Adipose differentiation-related protein regulates lipids and insulin in pancreatic islets.

The excess accumulation of lipids in islets is thought to contribute to the development of diabetes in obesity by impairing beta-cell function. However, lipids also serve a nutrient function in islets, and fatty acids acutely increase insulin secretion. A better understanding of lipid metabolism in islets will shed light on complex effects of lipids on beta-cells. Adipose differentiation-related protein (ADFP) is localized on the surface of lipid droplets in a wide range of cells and plays an important role in intracellular lipid metabolism. We found that ADFP was highly expressed in murine beta-cells. Moreover, islet ADFP was increased in mice on a high-fat diet (3.5-fold of control) and after fasting (2.5-fold of control), revealing dynamic changes in ADFP in response to metabolic cues. ADFP expression was also increased by addition of fatty acids in human islets. The downregulation of ADFP in MIN6 cells by antisense oligonucleotide (ASO) suppressed the accumulation of triglycerides upon fatty acid loading (56% of control) along with a reduction in the mRNA levels of lipogenic genes such as diacylglycerol O-acyltransferase-2 and fatty acid synthase. Fatty acid uptake, oxidation, and lipolysis were also reduced by downregulation of ADFP. Moreover, the reduction of ADFP impaired the ability of palmitate to increase insulin secretion. These findings demonstrate that ADFP is important in regulation of lipid metabolism and insulin secretion in beta-cells.

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.

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.

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.

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.

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.

In vivo distribution and metabolism of a phosphorothioate oligonucleotide within rat liver after intravenous administration.

In the rat, the liver represents a major site of phosphorothioate oligodeoxynucleotide deposition after i.v. administration. For this reason, we examined the intracellular fate of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, isolated from parenchymal and nonparenchymal cell types after systemic dosing using established perfusion and separation techniques followed by CGE. Isolated cells were further fractionated into nuclear, cytosolic and membrane constituents to assess the intracellular localization, distribution and metabolic profiles as a function of time and dose. After a 10-mg/kg i.v. bolus, intracellular drug levels where maximal after 8 hr and diminished significantly thereafter, suggesting an active efflux mechanism or metabolism. Nonparenchymal (i.e., Kupffer and endothelial) cells contained approximately 80% of the total organ cellular dose, and this was equivalently distributed between the two cell types, while the remaining 20% was associated with hepatocytes. Nonparenchymal cells contained abundant nuclear, cytosolic and membrane drug levels over a wide dose range. In contrast, at doses of less than 25 mg/kg, hepatocytes contained significantly less drug with no detectable nuclear-association. Doses at or above 25 mg/kg appeared to saturate nonparenchymal cell types, whereas hepatocytes continued to accumulate drug in all cellular compartments, including the nucleus. Our results suggest that although pharmacokinetic parameters vary as a function of hepatic cell type, significant intracellular delivery can be readily achieved in the liver after systemic administration.

Structural requirements for cationic lipid mediated phosphorothioate oligonucleotides delivery to cells in culture.

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (Cl4:0) and dioleyl (C18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C14:0) was more effective than dipalmityl (C16:0) lipid, which was more effective than distearyl (C18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56 degrees C for the distearyl lipid, 42 degrees C for the dipalmityl lipid and 24 degrees C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.

Effect of antisense oligonucleotides on cytokine release from human keratinocytes in an in vitro model of skin.

ISIS 1082, a phosphorothioate oligonucleotide 21 nucleotides in length targeted to the translation initiation codon of herpes simplex virus (HSV) type 1 and 2 virion capsid protein, has been shown to inhibit HSV-1 replication in vitro. The effects of ISIS 1082, its phosphodiester congener, ISIS 1049, and analogs consisting of 2' methoxy and 2' propoxy phosphodiesters and phosphorothioates on IL-1 alpha release and viability were evaluated in a three-dimensional in vitro skin model consisting of neonatal keratinocytes and fibroblasts. This in vitro system displays many of the functional and metabolic properties of a differentiated epidermis and can be induced to specifically release IL-1 alpha in response to a mixture of lipopolysaccharide and phorbol myristate acetate. Incubation of the skin model with 250 to 1000 microM concentrations of ISIS 1082 and its 2' methoxy and propoxy phosphorothioate analogs resulted in a concentration-dependent increase of cytokine release with minimal effects on cellular viability, as measured by the Neutral Red assay. This response was confirmed in primary keratinocytes, which were also shown to secrete IL-1 alpha into media supernatants after incubation with phosphorothioate oligomers. These data suggest that the IL-1 alpha released from keratinocytes in response to ISIS 1082 may contribute to the inflammatory and immune cell response seen in vivo.

In vitro pharmacokinetics of phosphorothioate antisense oligonucleotides.

ISIS 2105 (Afovirsen), a 20-mer phosphorothioate oligonucleotide that inhibits the production of a gene product essential to the growth of human papillomavirus, is in phase II clinical trials for the treatment of genital warts induced by human papillomavirus-6 and human papillomavirus-11. The uptake, subcellular distribution and metabolism of ISIS 2105 and three other similar length phosphorothioates have been studied in a variety of cell lines. Our experiments indicated that ISIS 2105 and other phosphorothioates are internalized and distributed in a time-, temperature-, concentration-, sequence- and cell line-dependent manner. Cell association was also influenced by the tissue culture medium. Several different analytical techniques revealed that phosphorothioates were more rapidly degraded in vitro than previously reported. These data suggest that phosphorothioate oligonucleotide uptake and stability observed in tissue culture can vary as a function of cellular assay conditions and analytical methods used. Comparison of these results with those obtained in vivo suggests that the pharmacokinetic behavior of this class of compounds cannot necessarily be predicted from in vitro studies.

Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region.

Phosphorothioate oligonucleotides complementary to mRNA of the human cytomegalovirus (HCMV) DNA polymerase gene or to RNA transcripts of the major immediate-early regions 1 and 2 (IE1 and IE2) of HCMV were evaluated for antiviral activity in a 96-well immunoassay with primary human dermal fibroblasts as host cells. Oligonucleotides complementary to RNA of the IE2 region exhibited the most potent antiviral activity. One of these oligonucleotides, ISIS 2922, was at least 30-fold more potent than the nucleoside analog, ganciclovir, with a 50% effective concentration of 0.37 microM in the 96-well immunoassay. In an infectious virus yield reduction assay, ISIS 2922 and ganciclovir reduced production of infectious virus by 2 log units at concentrations of 2.2 and 36 microM, respectively. A control oligonucleotide showed no inhibition of virus production at concentrations as high as 3 microM. ISIS 2922 reduced IE protein synthesis in HCMV-infected cells in a dose-dependent manner which correlated with antiviral activity. The antiviral activity of ISIS 2922 was not due to oligonucleotide-induced cytotoxicity since effects on cell viability or proliferation were observed only at concentrations well in excess of effective antiviral concentrations. The specificity and potency of ISIS 2922 suggest that it may be useful for the treatment of cytomegalovirus disease in humans.

Tritium labeling of antisense oligonucleotides by exchange with tritiated water.

We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (< 1 x 10(8) cpm/mumol) by hydrogen exchange with tritiated water at the C8 positions of purines in the presence of beta-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37 degrees C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodiester oligonucleotides.

In vitro toxicological evaluation of ISIS 1082, a phosphorothioate oligonucleotide inhibitor of herpes simplex virus.

ISIS 1082, a phosphorothioate oligonucleotide targeted to a translation initiation codon of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) virion capsid protein UL13 inhibits in vitro viral replication. To better understand the pharmacological properties of ISIS 1082, we examined its effects in nonvirally infected HeLa cells by using a number of cytotoxicity assays. Our data indicate that ISIS 1082 had no effect on HeLa cell viability as measured by cellular proliferation and clonogenic assays at concentrations as high as 100 microM. Additionally, DNA, RNA, and protein synthesis were only inhibited by 25% in cells treated with 100 microM ISIS 1082. The effects of ISIS 1082 on DNA synthesis were compared with those of acyclovir and trifluorothymidine, two clinically used antiherpetic agents. Acyclovir displayed effects similar to that of ISIS 1082. However, trifluorothymidine, which has been reported to be a potential mutagen and teratogen, significantly altered DNA replication at concentrations from 1 to 100 microM. Isolated HeLa DNA polymerases were inhibited by the compound, with a 50% inhibitory concentration of 2 microM. The in vitro antiviral (K. Draper and V. Brown-Driver, submitted for publication; K.G. Draper and V. Brown-Driver, Antiviral Res. Suppl. 1:106, 1991) and cytotoxicity studies suggest that ISIS 1082 is a selective, nontoxic, antiherpetic therapeutic agent.

In vitro toxicology and pharmacokinetics of antisense oligonucleotides.

The ability to rationally design antisense drugs and the theoretical selectivity of these compounds for specific genomic and viral mRNA targets make their use as therapeutic agents extremely attractive. Pharmacologic data emerging from our and other laboratories indicate that some unmodified and backbone modified oligonucleotides may be cytotoxic. In general, cytotoxicity was usually seen at concentrations higher than those required for specific antisense effects. Factors that may modulate the toxicity of phosphorothioates include cell type, various media components including serum, length and method of preparation. Pharmacokinetic experiments using phosphodiester, phosphorothioate and methylphosphonate oligonucleotides suggest that these compounds may be taken up and distributed within some cells. Uptake was generally time, temperature, concentration dependent, and required cellular energy. The mechanism of uptake varied according to oligonucleotide type. The in vitro data as well as preliminary in vivo studies demonstrating the safety, antiviral activity and bioavailability of a number of oligonucleotides suggest that these compounds represent a novel therapeutic modality.

Metastatic hibernomas in transgenic mice expressing an alpha-amylase-SV40 T antigen hybrid gene.

Mice transgenic for a hybrid gene containing the liver promoter of the mouse amylase gene (Amy-1a) fused to the SV40 tumor antigen coding region unexpected developed malignant brown adipose tissue tumors (malignant hibernomas). Expression of the alpha-amylase gene had previously been thought to be confined to the liver parotid, and pancreas; however, analysis of white and brown adipose tissue from nontransgenic mice revealed expression of the endogenous Amy-1a gene in these tissues. Gene constructs driven by the Amy-1a liver promoter thus provide a means of targeting gene expression to the adipocyte cell lineage in transgenic mice. Moreover the high frequency of metastases in the liver, lungs, spleen, heart, and adrenals of these mice provides an experimental system in which to study the development of disseminated malignancy.