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This study aims to compare the tracking algorithms provided by the OpenCV library to use on ultrasound video. Despite the widespread application of this computer vision library, few works describe the attempts to use it to track the movement of liver tumors on ultrasound video. Movements of the neoplasms caused by the patient`s breath interfere with the positioning of the instruments during the process of biopsy and radio-frequency ablation. The main hypothesis of the experiment was that tracking neoplasms and correcting the position of the manipulator in case of using robotic-assisted surgery will allow positioning the instruments more precisely. Another goal of the experiment was to check if it is possible to ensure real-time tracking with at least 25 processed frames per second for standard definition video. OpenCV version 4.5.0 was used with 7 tracking algorithms from the extra modules package. They are: Boosting, CSRT, KCF, MedianFlow, MIL, MOSSE, TLD. More than 5600 frames of standard definition were processed during the experiment. Analysis of the results shows that two algorithms-CSRT and KCF-could solve the problem of tumor tracking. They lead the test with 70% and more of Intersection over Union and more than 85% successful searches. They could also be used in real-time processing with an average processing speed of up to frames per second in CSRT and 100 + frames per second for KCF. Tracking results reach the average deviation between centers of neoplasms to 2 mm and maximum deviation less than 5 mm. This experiment also shows that no frames made CSRT and KCF algorithms fail simultaneously. So, the hypothesis for future work is combining these algorithms to work together, with one of them-CSRT-as support for the KCF tracker on the rarely failed frames.
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Procedimentos Cirúrgicos Robóticos , Humanos , Algoritmos , Computadores , MovimentoRESUMO
This study is aimed at the comparison of the process of manual and robotic positioning of the electrode performing radiofrequency ablation under the control of a surgical navigation system. The main hypothesis of this experiment was that the use of a collaborative manipulator (KUKA iiwa) will allow to position the active part of the electrode relative to the center of the tumor more accurately and from the first attempt. We also monitor the stability of the electrode's velocity during insertion and consider some advantages in ergonomics using the robotic manipulator. We use three more criteria to compare the surgeon's and robotic performance, unlike other studies, where only the target point's accuracy criterion is observed. The main idea is to examine the movement parameters of the electrode that can lead to potential patient trauma. Sphere-shaped tumor phantoms measuring 8 mm in diameter were filled with contrast and inserted in bovine livers. 10 livers were used for the robotic experiment and an equal quantity for manual surgery. The livers were encased in silicone phantoms designed to imitate the liver position in a real patient's abdominal cavity. Analysis of CT data gave the opportunity to find the entry and the target point for each tumor phantom. This data was loaded into a surgical navigation system that was used to track and record the position of the RF-electrode during the operation for further analysis. The standard deviation of points from the programmed linear trajectory totaled in the average 0.3 mm for the robotic experiment and 2.33 mm for the manual operation with a maximum deviation of 0.55 mm and 7.99 mm respectively. Standard deviation from the target point was 2.69 mm for the collaborative method and 2.49 mm for the manual method. The average velocity was 2.97 mm/s for the manipulator and 3.12 mm/s for the manual method, but the standard deviation of the velocity relative to the value of the average velocity was 0.66 mm/s and 3.05 mm/s respectively. Thus, in two criteria out of three, the manipulator is superior to the surgeon, and equality is established in one. Surgeons also noticed advantages in ergonomics performing the procedure using the manipulator. This experiment was produced as part of the work on the developing of a robotic multifunctional surgical complex. We can confirm the potential advantages of using collaborative robotic manipulators for minimally invasive surgery in case of practice for cancer treatment.
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A (001) SrTiO3 wafer has been investigated in situ at room temperature under application of a static electric field of varying polarity by fluorescence x-ray absorption near edge structure (XANES) analysis at the Sr-K and Ti-K absorption edges. The XANES spectra show a clear shift of the Ti-K absorption edge energy. The shift is attributed to a change of the Ti valence state in a volume invoked by diffusion of the oxygen ions and vacancies. No shift was observed for the Sr-K absorption edge energy. Theoretical calculations support these findings.
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AIM: To determine the utility of barium studies for diagnosing gastroparesis in patients with nausea, vomiting, or other related symptoms. MATERIALS AND METHODS: Radiology files revealed gastroparesis without gastric outlet obstruction on upper gastrointestinal tract barium studies in 50 patients with nausea, vomiting, and other related symptoms. Original reports and images were reviewed to determine whether gastric peristalsis was decreased/absent and to investigate gastric dilatation, fluid or debris, and delayed emptying of barium. Twenty patients (40%) had nuclear gastric emptying studies. Medical records were reviewed to determine the presentation, treatment, and course. The diagnosis of gastroparesis was considered accurate if patients with gastroparesis on barium studies responded to treatment. RESULTS: Forty-six patients (92%) had predisposing factors for gastroparesis, including narcotics and diabetes. Forty-five patients (90%) presented with nausea or vomiting, and 40 patients (80%) had one or more other symptoms, including bloating, early satiety, postprandial fullness, and abdominal pain. Barium studies revealed decreased gastric peristalsis in 46 (92%) of the 50 patients and absent peristalsis in four (8%); 46 patients (92%) had additional findings, including gastric dilatation in 30 (60%), delayed emptying of barium in 27 (54%), debris in 28 (56%; bezoars in three), and retained fluid in 13 (26%). Thirteen (65%) of 20 patients with nuclear gastric emptying studies had delayed emptying of solids and seven (35%) had normal emptying. Thirty-five (83%) of 42 patients treated for gastroparesis had symptomatic improvement versus two (25%) of eight patients not treated. CONCLUSION: Patients with nausea, vomiting, or other related symptoms who have gastroparesis without gastric outlet obstruction on barium studies can be treated for this condition on the basis of the clinical and radiographic findings.
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Radioisótopos de Bário , Gastroparesia/diagnóstico por imagem , Dor Abdominal/etiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Fluoroscopia/métodos , Esvaziamento Gástrico , Gastroparesia/complicações , Gastroparesia/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Náusea/etiologia , Estudos Retrospectivos , Vômito/etiologiaRESUMO
The steel of Damascus blades, which were first encountered by the Crusaders when fighting against Muslims, had features not found in European steels--a characteristic wavy banding pattern known as damask, extraordinary mechanical properties, and an exceptionally sharp cutting edge. Here we use high-resolution transmission electron microscopy to examine a sample of Damascus sabre steel from the seventeenth century and find that it contains carbon nanotubes as well as cementite nanowires. This microstructure may offer insight into the beautiful banding pattern of the ultrahigh-carbon steel created from an ancient recipe that was lost long ago.
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ISIS 2302, a phosphorothioate oligodeoxynucleotide with antisense activity against human ICAM-1 mRNA, was evaluated in a battery of tests to assess genotoxic potential. There was no evidence of genotoxicity in three in vitro studies performed: (i) a bacterial reverse mutation test; (ii) a chromosomal aberration test in Chinese hamster ovary cells; (iii) a mammalian cell gene mutation assay in L5187Y cells. Additionally, there was no in vivo evidence of genetic toxicity in a bone marrow micronucleus study in male and female mice. For all tests, top concentrations or doses assessed met harmonized regulatory guidelines. The cellular uptake of ISIS 2302 into target cells was confirmed using capillary gel electrophoresis and immunohistochemistry. Intracellular uptake into CHO cells, L5187Y cells, Salmonella typhimurium TA98 and bone marrow was concentration- and time-dependent. Consistent with what is known about the physical and chemical properties of phosphorothioate oligodeoxynucleotides, there was no evidence of genotoxicity in any of the assessed end-points. Furthermore, the absence of genotoxicity could not be ascribed to test system insensitivity or to an absence of exposure of the test system to ISIS 2302.
Assuntos
DNA/efeitos dos fármacos , Imunossupressores/toxicidade , Oligodesoxirribonucleotídeos Antissenso/toxicidade , Oligonucleotídeos/toxicidade , Tionucleotídeos/toxicidade , Animais , Medula Óssea/efeitos dos fármacos , Células CHO , Aberrações Cromossômicas , Cromossomos/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Eletroforese Capilar , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Modelos Químicos , Mutação , Oligonucleotídeos Fosforotioatos , RNA Mensageiro/metabolismo , Salmonella typhimurium/metabolismo , Fatores de TempoRESUMO
PURPOSE: To compare the antiviral activity and ocular distribution of first- and second-generation antisense oligonucleotides intended for the treatment of cytomegalovirus (CMV) retinitis. METHODS: The antiviral activity of ISIS 13312 and ISIS 2922 (Isis Pharmaceuticals, Inc., Carlsbad, CA) against 10 clinical CMV isolates was compared with a plaque-reduction assay. The ocular pharmacokinetics were compared after intravitreal injection in rabbits (36-90 microg) and monkeys (125-500 microg). Vitreous and/or retina were collected after single and multiple injections to characterize ocular distribution, clearance, and accumulation. Oligonucleotide concentrations were measured by capillary gel electrophoresis and immunohistochemical techniques. RESULTS: ISIS 13312 and ISIS 2922 demonstrated comparable antiviral activity that was consistent among the 10 clinical isolates examined (50% inhibitory concentration [IC(50)], <1 microM). Activity was independent of the resistance of CMV isolates to DNA polymerase inhibitors. After intravitreal injection, the kinetics of ISIS 2922 and ISIS 13312 were characterized by clearance from vitreous and distribution to the retina; however, ISIS 2922 was cleared more quickly from the retina than ISIS 13312. The half-life of ISIS 13312 in the monkey retina was approximately 2 months. Retinal concentrations of ISIS 13312 were dose dependent, with approximately a twofold increase in concentration after once-monthly doses compared with single-dose concentrations. Immunohistochemical analysis indicated that both oligonucleotides were efficiently distributed to numerous ocular tissues, including retina, ciliary body, and optic nerve. CONCLUSIONS: ISIS 13312 possesses antiviral activity and pharmacokinetic properties that favor its use as a therapeutic agent in treatment of CMV retinitis. The half-life of ISIS 13312 in retina is longer than that of ISIS 2922, potentially allowing for less frequent administration.
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Antivirais/farmacologia , Antivirais/farmacocinética , Citomegalovirus/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/farmacocinética , Retina/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Sequência de Bases , Disponibilidade Biológica , Corpo Ciliar/metabolismo , Citomegalovirus/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Desenho de Fármacos , Fibroblastos/virologia , Meia-Vida , Humanos , Macaca fascicularis , Dados de Sequência Molecular , Oligonucleotídeos Fosforotioatos , Coelhos , Tionucleotídeos/farmacocinética , Tionucleotídeos/farmacologia , Distribuição Tecidual , Ensaio de Placa Viral , Corpo Vítreo/metabolismoRESUMO
Phosphorothioate (PS) oligodeoxynucleotides represent the class of antisense drugs most advanced in development and clinical testing. Exploitation of antisense oligonucleotide technology for development of rationally designed therapeutic drugs has presented a unique set of challenges, some of which relate to their pharmacokinetic behavior in vivo. Pharmacokinetic studies of PS oligodeoxynucleotides demonstrate that they are well absorbed from parenteral sites, rapidly distributed broadly to all peripheral tissues, do not cross the blood-brain barrier, and are eliminated primarily by slow metabolism in tissues. In general, the pharmacokinetic properties of this class of compounds appear to be largely driven by chemistry rather than sequence.
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Oligodesoxirribonucleotídeos Antissenso/farmacocinética , Tionucleotídeos/farmacocinética , Animais , Proteínas Sanguíneas/metabolismo , Humanos , Injeções Intravenosas , Taxa de Depuração Metabólica , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Ligação Proteica , Tionucleotídeos/administração & dosagem , Distribuição TecidualRESUMO
ISIS 22023 is a modified phosphorothioate antisense oligonucleotide targeting murine Fas mRNA. Treatment of mice with ISIS 22023 reduced Fas expression in liver in a concentration-dependent and sequence-specific manner, which completely protected mice from fulminant death induced by agonistic Fas antibody. In this study, we characterized the relationships in mice between total dose administered, dose to the target organ, and ultimately, the intracellular concentration within target cell types to the pharmacologic activity of ISIS 22023. After subcutaneous injection, ISIS 22023 distributed to the liver rapidly and remained in the liver with the t(1/2) ranging from 11 to 19 days, depending on dose. There were apparent differences in patterns of uptake and elimination in different types of liver cells. Oligonucleotide appeared within hepatocytes rapidly, whereas the peak concentrations in Kupffer cells were delayed until 2 days after dose administration. Hepatocytes cleared oligonucleotide the most rapidly, whereas Kupffer cells appeared to retain oligonucleotide longer. The reduction of Fas mRNA levels (pharmacodynamic response) paralleled the increase of oligonucleotide concentration in mouse liver with maximum mRNA reduction of 90% at 2 days after a single 50 mg/kg subcutaneous administration. Moreover, the pharmacodynamics of ISIS 22023 correlated better with the pharmacokinetics in hepatocytes, supporting the concept that the presence of oligonucleotide in target cells results in reductions in mRNA and, ultimately, pharmacologic activity. These results provide a comprehensive understanding of the kinetics of an antisense drug at the site of action and demonstrate that the reductions in mRNA induced by this antisense oligonucleotide correlate with its concentrations in cell targets.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/genética , Receptor fas/genética , Algoritmos , Animais , Feminino , Meia-Vida , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ensaios de Proteção de Nucleases , Oligonucleotídeos Antissenso/sangue , Oligonucleotídeos Fosforotioatos , RNA Mensageiro/isolamento & purificaçãoRESUMO
Plasma pharmacokinetics, biodistribution, excretion, and metabolism of four modified 20-mer antisense oligonucleotides targeted to human intercellular adhesion molecule-1 mRNA have been characterized in rats and compared with a first-generation phosphorothioate oligodeoxynucleotide (PS ODN), ISIS 2302. The modified oligonucleotides contained 2'-O-(2-methoxyethyl) (2'-O-MOE) ribose sugar modifications on all or a portion of the nucleotides in the antisense sequence. The 2'-O-MOE-modified oligonucleotides were resistant to nuclease metabolism in both plasma and tissue. In general, plasma pharmacokinetics was not substantially altered by addition of the 2'-O-MOE modification to PS ODN. Thus, plasma clearance was dominated by distribution to tissues, broadly, with less than 10% of the administered dose excreted in urine or feces over 24 h. However, the 2'-O-MOE modification combined with the phosphodiester (PO) backbone exhibited 10-fold more rapid plasma clearance, with approximately 50% of the dose excreted in urine as intact oligonucleotide. Consistent with its rapid and extensive excretion, the PO 2'-O-MOE modification distributed to very few organs in any substantial amount with the exception of the kidney. Oligonucleotides that contained phosphorothioate backbones were highly bound to plasma proteins. Indeed, the primary characteristic that resulted in the most marked alterations in pharmacokinetics appeared to be the affinity and capacity of these compounds to bind plasma proteins. A balance of greater stability supplied by the 2'-O-MOE modification together with maintenance of plasma protein binding appears to be necessary to ensure favorable pharmacokinetics of this new generation of antisense oligonucleotides.
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Oligonucleotídeos Antissenso/farmacocinética , Tionucleotídeos/farmacocinética , Animais , Desoxirribonucleases/metabolismo , Estabilidade de Medicamentos , Masculino , Oligonucleotídeos Antissenso/sangue , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/urina , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Tionucleotídeos/sangue , Tionucleotídeos/química , Tionucleotídeos/urina , Distribuição TecidualRESUMO
Three modified 20-mer antisense oligonucleotides targeted to human intercellular adhesion molecule-1 mRNA were characterized for their presystemic stability and oral bioavailability compared with a first-generation phosphorothioate oligodeoxynucleotide (PS ODN), ISIS 2302. The three modified oligonucleotides contained 2'-O-(2-methoxyethyl) (2'-O-MOE) ribose sugar modifications on a portion, or on all of the nucleotides in the antisense sequence. In vitro metabolism studies conducted in various gastrointestinal and digestive tissue preparations indicated substantial improvement in stability of 2'-O-MOE-modified oligonucleotides. In addition, in vivo presystemic stability of these oligonucleotides was monitored in rats following intraduodenal administration. By 8 h after administration, only chain-shortened metabolites of the PS ODN were recovered in the gastrointestinal contents. In contrast, approximately 50% of the 2'-O-MOE ribose-modified (partial) compound remained intact (20-mer) by 8 h following administration. Both of the fully modified compounds (2'-O-MOE PO and PS) were completely stable with no measurable metabolites observed within 8 h of administration. The rank order of bioavailability was ISIS 11159 (full PS, full MOE) < ISIS 2302 (PS ODN) < ISIS 16952 (full PO, full MOE) < ISIS 14725 (full PS, partial MOE); the absolute plasma concentration bioavailability was measured in reference to intravenous dosing in the rat and was estimated at 0.3, 1.2, 2.1, and 5.5%, respectively. The optimal oligonucleotide chemistry for improved permeability and resulting bioavailability was the partially modified 3' hemimer 2'-O-MOE phosphorothioate oligonucleotide (ISIS 14725). Improved presystemic stability coupled with improved permeability were likely responsible for the remarkable improvement in the oral bioavailability of this compound.
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Oligonucleotídeos Antissenso/farmacocinética , Tionucleotídeos/farmacocinética , Animais , Disponibilidade Biológica , Estabilidade de Medicamentos , Masculino , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Permeabilidade , Ratos , Ratos Sprague-Dawley , Tionucleotídeos/química , Tionucleotídeos/metabolismoRESUMO
The plasma pharmacokinetics and tissue disposition of ISIS 2503 were studied in mice following single and multiple bolus intravenous (iv) injections of 1-50 mg/kg, and in monkeys following single and multiple 2-h iv infusions of 1-10 mg/kg and bolus iv injections of 1 mg/kg of ISIS 2503. ISIS 2503 and its metabolites were measured in plasma, urine, and tissues using solid-phase extraction followed by capillary gel electrophoresis (CGE). In both species, the plasma clearance of ISIS 2503 was characterized by rapid distribution to tissues, and to a lesser extent, metabolism. The plasma clearance in mice was at least two-fold more rapid than in monkeys at equivalent doses. The plasma disposition (t1/2) increased with dose. The highest concentrations of oligonucleotide were consistently observed in the kidney and liver in both species. At equivalent doses, tissue concentrations in monkeys were much higher than tissue concentrations in mice. Urinary excretion of total oligonucleotide was a minor elimination pathway in both species at doses < 10 mg/kg. However, urinary excretion of total oligonucleotide in mice was increased to 12-29% as dose increased from 20 to 50 mg/kg.
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Genes ras , Oligonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/genética , Tionucleotídeos/farmacocinética , Animais , Sequência de Bases , Proteínas Sanguíneas/metabolismo , Primers do DNA , Haplorrinos , Humanos , Camundongos , Oligonucleotídeos Antissenso/sangue , Oligonucleotídeos Antissenso/urina , Compostos Organofosforados/sangue , Compostos Organofosforados/farmacocinética , Compostos Organofosforados/urina , Tionucleotídeos/sangue , Tionucleotídeos/urina , Distribuição TecidualRESUMO
Antisense technology has progressed beyond the point of using only phosphorothioate oligodeoxynucleotides as therapeutic agents to looking at antisense molecules that contain additional chemical modifications as the next generation of therapeutic agents. These modifications are intended to improve the overall therapeutic properties by increasing potency, optimizing pharmacokinetic properties and improving the safety profile. This review will focus on the non-clinical pharmacokinetic and safety properties of 2'-O-methoxyethyl-modified oligonucleotides. Implications on the convenience and safe use of these compounds as therapeutic agents will be discussed.
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Oligonucleotídeos Antissenso/farmacologia , Animais , Haplorrinos , Humanos , Camundongos , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Fosforotioatos , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Antisense oligonucleotides are currently being investigated for the treatment of a variety of diseases. Antisense drugs are being administered primarily by parenteral injection. To explore more convenient patient delivery methods, we have characterized the tissue kinetics and tolerability of an inhaled aerosol formulation of a phosphorothioate oligonucleotide in mice. Concentrations of oligonucleotide in bronchioalveolar lavage fluid, plasma, and tissue and immunohistochemical localization were used to assess deposition and pharmacokinetic parameters. Significant concentrations of oligonucleotide in lung, as well as systemic tissues, were measured following a pulmonary dose of 12 mg/kg. Doses as low as 1-3 mg/kg also produced significant concentrations of oligonucleotide (>50 microg oligonucleotide per gram of tissue), and these were maintained in the lung with a halflife of 20 hours or greater. Oligonucleotide was localized to bronchiolar epithelium and alveolar epithelium and endothelium. Toxicity was mild at the 12 mg/kg level and minimal to absent at doses of 3 mg/kg or below. Based on a favorable pharmacokinetic profile and a relative lack of toxicity, inhalation delivery appears to be a therapeutic option for antisense oligonucleotides.
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Administração por Inalação , Pulmão/metabolismo , Oligodesoxirribonucleotídeos/farmacocinética , Oligodesoxirribonucleotídeos/toxicidade , Aerossóis/administração & dosagem , Animais , Líquido da Lavagem Broncoalveolar/química , Relação Dose-Resposta a Droga , Epitélio/química , Epitélio/metabolismo , Meia-Vida , Imuno-Histoquímica , Pulmão/química , Camundongos , Camundongos Endogâmicos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/análise , Tamanho da PartículaRESUMO
The pharmacokinetics of subcutaneous (s.c.) administration of a phosphorothioate oligodeoxynucleotide (PS-ODN) was evaluated in cynomolgus monkeys. In a single dose study, monkeys were injected s.c. or intravenously (i.v.) with doses of either 1 or 5 mg/kg ISIS 2302. The bioavailability of s.c. injection ranged from 26% to 55% and appeared to be dependent on the concentration of the dosing solution rather than the dose. The bioavailability of a subcutaneously administered 5 mg/kg dose of ISIS 2302 was 55% using a 50 mg/ml dosing solution and only 26% using a 10 mg/ml dosing solution. Slow absorption from the s.c. injection site significantly blunted the maximal concentration (Cmax) compared with i.v. administration. The time to peak plasma concentration (Tmax) increased slightly with increasing dose, from 0.5 to 1 hour for the 1 mg/kg dose to 1 to 2.5 hours for the 5 mg/kg dose. Plasma half-lives were prolonged after s.c. administration, indicating more dependence on absorption than elimination. The half-lives after s.c. administration averaged 3 hours, whereas after i.v. administration, the half-lives were <1 hour. Metabolism of the ISIS 2302 after s.c. injection was consistent with exonucleolytic cleavage, as previously observed after i.v. administration. In summary, s.c. administration of PS-ODN resulted in prolonged and extensive absorption of the ODN.
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Oligodesoxirribonucleotídeos Antissenso/farmacocinética , Tionucleotídeos/farmacocinética , Animais , Fármacos Gastrointestinais/sangue , Fármacos Gastrointestinais/metabolismo , Fármacos Gastrointestinais/farmacocinética , Injeções Intravenosas , Injeções Subcutâneas , Macaca fascicularis , Modelos Animais , Oligodesoxirribonucleotídeos Antissenso/sangue , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos , Tionucleotídeos/sangue , Tionucleotídeos/metabolismoRESUMO
Rigorous extraction methods coupled with capillary gel electrophoresis (CGE) provide a basis for a nonradiolabel assay for quantitation of intact antisense drug and its numerous chain-shortened metabolites. As part of the validation of the CGE method, we compared the quantitation of unlabeled ISIS 3521 (ISI 641A) and its chain-shortened metabolites with total radioactivity of [(35)S]-ISIS 3521. ISIS 3521 was labeled on the fifth nucleotide linkage from the 5'-end with (35)S by well-established methods. Multiple tissues collected from rats after administration of [(35)S]-ISIS 3521 were assayed by both radiolabel (liquid scintillation spectroscopy) and CGE methods. The CGE method provided accurate quantitation of the drug and its metabolites in kidney cortex and liver tissues. The correlation between methods for multiple tissues over time was excellent with 88.5% of the measurements being statistically equivalent. These data suggest that CGE is an accurate means of quantitating oligonucleotide in tissue and that it compares favorably with traditional radiochemical techniques. Clearance half-lives for total measurable oligonucleotides were equivalent to clearance of total radioactivity in both liver and kidney with the longest clearance half-life associated with the kidney.
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Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos/análise , Animais , Eletroforese Capilar/métodos , Meia-Vida , Córtex Renal/metabolismo , Fígado/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacocinética , Ratos , Tionucleotídeos/farmacocinéticaRESUMO
PURPOSE: This study examined the pharmacokinetics and tissue distribution of an antisense oligonucleotide ISIS 2503, formulated in stealth (pegylated) liposomes (encapsulated) or in phosphate-buffered saline (unencapsulated). METHODS: Encapsulated or unencapsulated ISIS 2503 was administered to rhesus monkeys by intravenous infusion. The concentrations of ISIS 2503 and metabolites in blood, plasma, and tissue samples were determined by capillary gel electrophoresis. RESULTS: Plasma concentrations of encapsulated ISIS 2503 decreased mono-exponentially after infusion with a mean half-life of 57.8 hours. In contrast, the concentration of unencapsulated ISIS 2503 in plasma decreased rapidly with a mean half-life of 1.07 hours. Both encapsulated and unencapsulated ISIS 2503 distributed widely into tissues. Encapsulated ISIS 2503 distributed primarily to the reticulo-endothelial system and there were few metabolites observed. In contrast, unencapsulated ISIS 2503 distributed rapidly to tissue with highest concentration seen in kidney and liver. Nuclease-mediated metabolism was extensive for unencapsulated oligonucleotide in plasma and tissues. CONCLUSIONS: The data suggest that stealth liposomes protect ISIS 2503 from nucleases in blood and tissues, slow tissue uptake, and slow the rate of clearance from the systemic circulation. These attributes may make these formulations attractive for delivering oligonucleotides to sites with increased vasculature permeability such as tumors or sites of inflammation.
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Oligonucleotídeos Antissenso/farmacocinética , Proteínas ras/antagonistas & inibidores , Animais , Proteínas Sanguíneas/metabolismo , Cápsulas/farmacocinética , Sistemas de Liberação de Medicamentos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Cinética , Lipossomos , Macaca mulatta , Masculino , Taxa de Depuração Metabólica , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos , Distribuição Tecidual , Proteínas ras/genéticaRESUMO
The aromatic retinoid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1 -propenyl] benzoic acid (TTNPB) is 1000-fold more teratogenic than all trans-retinoic acid (tRA) in several species. Factors that partially explain the potency of this retinoid include binding affinities to retinoid nuclear receptors (RARs) in the nanomolar range, reduced affinities for the cytosolic binding proteins (CRABPs), and slow rate of metabolism (M. A. Pignatello, F. C. Kauffman, and A. A. Levin, Toxicol. Appl. Pharmacol. 142, 319-327, 1997). The present work investigates the possible involvement of longer receptor occupancy and increased transcriptional activity of the ligand receptor complex in the greater toxicity of TTNPB. Ligand off-rates from nuclear receptors were determined in nucleosol fractions prepared from COS-1 cells transfected with cDNA encoding the appropriate RAR subtype. When assayed at 10 degrees C, [3H]TTNPB was displaced from the RARs at a significantly faster rate than that of [3H]tRA. The difference in displacement was reduced at 4 degrees C. These observations are consistent with the 10-fold lower affinity of TTNPB vs tRA for RARs and, therefore, do not explain the greater potency of TTNPB. The ability of TTNPB and tRA to activate the RARs was determined using a luciferase reporter gene transfected into JEG-3 cells with the appropriate RAR subtype. The expression of the reporter was driven by a retinoic acid response element (RARE) from the RAR beta gene, which was incorporated into the reporter plasmid. Dose-response for gene activation indicated that the potency of TTNPB and tRA in activating mRAR alpha, beta, and gamma was similar after 24 h with comparable EC50s in the nanomolar range. However, after 72 h, activation by TTNPB was greater than that of tRA as indicated by EC50s and threshold for activation. This study indicates that the higher potency of TTNPB in activating the RARs may be due to slower disappearance of the retinoid and, therefore, is a contributing factor to its greater toxicity.
Assuntos
Antineoplásicos/toxicidade , Benzoatos/toxicidade , Receptores do Ácido Retinoico/metabolismo , Retinoides/toxicidade , Transcrição Gênica/efeitos dos fármacos , Animais , Ligação Competitiva , Células COS , Células Cultivadas , Enzimas/metabolismo , Ligantes , Luciferases/metabolismo , Plasmídeos , Ligação Proteica , Receptores do Ácido Retinoico/classificação , Fatores de Risco , Temperatura , Fatores de Tempo , Transfecção , beta-Galactosidase/metabolismoRESUMO
Antisense therapeutics using synthetic oligodeoxynucleotides (ODNs) are currently being evaluated in clinical trials for cancer, inflammation, and viral diseases. These macromolecules afford a unique opportunity to treat disease at the molecular level. The specificity of these compounds is derived from the genetic code and Watson-Crick base pairing, utilizing an antisense paradigm for the inhibition of translation and the regulation of protein expression. Currently, most antisense ODNs in development contain a phosphorothioate (P=S) backbone. Additional modifications primarily involve the 2' position on the ribose or modification of the nucleotide linkages of the backbone. To date, no toxicities in animal models appear related to inhibition of the pharmacologic target, rather toxicities induced by P=S ODNs appear similar and are independent of pharmacologic target. In general, toxicities correlate well with pharmacokinetic or tissue distribution parameters. In primates, the primary acute effects are associated with complement activation and the systemic effects associated with accumulation of high concentrations of P=S ODNs in the kidneys. In rodents, the primary effect is an immune stimulation characterized by splenomegaly, lymphoid hyperplasia, and mononuclear cell infiltrates in multiple tissues. At extraordinarily high doses (15-50 times the targeted clinical doses), hepatocellular and renal tubular degeneration are evident in rodents. Second generation antisense compounds, new routes of administration, and new formulations appear to broaden and improve the application of antisense technology.