","doi":"10.1089/108729001750171335","DOIUrl":"https://doi.org/10.1089/108729001750171335","url":null,"abstract":"We wished to assess the clinical safety and pharmacokinetics of ascending doses of a synthetic oligodeoxynucleotide (LR-3280) administered after coronary angioplasty. Antisense oligodeoxynucleotides designed to hybridize with target messenger ribonucleic acid (mRNA) in a complementary fashion to inhibit the expression of corresponding protein also have the ability to bind to extracellular growth factors. LR-3280 has been shown to reduce c-myc expression, inhibit growth and collagen biosynthesis in human vascular cells, and reduce neointimal formation in animal models of vascular injury. After successful percutaneous transluminal coronary angioplasty (PTCA), 78 patients were randomized to receive either standard care (n = 26) or standard care and escalating doses of LR-3280 (n = 52) (doses from 1 to 24 mg), administered into target vessel through a guiding catheter. Overall safety was evaluated by clinical adverse events, laboratory tests, and electrocardiograms. Patency was evaluated by quantitative coronary angiography. There were no clinically significant differences between treated and control patients. No adverse effects of LR-3280 on the patency of dilated coronary arteries were observed. Pharmacokinetic data revealed that peak plasma concentrations of LR-3280 occurred at 1 minute over the studied dose range and rapidly decreased after approximately1 hour, with little LR-3280 detected in the urine between 0-6 hours and 12-24 hours. The intracoronary administration of LR-3280 is well tolerated at doses up to 24 mg and produces no adverse effects in dilated coronary arteries. These results provide the basis for the evaluation of local delivery of this phosphorothioate oligodeoxynucleotide for the prevention of human vasculoproliferative disease.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"63 1","pages":"99-106"},"PeriodicalIF":0.0,"publicationDate":"2001-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77620566","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-02-01DOI: 10.1089/108729001750072092
B. Peng, J. Andrews, I. Nestorov, B. Brennan, P. Nicklin, M. Rowland
The aim of this study was to develop a whole body physiologically based model of the pharmacokinetics (PBPK) of the phosphorothioate oligonucleotide (PS-ODN) ISIS 1082 in vivo. Rats were administered an intravenous (i.v.) bolus dose of ISIS 1082 (10 mg/kg plus 3H tracer), and arterial blood and tissues were taken at specific times up to 72 hours. Radioactivity was measured in all samples. The parent compound was determined specifically in blood and tissues at 90 minutes and in liver and kidney also at 24 hours, using capillary gel electrophoresis (CGE). A whole body PBPK model was fitted to the combined blood and tissue radioactivity data using nonlinear regression analysis. CGE analysis indicated that the predominant species in plasma and all tissues is ISIS 1082, together with some n-1 and n-2 metabolites. Total radioactivity primarily reflects these species. The whole body model successfully described temporal events in all tissues. However, to adequately model the experimental data, all tissues had to be partitioned into vascular and extravascular spaces to accommodate the relatively slow distribution of ISIS 1082 out of blood because of a permeability rate limitation. ISIS 1082 distributes extensively into tissues, but the relative affinity varies enormously, being highest for kidney and liver and lowest for muscle and brain. A whole body PBPK model with a permeability rate limited tissue distribution was developed that adequately described events in both blood and tissue for an oligonucleotide. This model has the potential not only to characterize the events in individual tissues throughout the body for such compounds but also to scale across animal species, including human.
{"title":"Tissue distribution and physiologically based pharmacokinetics of antisense phosphorothioate oligonucleotide ISIS 1082 in rat.","authors":"B. Peng, J. Andrews, I. Nestorov, B. Brennan, P. Nicklin, M. Rowland","doi":"10.1089/108729001750072092","DOIUrl":"https://doi.org/10.1089/108729001750072092","url":null,"abstract":"The aim of this study was to develop a whole body physiologically based model of the pharmacokinetics (PBPK) of the phosphorothioate oligonucleotide (PS-ODN) ISIS 1082 in vivo. Rats were administered an intravenous (i.v.) bolus dose of ISIS 1082 (10 mg/kg plus 3H tracer), and arterial blood and tissues were taken at specific times up to 72 hours. Radioactivity was measured in all samples. The parent compound was determined specifically in blood and tissues at 90 minutes and in liver and kidney also at 24 hours, using capillary gel electrophoresis (CGE). A whole body PBPK model was fitted to the combined blood and tissue radioactivity data using nonlinear regression analysis. CGE analysis indicated that the predominant species in plasma and all tissues is ISIS 1082, together with some n-1 and n-2 metabolites. Total radioactivity primarily reflects these species. The whole body model successfully described temporal events in all tissues. However, to adequately model the experimental data, all tissues had to be partitioned into vascular and extravascular spaces to accommodate the relatively slow distribution of ISIS 1082 out of blood because of a permeability rate limitation. ISIS 1082 distributes extensively into tissues, but the relative affinity varies enormously, being highest for kidney and liver and lowest for muscle and brain. A whole body PBPK model with a permeability rate limited tissue distribution was developed that adequately described events in both blood and tissue for an oligonucleotide. This model has the potential not only to characterize the events in individual tissues throughout the body for such compounds but also to scale across animal species, including human.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"15 1","pages":"15-27"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89812403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-02-01DOI: 10.1089/108729001750072100
H. Engdahl, Magnus Lindell, E. Wagner
This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system. This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA. One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA. As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA. One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability. To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends. The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo. As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments). In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.
{"title":"Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation.","authors":"H. Engdahl, Magnus Lindell, E. Wagner","doi":"10.1089/108729001750072100","DOIUrl":"https://doi.org/10.1089/108729001750072100","url":null,"abstract":"This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system. This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA. One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA. As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA. One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability. To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends. The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo. As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments). In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"15 1","pages":"29-40"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83098789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-02-01DOI: 10.1089/108729001750072128
N. Galyam, D. Grisaru, M. Grifman, N. Melamed‐Book, F. Eckstein, S. Seidman, A. Eldor, H. Soreq
3'-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected with 2'-O-methyl (Me) or phosphorothioate (PS) substitutions were targeted to acetylcholinesterase (AChE) mRNA and studied in PC12 cells. Me-modified AS-ODN suppressed AChE activity up to 50% at concentrations of 0.02-100 nM. PS-ODN was effective at 1-100 nM. Both AS-ODN displayed progressively decreased efficacy above 10 nM. In situ hybridization and confocal microscopy demonstrated dose-dependent decreases, then increases, in AChE mRNA. Moreover, labeling at nuclear foci suggested facilitated transcription or stabilization of AChE mRNA or both under AS-ODN. Intracellular concentrations of biotinylated oligonucleotide equaled those of target mRNA at extracellular concentrations of 0.02 nM yet increased only 6-fold at 1 microM ODN. Above 50 nM, sequence-independent swelling of cellular, but not nuclear, volume was observed. Our findings demonstrate suppressed AChE expression using extremely low concentrations of AS-ODN and attribute reduced efficacy at higher concentrations to complex host cell feedback responses.
{"title":"Complex host cell responses to antisense suppression of ACHE gene expression.","authors":"N. Galyam, D. Grisaru, M. Grifman, N. Melamed‐Book, F. Eckstein, S. Seidman, A. Eldor, H. Soreq","doi":"10.1089/108729001750072128","DOIUrl":"https://doi.org/10.1089/108729001750072128","url":null,"abstract":"3'-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected with 2'-O-methyl (Me) or phosphorothioate (PS) substitutions were targeted to acetylcholinesterase (AChE) mRNA and studied in PC12 cells. Me-modified AS-ODN suppressed AChE activity up to 50% at concentrations of 0.02-100 nM. PS-ODN was effective at 1-100 nM. Both AS-ODN displayed progressively decreased efficacy above 10 nM. In situ hybridization and confocal microscopy demonstrated dose-dependent decreases, then increases, in AChE mRNA. Moreover, labeling at nuclear foci suggested facilitated transcription or stabilization of AChE mRNA or both under AS-ODN. Intracellular concentrations of biotinylated oligonucleotide equaled those of target mRNA at extracellular concentrations of 0.02 nM yet increased only 6-fold at 1 microM ODN. Above 50 nM, sequence-independent swelling of cellular, but not nuclear, volume was observed. Our findings demonstrate suppressed AChE expression using extremely low concentrations of AS-ODN and attribute reduced efficacy at higher concentrations to complex host cell feedback responses.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"4 1","pages":"51-7"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87192987","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-02-01DOI: 10.1089/108729001750072137
U. Maus, W. Seeger, J. Lohmeyer
Phosphorothioate oligodeoxynucleotides (PS-ODN) are widely used prototypic antisense oligomers for sequence-specific suppression of normal and diseased gene expression. As polyanionic molecules, however, PSODN may also evoke nonsequence-specific side effects. The objective of the present study was to evaluate the impact of PS-ODN treatment of human pulmonary artery endothelial cells (HPAEC) and microvascular endothelial cells of the lung (HMVEC-L) on the cellular pool of the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) as well as Sp1, using gel shift assays. In addition, by performing supershift assays, we investigated whether antisense treatment of endothelial cells affected the subunit composition of NF-κB and AP-1. Our data show that pretreatment of HPAEC and HMVEC-L with PS-ODN doses ranging from 50 to 5000 nM did not affect the total NF-κB, AP-1, or Sp1 pool in tumor necrosis factor-α (TNF-α)-activated endothelial cells (EC) or the subunit composition of the transcri...
{"title":"Impact of a Phosphorothioate Oligodeoxynucleotide MCP-1 on NF-κB, AP-1, SP1 and NF-κB, and AP-1 Subunit Composition in Human Pulmonary Endothelial Cells","authors":"U. Maus, W. Seeger, J. Lohmeyer","doi":"10.1089/108729001750072137","DOIUrl":"https://doi.org/10.1089/108729001750072137","url":null,"abstract":"Phosphorothioate oligodeoxynucleotides (PS-ODN) are widely used prototypic antisense oligomers for sequence-specific suppression of normal and diseased gene expression. As polyanionic molecules, however, PSODN may also evoke nonsequence-specific side effects. The objective of the present study was to evaluate the impact of PS-ODN treatment of human pulmonary artery endothelial cells (HPAEC) and microvascular endothelial cells of the lung (HMVEC-L) on the cellular pool of the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) as well as Sp1, using gel shift assays. In addition, by performing supershift assays, we investigated whether antisense treatment of endothelial cells affected the subunit composition of NF-κB and AP-1. Our data show that pretreatment of HPAEC and HMVEC-L with PS-ODN doses ranging from 50 to 5000 nM did not affect the total NF-κB, AP-1, or Sp1 pool in tumor necrosis factor-α (TNF-α)-activated endothelial cells (EC) or the subunit composition of the transcri...","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"4 1","pages":"59-64"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78668835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-02-01DOI: 10.1089/108729001750072119
S. Mistry, C. Benham, G. Atweh
Stathmin is a major cytosolic phosphoprotein that plays an important role in the control of cellular proliferation by regulating the dynamics of the microtubules that make up the mitotic spindle. Because stathmin is expressed at high levels in all human cancers, it is an attractive molecular target for anticancer interventions. We had shown previously that antisense stathmin inhibition results in marked abrogation of the transformed phenotype of leukemic cells in vitro and in vivo. Unlike the antisense approach, ribozymes can catalytically cleave several molecules of target RNA. This may provide a more efficient strategy for downregulating genes, such as stathmin, that are expressed at very high levels in cancer cells. We designed several antistathmin hammerhead ribozymes and tested their cleavage activity against short synthetic stathmin RNA substrates. In vitro cleavage studies demonstrated site-specific cleavage of stathmin RNA that was dependent on ribozyme concentration and duration of exposure to ribozyme. The most active antistathmin ribozyme was capable of cleaving >90% stathmin RNA in a catalytic manner, cleaving multiple substrate molecules per ribozyme molecule. We also demonstrated that the designed antistathmin ribozymes are capable of selectively cleaving native stathmin RNA in a mixture of total RNA isolated from leukemic cells. These antistathmin ribozymes may provide a novel and effective form of gene therapy that may be applicable to a wide variety of human cancers.
{"title":"Development of ribozymes that target stathmin, a major regulator of the mitotic spindle.","authors":"S. Mistry, C. Benham, G. Atweh","doi":"10.1089/108729001750072119","DOIUrl":"https://doi.org/10.1089/108729001750072119","url":null,"abstract":"Stathmin is a major cytosolic phosphoprotein that plays an important role in the control of cellular proliferation by regulating the dynamics of the microtubules that make up the mitotic spindle. Because stathmin is expressed at high levels in all human cancers, it is an attractive molecular target for anticancer interventions. We had shown previously that antisense stathmin inhibition results in marked abrogation of the transformed phenotype of leukemic cells in vitro and in vivo. Unlike the antisense approach, ribozymes can catalytically cleave several molecules of target RNA. This may provide a more efficient strategy for downregulating genes, such as stathmin, that are expressed at very high levels in cancer cells. We designed several antistathmin hammerhead ribozymes and tested their cleavage activity against short synthetic stathmin RNA substrates. In vitro cleavage studies demonstrated site-specific cleavage of stathmin RNA that was dependent on ribozyme concentration and duration of exposure to ribozyme. The most active antistathmin ribozyme was capable of cleaving >90% stathmin RNA in a catalytic manner, cleaving multiple substrate molecules per ribozyme molecule. We also demonstrated that the designed antistathmin ribozymes are capable of selectively cleaving native stathmin RNA in a mixture of total RNA isolated from leukemic cells. These antistathmin ribozymes may provide a novel and effective form of gene therapy that may be applicable to a wide variety of human cancers.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"82 1","pages":"41-9"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81236034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-02-01DOI: 10.1089/108729001750072083
Y. Liu, R. Bergan
Prior studies have shown that electroporation is a simple and effective method for the introduction of oligonucleotides (ODN) into cells. In ex vivo bone marrow purging models, electroporation of ODN into cells has been associated with selective killing of human neoplastic cells while sparing hematopoietic stem cells. Prior studies used conventional electroporation methods (i.e., exponential decay) to introduce ODN into cells. Square wave electroporation allows the delivery of a more defined and regulated electrical pulse and is associated with high transfection efficiencies in a variety of systems. The current study was undertaken to determine whether square wave electroporation was more effective than exponential decay electroporation for the delivery of ODN into hematopoietic cells. Using fluorescein-tagged ODN and K562, chronic myelogenous leukemia (CML) cells, higher transfection rates were observed after square wave electroporation. In addition, c-myc antisense ODN were more effective in reducing c-myc protein when introduced by square wave electroporation, as compared with introduction by exponential decay electroporation. Square wave electroporation is thus identified as the optimal method for delivering ODN into hematopoietic cells.
{"title":"Improved intracellular delivery of oligonucleotides by square wave electroporation.","authors":"Y. Liu, R. Bergan","doi":"10.1089/108729001750072083","DOIUrl":"https://doi.org/10.1089/108729001750072083","url":null,"abstract":"Prior studies have shown that electroporation is a simple and effective method for the introduction of oligonucleotides (ODN) into cells. In ex vivo bone marrow purging models, electroporation of ODN into cells has been associated with selective killing of human neoplastic cells while sparing hematopoietic stem cells. Prior studies used conventional electroporation methods (i.e., exponential decay) to introduce ODN into cells. Square wave electroporation allows the delivery of a more defined and regulated electrical pulse and is associated with high transfection efficiencies in a variety of systems. The current study was undertaken to determine whether square wave electroporation was more effective than exponential decay electroporation for the delivery of ODN into hematopoietic cells. Using fluorescein-tagged ODN and K562, chronic myelogenous leukemia (CML) cells, higher transfection rates were observed after square wave electroporation. In addition, c-myc antisense ODN were more effective in reducing c-myc protein when introduced by square wave electroporation, as compared with introduction by exponential decay electroporation. Square wave electroporation is thus identified as the optimal method for delivering ODN into hematopoietic cells.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"26 1","pages":"7-14"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77579033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-02-01DOI: 10.1089/108729001750072074
R. Brand, T. L. Hannah, J. Norris, P. Iversen
The potential for using antisense compounds as therapeutic agents has generated great enthusiasm. Strategies for delivery of these compounds are, therefore, of great interest. Transdermal iontophoresis has been used successfully as an enhancement technique for the transdermal delivery of these compounds in vitro. The effectiveness of using percutaneous penetration as a means to deliver therapeutic levels of these compounds in vivo, however, remains to be demonstrated. The purpose of this work was to demonstrate the ability of iontophoretically delivered compounds to alter enzyme levels in the intact rat. A C5 propyne-modified phosphorothioate oligonucleotide (PS-ODN) targeted to the cytochrome p450-3A2 (CYP3A2) mRNA translational start site and the reverse sequence, used as a control, were synthesized. A patch containing either an oligonucleotide or a buffer control was placed on the animal's back, and an iontophoretic current of 0.5 mA/cm2 was applied for 3.5 hours. Twenty-four hours later, CYP3A2 levels were measured noninvasively using the midazolam-induced sleeping rat model. Liver and small intestinal microsomes were made after completion of sleep studies and assayed for CYP3A2, CYP1A1/2, CYP2B1/2, and CYP2E1. Midozolam-treated animals with antisense to CYP3A2 slept significantly longer than did the controls (p < 0.05). CYP3A2 levels were significantly lower in liver microsomes from antisense-treated animals than in either buffer control (p < 0.001) or reverse sequence animals (p < 0.05). The reverse sequence was also significantly different from the buffer control (p < 0.01), indicating a nonspecific effect of the PS background. Nontarget cytochrome levels were not altered by treatment. There were no significant differences in small intestine CYP3A2 levels between treatment groups. These data demonstrate that transdermally delivered PS-ODN can reach concentrations sufficient to induce changes in specific target enzymes in vivo. Further studies are warranted to investigate potential uses for these molecules.
{"title":"Transdermal delivery of antisense oligonucleotides can induce changes in gene expression in vivo.","authors":"R. Brand, T. L. Hannah, J. Norris, P. Iversen","doi":"10.1089/108729001750072074","DOIUrl":"https://doi.org/10.1089/108729001750072074","url":null,"abstract":"The potential for using antisense compounds as therapeutic agents has generated great enthusiasm. Strategies for delivery of these compounds are, therefore, of great interest. Transdermal iontophoresis has been used successfully as an enhancement technique for the transdermal delivery of these compounds in vitro. The effectiveness of using percutaneous penetration as a means to deliver therapeutic levels of these compounds in vivo, however, remains to be demonstrated. The purpose of this work was to demonstrate the ability of iontophoretically delivered compounds to alter enzyme levels in the intact rat. A C5 propyne-modified phosphorothioate oligonucleotide (PS-ODN) targeted to the cytochrome p450-3A2 (CYP3A2) mRNA translational start site and the reverse sequence, used as a control, were synthesized. A patch containing either an oligonucleotide or a buffer control was placed on the animal's back, and an iontophoretic current of 0.5 mA/cm2 was applied for 3.5 hours. Twenty-four hours later, CYP3A2 levels were measured noninvasively using the midazolam-induced sleeping rat model. Liver and small intestinal microsomes were made after completion of sleep studies and assayed for CYP3A2, CYP1A1/2, CYP2B1/2, and CYP2E1. Midozolam-treated animals with antisense to CYP3A2 slept significantly longer than did the controls (p < 0.05). CYP3A2 levels were significantly lower in liver microsomes from antisense-treated animals than in either buffer control (p < 0.001) or reverse sequence animals (p < 0.05). The reverse sequence was also significantly different from the buffer control (p < 0.01), indicating a nonspecific effect of the PS background. Nontarget cytochrome levels were not altered by treatment. There were no significant differences in small intestine CYP3A2 levels between treatment groups. These data demonstrate that transdermally delivered PS-ODN can reach concentrations sufficient to induce changes in specific target enzymes in vivo. Further studies are warranted to investigate potential uses for these molecules.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"155 1","pages":"1-6"},"PeriodicalIF":0.0,"publicationDate":"2001-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73416128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-12-01DOI: 10.1089/OLI.1.2000.10.443
O. Sedelnikova, I. Panyutin, A. N. Luu, M. Reed, T. Licht, M. Gottesman, R. Neumann
Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.
{"title":"Targeting the human mdr1 gene by 125I-labeled triplex-forming oligonucleotides.","authors":"O. Sedelnikova, I. Panyutin, A. N. Luu, M. Reed, T. Licht, M. Gottesman, R. Neumann","doi":"10.1089/OLI.1.2000.10.443","DOIUrl":"https://doi.org/10.1089/OLI.1.2000.10.443","url":null,"abstract":"Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"8 1","pages":"443-52"},"PeriodicalIF":0.0,"publicationDate":"2000-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73147553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-12-01DOI: 10.1089/OLI.1.2000.10.463
C. Malchere, J. Verheijen, S. van der Laan, L. Bastide, J. V. van Boom, B. Lebleu, I. Robbins
The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H. The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions. It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay.
使用肽核酸(PNA)作为反义剂的潜在药理学效益因其无法激活RNase h而受到影响。混合骨干寡核苷酸(ON)(或gapmer)方法，将RNase h -胜任残基的短内部窗口嵌入RNase h -不胜任的ON中，以前没有应用于PNA，因为PNA和DNA杂交成具有非常不同螺旋结构的RNA，在两个PNA-DNA连接处产生结构扰动。这是第一次证明PNA内部的短磷酸二酯窗口足以唤起RNase h依赖性的靶向RNA切割，并在体外实验中消除翻译延伸。
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