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A A Levin

Publications and source records attributed to A A Levin.

At least 19 recordsLinked to original sources
PubMed2001

Comparison of pharmacokinetics and tissue disposition of an antisense phosphorothioate oligonucleotide targeting human Ha-ras mRNA in mouse and monkey.

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.

Animals↗
PubMed2001

Pharmacokinetics and pharmacodynamics of an antisense phosphorothioate oligonucleotide targeting Fas mRNA in mice.

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.

Algorithms↗
PubMed2001

Pharmacokinetic properties of 2'-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats.

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.

Animals↗
PubMed2001

Absolute bioavailability of 2'-O-(2-methoxyethyl)-modified antisense oligonucleotides following intraduodenal instillation in rats.

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.

Animals↗
PubMed2001

Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides.

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.

Animals↗
PubMed2001

Antiviral activity and ocular kinetics of antisense oligonucleotides designed to inhibit CMV replication.

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.

Animals↗
PubMed2000

Pharmacokinetic and toxicity profile of a phosphorothioate oligonucleotide following inhalation delivery to lung in mice.

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.

Administration, Inhalation↗
PubMed2000

Comparison of the pharmacokinetics of subcutaneous and intravenous administration of a phosphorothioate oligodeoxynucleotide in cynomolgus monkeys.

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.

Animals↗
PubMed1999

A nonradioisotope biomedical assay for intact oligonucleotide and its chain-shortened metabolites used for determination of exposure and elimination half-life of antisense drugs in tissue.

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.

Animals↗
PubMed1999

Multiple factors contribute to the toxicity of the aromatic retinoid TTNPB (Ro 13-7410): interactions with the retinoic acid receptors.

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.

Animals↗
PubMed1999

Pharmacokinetics and tissue disposition in monkeys of an antisense oligonucleotide inhibitor of Ha-ras encapsulated in stealth liposomes.

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.

Animals↗
PubMed1998

Preclinical evaluation of the effects of a novel antisense compound targeting C-raf kinase in mice and monkeys.

CGP 69846A (ISIS 5132) is an antisense phosphorothioate oligodeoxynucleotide which targets human C-raf kinase and is currently being developed as an antineoplastic agent. The toxicity of this compound was evaluated in mice and monkeys following repeated i.v. injections or infusions for 4 weeks at doses up to 100 mg/kg. Because CGP 69846A is inactive in the mouse, ISIS 11061, the murine-specific homologue targeting C-raf kinase mRNA was evaluated concurrently with CGP 69846A to assess the potential toxicity associated with reduced C-raf expression. There were no toxicities that differentiated ISIS 11061 from CGP 69846A in mice. Effects in mice included hepatomegaly and hepatocellular degeneration at the high dose of 100 mg/kg CGP 69846A that potentially resulted in lethality. Other effects which were observed at 20 and 100 mg/kg included mononuclear cell infiltrates in multiple organs, extramedullary hematopoiesis in the spleen and liver, an increase in bone marrow cellularity, an increase in white blood cells, a decrease in platelet counts, and Kupffer cell hyperplasia. These alterations were reversible following a recovery period. No adverse effects in mice were observed with doses < or = 10 mg/kg. In monkeys, administration of 10 mg/kg of CGP 69846A was associated with effects observed with other P = S ODNs, namely, prolongation of activated partial thromboplastin time (APTT) and activation of complement. These effects were transient and correlated with plasma concentrations of CGP 69846A. Below a concentration of 35 micrograms/ml of intact CGP 69846A the prolongation of APTT was less than 50% and levels of complement split products were not increased. All monkeys tolerated complement activation with no evidence of treatment-related clinical signs. Complement and coagulation were not affected by the lower doses of 1 and 3 mg/kg. No histopathology or alteration in hematology or serum chemistry was induced by doses up to 10 mg/kg in monkeys. The plasma and tissue deposition of CGP 69846A were characterized in mice and monkeys and toxicity was dependent on dose of CGP 69846A. In the present preclinical evaluation of toxicity in mice and monkeys, CGP 69846A is well tolerated at doses targeted for clinical trials. Toxicities induced by CGP 69846A in monkeys and mice occurred at doses of 10 mg/kg and greater. Effects induced by CGP 69846A were not unique and have been observed previously with other phosphorothioate oligodeoxynucleotides.

Animals↗
PubMed1998

Pharmacokinetics of an antisense oligonucleotide injected intravitreally in monkeys.

The kinetics of an intravitreally administered phosphorothioate oligonucleotide, ISIS 2922, were studied in cynomolgus monkeys. Vitreal and retinal concentrations were measured after administration of 11, 57, or 115 microg/eye. ISIS 2922 concentrations in vitreous and retina were compared, after single, weekly, or biweekly doses, for potential accumulation. ISIS 2922 levels were quantified using solid-phase extraction followed by capillary gel electrophoresis. Concentrations of ISIS 2922 in the vitreous were proportional to the dose and were nearly linear with respect to the dose. The ISIS 2922 concentrations 3 days after dosing ranged from 80 nM to approximately 1.5 microM. By 14 days after intravitreal injection, the concentrations were below the limit of quantitation (<10 nM) for all dose groups. There was no accumulation in the vitreous after multiple weekly or biweekly doses. The concentrations of ISIS 2922 in the retina 2 days after a single intravitreal injection ranged from 50 nM to 1.1 microM. The uptake and disposition of ISIS 2922 in the retina appeared to have been saturated between the 57- and 115-microg doses; the average concentrations were 0.71 +/- 0.24 microM (N = 4) and 0.88 +/- 0.27 microM (N = 3) for the two doses, respectively. Electrophoretic profiles of extracts revealed multiple chain-shortened oligonucleotides in the vitreous and retina, suggesting extensive metabolism in both compartments. Analyses from the multiple-dose study suggested that accumulation was dependent on the total administered dose, with accumulation occurring after biweekly dosing in the 115-microg dose group and only after weekly dosing in the 57-microg dose group.

Animals↗
PubMed1997

Characterization of the retinoid binding properties of the major fusion products present in acute promyelocytic leukemia cells.

The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are present in different lengths of PML (amino acids 1-552 and 1-394, respectively), contain most of the functional domains of PML and RAR alpha, bind all-trans-retinoic acid (t-RA), and act as t-RA-dependent transcription factors. T-RA is an effective inducer of clinical remission only in patients carrying the t(15;17) and expressing the PML/RAR alpha products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RAR alpha product has been found associated with a poorer prognosis than bcr1-PML/RAR alpha. In the present study we have investigated the structural and functional properties of the bcr3-PML/RAR alpha in comparison to the previously characterized bcr1-PML/RAR alpha. In particular, we have measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to both of these isoforms. T-RA binding analysis of nuclear and cytosolic extracts prepared from bcr3-PML/RAR alpha APL patients and from bcr3-PML/RAR alpha COS-1 transfected cells indicates that this protein is present only as high-molecular-weight nuclear complexes. Using saturation binding assays and Scatchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RAR alpha receptor than to bcr1-PML/RAR alpha or RAR alpha (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). Moreover, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 and 0.075 nmol/L) were detectable in the bcr3-PML/RAR alpha product but not in the bcr1-PML/RAR alpha product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RAR alpha isoform than to the bcr1-PML/RAR alpha or RAR alpha. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RAR alpha product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the betaRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RAR alpha products can be measured.

Alitretinoin↗
PubMed1997

Didehydroretinoic acid: retinoid receptor-mediated transcriptional activation and binding properties.

All-trans-3,4-Didehydroretinoic acid (vitamin A2 acid; DDRA) is one of the retinoids present in human skin, the most responsive tissue to retinoid treatment. To understand the mechanism of action of DDRA in the control of differentiation and tumorigenesis, we studied its interaction with cellular retinoic acid-binding proteins (CRABPs) and nuclear all-trans-retinoic acid (RA) receptors (RARs), and 9-cis-retinoic acid receptors (RXRs). The IC50 plots of DDRA for inhibition of [3H]RA binding to CRABP I and II and to RAR alpha, beta and gamma illustrate that this retinoid binds with the same affinity as RA to these proteins. DDRA, however, showed higher affinity than RA for RXR alpha. Evaluation of the transcriptional activation potential of DDRA in CV-1 cells showed that this retinoid induced RAR alpha-mediated transcription to the same magnitude as RA in the 10(-9) to 10(-6) M concentration range. However, in comparison to RA, DDRA produced a 2- to 3-fold higher activation of the transcription mediated by RXR alpha homodimers, as well as RAR beta-RXR alpha heterodimers. These results suggest that the biological activity of retinoids in the skin may be attained through the joint potential of both RA and DDRA.

Animals↗