Metabolism of antisense oligonucleotides in rat liver homogenates

Phosphorothioate antisense oligodeoxynucleotides are novel therapeutic agents designed to selectively and specifically inhibit production of various disease-related gene products. In vivo pharmacokinetic experiments indicate that these molecules are widely distributed in many species, with the majority of oligomers accumulating within liver and kidney. To better understand the metabolism of these agents, we studied the stability of several phosphorothioate oligodeoxynucleotides, their congeners, and second generation oligomer chemistries in rat liver homogenates. To examine metabolism, background nuclease activity was characterized in whole liver homogenates by using ISIS 1049, a 21-mer phosphodiester oligodeoxynucleotide. Nuclease activity could readily be detected in liver homogenates. Under optimized conditions, the predominant enzymatic activity was 3'-exonucleolytic and could be influenced by pH and ionic conditions. However, in addition to 3' exonucleases, 5' exo- and endonuclease activities were also observed. Our data indicate that metabolism of phosphorothioate oligodeoxynucleotides was more complex than that of phosphodiesters for many reasons, including phosphorothioate oligodeoxynucleotide inhibition of nucleases and the presence of R(p) and S(p) stereoisomers. The rate of phosphorothioate metabolism also appeared to be influenced by sequence, with pyrimidine-rich compounds being metabolized to a greater extent than purine-rich oligomers. Other factors affecting stability included oligomer chemistry and length. Concomitant experiments performed in rats dosed systemically with the same compounds mimic the activities seen in vitro and suggest that this liver homogenate system is a valuable model with which to study the mechanism of metabolism of antisense oligonucleotides.     

Efficacy and safety of a low monthly dose of intravenous iron sucrose in peritoneal dialysis patients

Purpose

Scientific data regarding intravenous iron supplementation in peritoneal dialysis (PD) patients are scarce. In attempting to administer the minimum monthly IV iron dose that could improve erythropoiesis, we wanted to assess the safety and efficacy of monthly maintenance intravenous administration of 100 mg iron sucrose in PD patients.

Methods

In a 9-month prospective study, all clinically stable PD patients received intravenously 200 mg of iron sucrose as a loading dose, followed by monthly doses of 100 mg for five consecutive months. Levels of hemoglobin (Hb), ferritin, transferrin saturation (TSAT), reticulocyte hemoglobin content (CHr) and C-reactive protein (CRP) were measured before each administration and 3 months after the last iron infusion. Also, doses of concurrent erythropoietin administration were recorded.

Results

Eighteen patients were eligible for the study. Mean levels of Hb and ferritin increased significantly (from 10.0 to 10.9 mg/dL, p?=?0.01 and from 143 to 260 ng/mL, p?=?0.005), as well as the increase in TSAT levels approached borderline significance (from 26.2 to 33.1%, p?=?0.07). During the 6 months of iron administration, the erythropoietin dose was reduced in five patients and discontinued in one. During the 3 months following the last iron infusion, three of them again raised the erythropoietin dose to previous levels. None of the patients experienced any side effects related to IV iron administration.

Conclusions

A monthly maintenance intravenous dose of 100 mg iron sucrose may be a practical, effective, and safe in the short term, treatment of anemia in PD patients resulting in improved hemoglobin levels, iron indices, and erythropoietin response.

     

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.     

Cross-species comparison of in vivo PK/PD relationships for second-generation antisense oligonucleotides targeting apolipoprotein B-100

The in vivo pharmacokinetics/pharmacodynamics of 2′-O-(2-methoxyethyl) (2′-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2′-MOE modified apoB ASOs studied in mice (t_1/2 ≅ 20 days) and humans (t_1/2 ≅ 30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory E_max model, the ASO liver EC_50s were 101 ± 32, 119 ± 15, and 300 ± 191 μg/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC₅₀ in man, extrapolated from trough plasma exposure, was 81 ± 122 μg/g. Therefore, extraordinary consistency of the exposure-response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver.     

Circulating human B lymphocytes are deficient in nucleotide excision repair and accumulate mutations upon proliferation

Faithful repair of DNA lesions is a crucial task that dividing cells must actively perform to maintain genome integrity. Strikingly, nucleotide excision repair (NER), the most versatile DNA repair system, is specifically down-regulated in terminally differentiated cells. This prompted us to examine whether NER attenuation might be a common feature of all G?-arrested cells, and in particular of those that retain the capacity to reenter cell cycle and might thus convert unrepaired DNA lesions into mutations, a prerequisite for malignant transformation. Here we report that quiescent primary human B lymphocytes down-regulate NER at the global genome level while maintaining proficient repair of constitutively expressed genes. Quiescent B cells exposed to an environment that causes both DNA damage and proliferation accumulate point mutations in silent and inducible genes crucial for cell replication and differentiation, such as BCL6 and Cyclin D2. Similar to differentiated cells, NER attenuation in quiescent cells is associated with incomplete phosphorylation of the ubiquitin activating enzyme Ube1, which is required for proficient NER. Our data establish a mechanistic link between NER attenuation during quiescence and cell mutagenesis and also support the concept that oncogenic events targeting cell cycle- or activation-induced genes might initiate genomic instability and lymphomagenesis.     

Selective killing of cancer cells based on loss of heterozygosity and normal variation in the human genome: a new paradigm for anticancer drug therapy.

Most drugs for cancer therapy are targeted to relative differences in the biological characteristics of cancer cells and normal cells. The therapeutic index of such drugs is theoretically limited by the magnitude of such differences, and most anticancer drugs have considerable toxicity to normal cells. Here we describe a new approach for developing anticancer drugs. This approach, termed variagenic targeting, exploits the absolute difference in the genotype of normal cells and cancer cells arising from normal gene sequence variation in essential genes and loss of heterozygosity (LOH) occurring during oncogenesis. The technology involves identifying genes that are: 1) essential for cell survival; 2) are expressed as multiple alleles in the normal population because of the presence of one or more nucleotide polymorphisms; and 3) are frequently subject to LOH in several common cancers. An allele-specific drug inhibiting the essential gene remaining in cancer cells would be lethal to the malignant cell and would have minimal toxicity to the normal heterozygous cell that retains the drug-insensitive allele. With antisense oligonucleotides designed to target two alternative alleles of replication protein A, 70-kDa subunit (RPA70) we demonstrate in vitro selective killing of cancer cells that contain only the sensitive allele of the target gene without killing cells expressing the alternative RPA70 allele. Additionally, we identify several other candidate genes for variagenic targeting. This technology represents a new approach for the discovery of agents with high therapeutics indices for treating cancer and other proliferative disorders.     

Phosphorothioate oligodeoxynucleotides distribute similarly in class A scavenger receptor knockout and wild-type mice

It has been suggested that binding of phosphorothioate oligodeoxynucleotides (P=S ODNs) to macrophage scavenger receptors (SR-AI/II) is the primary mechanism of P=S ODN uptake into cells in vivo. To address the role of scavenger receptors in P=S ODN distribution in vivo, several pharmacokinetic and pharmacological parameters were compared in tissues from scavenger receptor knockout mice (SR-A-/-) and their wild-type counterparts after i.v. administration of 5- and 20-mg/kg doses of P=S ODN. With an antibody that recognizes P=S ODN, no differences in cellular distribution or staining intensity in livers, kidneys, lungs, or spleens taken from SR-A-/- versus wild-type mice could be detected at the histological level. There were no significant differences in P=S ODN concentrations in these organs as measured by capillary gel electrophoresis as well, although the concentration of P=S ODN in isolated Kupffer cells from livers of SR-A-/- mice was 25% lower than that in Kupffer cells from wild-type mice. Furthermore, a P=S ODN targeting murine A-raf reduced A-raf RNA levels to a similar extent in livers from SRA-/- (92.8%) and wild-type (88.3%) mice. Finally, in vitro P=S ODN uptake studies in peritoneal macrophages from SR-A-/- versus wild-type mice indicate that other high- and low-affinity uptake mechanisms predominate. Taken as a whole, our data suggest that, although there may be some contribution to P=S ODN uptake by the SR-AI/II receptor, this mechanism alone cannot account for the bulk of P=S ODN distribution into tissues and cells in vivo, including macrophages.     

In vitro genetically aberrant T-cell clones with continuous growth are associated with atopic dermatitis

Atopic dermatitis is a disease with a genetic predisposition affecting the immune system, with T lymphocytes participating in the immune dysregulation. Most in vitro T lymphocyte studies of atopic dermatitis have focused on antigen-specific T-cell clones. However, antigen-non-specific regulatory T lymphocytes may also take part in the pathway leading to antigen-specific clonal T-lymphocyte proliferation. T lymphocytes from skin biopsy specimens from three patients with severe atopic dermatitis were cultured in the presence of IL-2 and IL-4, but without antigen added. Initially, proliferation was oligo- or polyclonal, but in all cases overgrowth by T cells with clonal chromosomal aberrations was subsequently observed. These abnormal T-cell clones demonstrated continuous growth and complete or partial phenotypic loss of the T-cell antigen receptor complex. In summary, these findings suggest that a subset of aberrant skin-homing T lymphocytes is associated with atopic dermatitis.     

Hepatic distribution of a phosphorothioate oligodeoxynucleotide within rodents following intravenous administration.

The pharmacokinetics of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, were characterized within rodent whole liver, and cellular and subcellular compartments. Cross-species comparisons were performed using Sprague-Dawley rat and CD-1 mouse strains. Although whole liver oligonucleotide deposition and the proportion of drug found within parenchymal and nonparenchymal cells were similar between the two rodent species as a function of both time and dose, dramatic differences in subcellular pharmacokinetics were observed. Specifically, within murine hepatocyte nuclei, drug was observed at the 10 mg/kg dose, whereas in the rat nuclear-associated levels required the administration of 25 mg/kg. Under all experimental regimens, murine hepatic nuclear-associated drug concentrations were at least 2-fold higher than those found in rat liver cells. More detailed metabolic analysis was also performed using high performance liquid chromatography/electrospray-mass spectrometry (HPLC/ES-MS) and demonstrated that although the extent of metabolism was similar for rat and mouse, the pattern of n-1 metabolites varied as a function of both species and cell type. While rat and mouse hepatocytes and rat nonparenchymal cellular metabolites were predominantly products of 3'-exonuclease degradation, mouse nonparenchymal cells contained a majority of n-1 metabolites produced by 5'-exonucleolytic activity. Based upon these data, it would appear that subcellular oligonucleotide disposition and metabolism among rodent species are more divergent than whole organ pharmacokinetics might predict.