Phenotypic variation in the response to the human immunodeficiency virus among derivatives of the CEM T and WIL-2 B cell lines.

Derivatives of the CEM T and WIL-2 B cell lines showed striking diversity in their responses to the HTLV-IIIB strain of the human immunodeficiency virus (HIV). Several stable phenotypic patterns could be defined, based on whether cells were permissive (P+, P-) for virus production, were sensitive or insensitive to cytopathic effects after infection by free virus (C+, C-), and whether they underwent fusion on contact with virus-infected cells (F+, F-). Although expression of CD4 was essential for infection by HTLV-IIIB, very low levels were sufficient for productive infection of WIL-2 derivatives. Conversely, some CEM T cell lines that expressed ample CD4, and which were able to bind virus gp120 and undergo fusion, did not support productive infection by free virus. One nonpermissive, CD4+ derivative of CEM could bind gp120 but failed to undergo fusion, suggesting an alteration in some membrane protein other than CD4 that is essential for virus entry and HIV-induced cell fusion. The AA2 derivative of the WIL-2 cell line is also described, which is remarkably permissive for HIV replication and exquisitely sensitive to virus cytopathic effect. The panel of related cell lines with different host-virus phenotypes could be useful for more precisely defining steps in the infectious cycle of HIV, and for identifying host cell genes and gene products that determine the outcome of HIV infection.

Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells.

Nuclear and whole-cell deoxynucleoside triphosphate (dNTP) pools were measured in HeLa cells at different densities and throughout the cell cycle of synchronized CHO cells. Nuclei were prepared by brief detergent (Nonidet P-40) treatment of subconfluent monolayers, a procedure that solubilizes plasma membranes but leaves nuclei intact and attached to the plastic substratum. Electron microscopic examination of monolayers treated with Nonidet P-40 revealed protruding nuclei surrounded by cytoskeletal remnants. Control experiments showed that nuclear dNTP pool sizes were stable during the time required for isolation, suggesting that redistribution of nucleotides during the isolation procedure was minimal. Examination of HeLa whole-cell and nuclear dNTP levels revealed that the nuclear proportion of each dNTP was distinct and remained constant as cell density increased. In synchronized CHO cells, all four dNTP whole-cell pools increased during S phase, with the dCTP pool size increasing most dramatically. The nuclear dCTP pool did not increase as much as the whole-cell dCTP pool during S phase, lowering the relative nuclear dCTP pool. Although the whole-cell dNTP pools decreased after 30 h of isoleucine deprivation, nuclear pools did not decrease proportionately. In summary, nuclear dNTP pools in synchronized CHO cells maintained a relatively constant concentration throughout the cell cycle in the face of larger fluctuations in whole-cell dNTP pools. Ribonucleotide reductase activity was measured in CHO cells throughout the cell cycle, and although there was a 10-fold increase in whole-cell activity during S phase, we detected no reductase in nuclear preparations at any point in the cell cycle.

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.

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.

Two steps in the intracellular transport of IgD are sensitive to energy depletion.

The secretory pathway of murine IgD can be dissected by the use of carbonylcyanide m-chlorophenylhydrazone (CCCP), which inhibits two distinct steps of intracellular transport. The newly synthesized IgD that accumulates at the first step contains high mannose type oligosaccharides which are partially trimmed. The IgD arrested at this step is less processed than the IgD arrested by treatment with monensin. The properties of this biosynthetic intermediate are consistent with inhibition of Ig passage from the endoplasmic reticulum to the Golgi complex. A second CCCP-sensitive step exists in the biosynthesis of IgD, and is characterized by delta-chains that are resistant to endoglycosidase H and contain galactose. This indicates that this second step occurs during or after the passage through the trans-Golgi compartment. The galactose-containing oligosaccharides of the delta-chains arrested at this step do not contain fucose (as do mature, secreted delta-chains). Fucosylation is not inhibited by CCCP, nor is the secretion of fucose-containing delta-chains. These results show that terminal sugars are added to secretory IgD in at least two transport compartments, separable by their sensitivity to CCCP. The inhibition of the secretory pathway at both steps is reversible; upon removal of the drug the arrested IgD is processed normally and is secreted. The sensitivity to CCCP probably reflects transport steps that are sensitive to even partial depletion of ATP, because treatments with other inhibitors of oxidative phosphorylation yield similarly arrested Ig molecules. Thus, by using the protonophore CCCP, we demonstrate two energy-requiring steps in IgD transport which seem to be at two transitions in the secretory pathway. One step is during the passage from the endoplasmic reticulum to the mid-Golgi compartment and the other step is during Ig passage through the trans-Golgi, or subsequent transport to the cell surface.

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.

Quantitation of phosphorothioate oligonucleotides in human plasma.

Methods are presented for the extraction of phosphorothioate oligonucleotides from human plasma to permit quantitation by capillary gel electrophoresis. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reverse-phase column to remove salts. A second desalting step, achieved by dialysis utilizing a membrane with a molecular weight cutoff of 2500 Da floating on distilled water, was required to remove residual ionic material from the extracted sample. This method should be generally applicable to the analysis and quantitation of phosphorothioate oligonucleotides.

Ribonucleotide reductase: evidence for specific association with HeLa cell mitochondria.

Mammalian mitochondria contain pools of deoxyribonucleoside 5'-triphosphates that behave differently from the much larger whole-cell pools. To investigate the origins of these pools, we analyzed HeLa cell mitochondria for ribonucleotide reductase activity. Three findings suggest specific association of a reductase with mitochondria: (1) enzyme activity in extracts of washed mitochondria, (2) stimulation of that activity by dATP at levels inhibitory to the major cellular activity, and (3) association of immunoreactive material with washed and fractionated mitochondria.

Pharmacokinetics of a potential human cytomegalovirus therapeutic, a phosphorothioate oligonucleotide, after intravitreal injection in the rabbit.

The disposition of ISIS 2922, a phosphorothioate oligonucleotide for treatment of cytomegalovirus associated retinitis, was evaluated in rabbits. Vitreous humor and retina samples were collected from rabbits that received a single intravitreal injection of 66 microg [14C]-labeled ISIS 2922 and were analyzed using anion exchange HPLC. Four hr postdosing, the concentration of ISIS 2922 in vitreous humor was 3.3 microM. The elimination of ISIS 2922 from the vitreous humor exhibited first-order kinetics with a t1/2 of 62 hr. By 10 days postdosing, the mean concentration of ISIS 2922 in rabbit vitreous humor had decreased to 0.17 microM, which represented 22% of the total radioactivity remaining in the vitreous. The remaining 78% coeluted on anion exchange HPLC with shorter oligonucleotides. In retina, ISIS 2922 accumulated over the first 5 days postdosing, reaching a maximum concentration of 3.5 microM, and then declined thereafter with an estimated t1/2 of 79 hr. By 10 days postdosing when only 24% of the total radioactivity in the retina was parent compound, the concentration of ISIS 2922 remained at 1.6 microM, which was 10 times higher than the concentration in the vitreous humor. Whereas the elimination of full-length ISIS 2922 and total radioactivity from the vitreous humor occurred at nearly equal rates, ISIS 2922 disappeared more rapidly than did total radioactivity from the retina, suggesting a greater role for metabolism in the clearance process from retina than the vitreous. Alternatively, the results are consistent with metabolites being cleared from the vitreous at approximately the same rate as parent compound while in the retina metabolites may be cleared more slowly. The data were analyzed with a user-defined pharmacokinetic model, which was then used to predict the potential for accumulation of ISIS 2922 during clinical dosing.

Isotope effects and alternative substrate reactivities for tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a hemoprotein which catalyzes the first step in the oxidative degradation of tryptophan. The reaction is believed to proceed by addition of O2 across the 2,3-bond of the indole ring, followed by decomposition of the resultant dioxetane to give N-formylkynurenine. A primary D2O isotope effect of 4.4 on Vmax/Km was observed at the pH optimum, pH 7.0. This implies that abstraction of the indole proton is at least partially rate-determining. An inverse secondary isotope effect of 0.96 was observed for L-[2-3H]tryptophan at this pH. The secondary isotope effect signals the formation of the C-O bond at C-2. As the rate of proton abstraction increased with increasing pH, the D2O isotope effect decreased to 1.2 at pH 8.5 and the secondary isotope effect increased to 0.92. The rate-determining steps therefore change with increasing pH, and bond formation at C-2 becomes more rate-limiting. The secondary isotope effect did not change significantly with varying O2 concentration so that substrate binding is primarily ordered with O2 binding first. The specificity of the enzyme towards substituted tryptophans shows that substitution of the phenyl ring of the indole is sterically unfavorable. Steric hindrance is highest at the 4- and 7-positions, while the 5- and 6-positions are less sensitive. 6-Fluoro-L-tryptophan was more reactive than tryptophan, and the increased reactivity can be explained by an electronic effect that enhances of the rate of C-O bond formation at C-2.

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.

Pharmacokinetics and metabolism in mice of a phosphorothioate oligonucleotide antisense inhibitor of C-raf-1 kinase expression.

The plasma and tissue disposition of CGP 69846A (ISIS 5132) was characterized in male CD-1 mice following iv bolus injections administered every other day for 28 days (total of 15 doses). The doses ranged from 0.8 mg/kg to 100 mg/kg. Urinary excretion of oligonucleotide was also monitored over a 24-hr period following single dose administration over the same dose range. Pharmacokinetic plasma profiles were determined following single dose administration (dose 1) and after multiple doses (dose 15) at doses of 4 and 20 mg/kg. Concentrations in kidney, liver, spleen, heart, lung, and lymph nodes were characterized following doses 1, 8, and 15 for all doses. Capillary gel electrophoresis was used to quantitate intact (full-length) oligonucleotide and its metabolites (down to N - 11 base deletions) in both plasma and tissue at all time points. The plasma and tissue disposition of CGP 69846A was characterized by a rapid distribution into all tissues analyzed. Rapid plasma clearance of the parent oligonucleotide (9.3-14.3 ml/min/kg) was predominantly the result of distribution to tissue and, to a lesser extent, metabolism. Appearance and pattern of chain-shortened metabolites seen in plasma and tissue were consistent with predominantly exonuclease-mediated base deletion. No measurable accumulation of oligonucleotide was observed in plasma following multiple-dose administration, but both the liver and the kidney exhibited 2-3-fold accumulations. In general, the tissues exhibited half-lives for the elimination of parent oligonucleotide of 16-60 hr compared with plasma half-lives of 30-45 min. After repeated administrations, significant decreases in plasma clearance and volume of distribution at steady state (Vss) were observed following dose 15 at the dose of 20 mg/kg but not at the dose of 4 mg/kg. Changes in tissue accumulation and evidence for saturation of tissue distribution at the high doses may explain the plasma disposition changes observed in the absence of alteration of metabolism or plasma accumulation. Urinary excretion was a minor pathway for elimination of oligonucleotide over the 24-hr period immediately following iv administration. However, the amount of oligonucleotide excreted in the urine increased as a function of dose from less than 1% to approximately 13% of the administered dose over a dose range of 0.8 mg/kg to 100 mg/kg.

Phase I safety and pharmacokinetic profile of an intercellular adhesion molecule-1 antisense oligodeoxynucleotide (ISIS 2302).

Healthy male volunteers received single or multiple intravenous infusions of an intercellular adhesion molecule-1 antisense phosphorothioate oligodeoxynucleotide, ISIS 2302, in a rising-dose (0.06-2.00 mg/kg infused over 2 hr), double-blind, placebo-controlled trial. Brief, dose-related increases in activated partial thromboplastin time were seen at the time of peak plasma concentration (C(max)). Clinically insignificant increases in C3a were seen after higher, repeated doses, but C5a, blood pressure and pulse were unaffected. No adverse events or other laboratory abnormalities were related to treatment with the drug. ISIS 2302 C(max) was linearly related to dose and occurred at the end of infusion. Plasma half-life for intact drug (53-54 min) and total oligonucleotide (67-74 min) were similar at the two doses (0.5 and 2.0 mg/kg) at which extensive pharmacokinetic data were collected. Nonlinear changes in area under the plasma concentration/time curve and steady-state volume of distribution with increasing dose suggested a saturable component to disposition. Metabolites co-migrating with n-1, n-2 and n-3 chain-shortened versions of ISIS 2302 appeared very rapidly in plasma, and disposition and metabolism appeared unaltered by repeated dosing. ISIS 2302 was well tolerated and behaved reproducibly with respect to plasma pharmacokinetics and expected side effects.

Inhibition of human immunodeficiency virus type 1 infection in SCID-hu Thy/Liv mice by the G-quartet-forming oligonucleotide, ISIS 5320.

Viral replication was inhibited in a dose-dependent manner after administration of the phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) to human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu Thy/Liv mice. Potent in vivo antiviral activity was observed against the T-cell-tropic molecular clone NL4-3; the agent was found to have weak activity against one primary HIV-1 isolate, and the agent was inactive against a second primary isolate.

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.