Tissue distribution and physiologically based pharmacokinetics of antisense phosphorothioate oligonucleotide ISIS 1082 in rat

The aim of this study was to develop a whole body physiologically based model of the pharmacokinetics (PBPK) of the phosphorothioate oligonucleotide (PS-ODN) ISIS 1082 in vivo. Rats were administered an intravenous (i.v.) bolus dose of ISIS 1082 (10 mg/kg plus 3H tracer), and arterial blood and tissues were taken at specific times up to 72 hours. Radioactivity was measured in all samples. The parent compound was determined specifically in blood and tissues at 90 minutes and in liver and kidney also at 24 hours, using capillary gel electrophoresis (CGE). A whole body PBPK model was fitted to the combined blood and tissue radioactivity data using nonlinear regression analysis. CGE analysis indicated that the predominant species in plasma and all tissues is ISIS 1082, together with some n-1 and n-2 metabolites. Total radioactivity primarily reflects these species. The whole body model successfully described temporal events in all tissues. However, to adequately model the experimental data, all tissues had to be partitioned into vascular and extravascular spaces to accommodate the relatively slow distribution of ISIS 1082 out of blood because of a permeability rate limitation. ISIS 1082 distributes extensively into tissues, but the relative affinity varies enormously, being highest for kidney and liver and lowest for muscle and brain. A whole body PBPK model with a permeability rate limited tissue distribution was developed that adequately described events in both blood and tissue for an oligonucleotide. This model has the potential not only to characterize the events in individual tissues throughout the body for such compounds but also to scale across animal species, including human.     

Metabolism of sodium oestrone [35S]sulphate in the guinea pig

Intraperitoneal administration of sodium oestrone [(35)S]sulphate to male and female free-ranging guinea pigs is followed by excretion of most of the radioactivity mainly as inorganic [(35)S]sulphate in the urine within 72h. The remainder of the radioactivity in the urine was found in oestrone [(35)S]sulphate, two unidentified metabolites (A and B) and traces of oestradiol-17beta 3-[(35)S]sulphate. When injected intraperitoneally into animals with bile-duct and bladder cannulae, most of the dose was excreted in the bile. Unchanged oestrone [(35)S]sulphate was the main biliary component excreted in males and females, but the latter also excreted appreciable amounts of oestradiol-17beta 3-[(35)S]sulphate and metabolites A and B. The urine from these animals also contained these metabolites, inorganic [(35)S]sulphate and also oestrone [(35)S]sulphate, but in small amounts. Metabolite A was present only in samples from males. Whole body radioautography pinpointed the liver and kidney as the possible sites of metabolism of the ester. The ester underwent little desulphation in the isolated perfused female guinea-pig liver and in animals in which kidney function had been eliminated, and was excreted unchanged in the bile. These results and the observed low oestrogen sulphatase and arylsulphatase C activities found in guinea-pig liver and kidney support the view that the two enzymes are identical.     

Distribution and excretion of a phosphorothioate oligonucleotide in rats with experimentally induced renal injury

The effects of renal injury on the urinary excretion and tissue distribution of a 20-mer phosphorothioate oligonucleotide were investigated in male Sprague-Dawley rats. Renal injury was produced by treating the rats with either 5.0 mg/kg cisplatin or 2.5 mg/kg of a monoclonal antibody (mAb) directed toward Thy1.1. Controls received saline. Three days after cisplatin treatment or 2 days after anti- Thy1.1 treatment, the rats received 10 mg/kg ISIS 3521. Blood was collected at various times to assess the plasma concentrations of ISIS 3521, and rats were killed at various times from 6 to 48 hours after intravenous (i.v.) infusion of oligonucleotide to assess tissue concentrations by capillary gel electrophoresis (CGE). Cisplatin and anti-Thy1.1 antibody produced histologic and biochemical changes consistent with proximal tubular damage and glomerular damage, respectively. Urinary excretion of oligonucleotides was increased 2- to 4-fold of control; however, this amount accounted for only 1% to 2% of dose compared to 0.5% in controls. Proximal tubular damage reduced renal accumulations of ISIS 3521 and other oligonucleotide metabolites, but there were no obvious compensatory increases in concentrations in other organs except for a slight increase in spleen levels of total oligonucleotide. Glomerular damage was not associated with any change in oligonucleotide disposition. Immunohistochemical studies showed no evidence of alterations in the pattern of distribution within the injured kidney. The data suggest that acute renal dysfunction, either renal tubular or glomerular, does not markedly alter the urinary elimination and tissue deposition of a phosphorothioate oligonucleotide.     

Tissue distribution of a menthyl-conjugated oligodeoxyribonucleotide antisense to PAI-1 mRNA

The inhibitory effect of numerous analogues of PO-16, an hexadecadeoxyribonucleotide antisense to sequences -22 to -17 of PAI-1 mRNA coding for a fragment of the signal peptide, on the expression of PAI-1 in endothelial cells, and physiological consequences of the subsequently reduced PAI-1 activity tested in vitro and in vivo, were described in our previous studies. Of particular interest was PO-16 5'-O-conjugated with menthyl phosphorothioate (MPO-16R). In this work, tissue localisation of MPO-16R labelled with [(35)S] phosphorothioate at the 3'-end, was determined. [(35)S]MPO-16R and control [(35)S]MPO-16R-SENSE oligonucleotides were administered intravenously into 22 rats and organ distribution of the labelled bioconjugates was assessed after 24 and 48 h. For this purpose, tissue sections were subjected to autoradiography, and quantitated by liquid scintillation after solubilisation. Overall clearance of radioactivity was already seen after 24 h, with the radioactivity recovered mainly in the kidney and liver. A smaller fraction of radioactivity was also retained in the spleen and heart. The kidney concentration of the labelled probe was higher than that of liver by 50%. The distribution of PAI-1 mRNA in untreated rat kidney, liver, spleen and heart established by two independent techniques: Ribonuclease Protection Assay and Real-Time PCR, shows the same pattern as that observed for [(35)S]MPO-16R antisense.     

Metabolism of sodium oestrone [35S]sulphate in the rat

Intraperitoneal, intravenous or oral administration of sodium oestrone [(35)S]-sulphate to male and female Medical Research Council hooded rats is followed by the rapid excretion of the bulk of the radioactivity in urine in the form of inorganic [(35)S]sulphate. Pre-treatment of rats with an antibiotic regimen does not affect the results except in the case of oral administration, when relatively large amounts of the dose are recovered as ester [(35)S]sulphate in faeces. Intravenous administration of the labelled ester to male and female rats with cannulae in bile duct and ureter gave results similar to those obtained with free-range animals. Only small amounts of radioactivity appeared in bile and this was mainly in the form of ester sulphate, including both oestrone [(35)S]sulphate and oestradiol-17beta 3[(35)S]-sulphate. Whole-body radioautography pinpointed the liver as the probable site of the desulphation of the sulphate ester and this was confirmed by liver and kidney perfusion experiments and by studies with rats in which kidney function had been eliminated by ligation of the renal pedicles.     

The intracellular ratio of cysteine and cystine in various tissues

1. The cysteine-cystine ratio was measured in rat kidney cortex, diaphragm, jejunum, liver and brain. 2. This ratio was determined by incubating these tissues in buffer containing [(35)S]cystine and then homogenizing the tissue in a buffered solution of N-ethylmaleimide. The products of this reaction were separated by high-voltage electrophoresis and the radioactivity in the cystine and 2-(l-2'-amino-2'-carboxyethylthio)-N-ethylsuccinimide regions was determined. 3. In these tissues cyst(e)ine was mainly present in the reduced form. 4. After incubation of [(35)S]cystine with rat jejunal segments it was found that 36% of the cystine in the medium has been reduced. 5. Anaerobiosis, Na(+)-free media, glucose and high concentrations of cystine and lysine were found not to affect significantly the cysteine-cystine ratio in rat kidney-cortex slices.     

Metabolism of 2,4',5-trichlorobiphenyl: role of the intestinal microflora in the formation of bronchial-seeking methyl sulphone metabolites in mice

Groups of germ-free and conventional mice were treated with 2,4',5-trichlorobiphenyl (triCB) and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. Conventional mice accumulated significantly more extractable radioactivity both in lung and kidney tissues when compared to germ-free mice. The extracted radioactivity in lung and kidney tissues was shown to be due to the accumulation of methyl-[35S]sulphonyl-triCB. The low radioactivity in lungs of the germ-free mice was also shown to be due to the accumulation of small amounts of the sulphones. The results indicate an involvement of the intestinal flora in the formation of methyl sulphone metabolites of triCB.     

Distribution and breakdown of labeled coenzyme Q10 in rat

Radioactive coenzyme Q(10) ([(3)H]CoQ) was synthesized in a way that the metabolites produced retained the radioactivity. Administration of the lipid to rats intraperitoneally resulted in an efficient uptake into the circulation, with high concentrations found in spleen, liver, and white blood cells; lower concentrations in adrenals, ovaries, thymus, and heart; and practically no uptake in kidney, muscle, and brain. In liver homogenate most [(3)H]CoQ appeared in the organelles, but it was also present in the cytosol and transport vesicles. Mitochondria, purified on a metrizamide gradient, had a very low concentration of [(3)H]CoQ, which was mainly present in the lysosomes. All organs that took up the labeled lipid also contained water-soluble metabolites. The majority of metabolites excreted through the kidney and appeared in the urine. Some metabolites were also present in the feces, which further contained nonmetabolized [(3)H]CoQ, excreted through the bile. The major metabolites were purified from the urine, and the mass spectrometric fragmentation showed that these compounds, containing the ring with a short side chain, are phosphorylated. Thus, the results demonstrate that CoQ is metabolized in all tissues, the metabolites are phosphorylated in the cells, transported in the blood to the kidney, and excreted into the urine.     

The liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys

Infection with human or simian immunodeficiency virus (SIV) is characterized by the rapid turnover of both viral particles and productively infected cells. It has recently been reported that the clearance of SIV in vivo is exceedingly fast, with half-lives on the order of minutes. The underlying mechanism or site responsible for this rapid clearance, however, remains unknown. To investigate this issue, we chose to infuse infectious SIVmac239 grown from autologous peripheral blood mononuclear cells that were radioactively labeled by [(35)S]methionine and [(35)S]cysteine. This approach eliminates from the viral membrane alloantigens that may have a significant impact on viral clearance. In addition, this approach also permits identification of the sites of viral clearance by measuring the radioactive intensity, even if degradation of SIV RNA occurs in tissues. We now report that the half-life of infused SIV in blood is extremely close to estimates from a previous study, in which unlabeled SIV grown in a heterologous cell line was used. The allogeneic effect due to the presence of human antigens on the surfaces of virions may, therefore, play a minimal role in the high rate of virion clearance. Moreover, close to 30% of infused radioactivity was found in the liver and measureable amounts were detected in the lungs (5.4%), lymph nodes (3.0%), and spleen (0.4%). The detection of a significant proportion of infused virus in the liver suggests that viral clearance from circulation is mediated by a common, nonspecific mechanism, such as the phagocytic functions of the reticuloendothelial system. The rapid clearance and degradation of exogenously infused virions may pose a major obstacle for gene therapy with viral vectors, unless strategies to overcome the rapid in vivo elimination of these particles are developed.     

A method for the determination of betaine in tissues using high performance liquid chromatography

Betaine is a major metabolite of choline in liver and kidney and may be an important product of choline metabolism in other tissues. The available methods for assay of betaine, however, are time consuming and relatively insensitive. We describe a modification of published methods that provides a sensitive and specific assay for betaine by derivatization and HPLC separation with UV quantitation. Betaine and other water-soluble choline metabolites are extracted from biological samples and separated by HPLC based on mobility of 14C-labeled internal standards. The betaine fraction is collected and derivatized with 4'-bromo-phenacyl triflate. The betaine-triflate derivative is quantitated by UV absorbance and the result is corrected for possible losses due to incomplete extraction recovery and incomplete derivatization by simultaneous measurement of radioactivity from the derivatized 14C-betaine internal standard. Betaine concentrations determined with this procedure are reported for several adult and fetal rat tissues.     

Studies on distribution and metabolism of valproate in rat brain,liver, and kidney

Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [¹⁴C]VPA and [³H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the¹⁴C/³H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and synaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg²⁺-ATPase and (Na⁺+K⁺)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action on the Na⁺ pump.Contribution No. 0601 from the Department of Cell and Molecular Biology, the Medical College of Georgia, Augusta, Georgia 30912.     

Taurine transport rates between plasma and tissues in adult and 7-day-old mice.

Transport rates for taurine from plasma to liver, kidney, heart, spleen and femoral muscle were evaluated in adult and 7-day-old mice in vivo. The mice were injected with [35S]taurine and the specific radioactivity of taurine was determined in the above tissues at varying intervals from 10 min up to 48 hr after the injection. A multicompartment model was fitted to the data and the transport rates with their confidence limits were estimated using a digital computer. The tissue-plasma exchange rate was generally faster in adult mice than in 7-day-old mice. The transport rates between the plasma and the brain or muscle were low, while taurine penetrated into the liver and kidneys very rapidly. There was no distinct correlation between the calculated transport rates and the tissue taurine concentrations. The metabolic breakdown of taurine in the tissues was slow, since only negligible amounts of radioactivity were recovered in the metabolites of taurine, isethionic acid and inorganic sulphate. It seems unlikely that either the magnitudes of the transport rates between the plasma and the tissues or taurine breakdown rates in situ act as the primary factor determining the taurine levels in tissues.     

Tissue kallikrein synthesis and its modification by testosterone or low dietary sodium.

A method has been developed to measure the relative rate of rat tissue kallikrein synthesis which employs a specific antiserum raised against a purified rat urinary kallikrein. Incorporation of [35S]methionine into kallikrein and protein 20 min after intraperitoneal injection was measured in submaxillary gland, pancreas, kidney and descending colon. Kallikrein content was measured with a direct radioimmunoassay, and kallikrein-specific incorporation of [35S]methionine measured after immunoprecipitation. Kallikrein specific radioactivity (c.p.m./mg of enzyme) was about 100-fold greater than that in total protein in both kidney and colon. In contrast, in pancreas the incorporation into the enzyme was only 5-fold higher than into protein, and in submaxillary gland the incorporation was equivalent. Measured as kallikrein-specific radioactivity relative to total protein radioactivity incorporated in 20 min, kallikrein represents 0.18% of total protein synthesis in the kidney, 0.34% in the pancreas, 0.41% in the colon, but 7.29% in the submaxillary gland. Dietary Na+ restriction increased the relative rate of kallikrein synthesis 1.8-fold in the kidney without a comparable effect in submaxillary gland. In contrast, testosterone increased the relative rate of synthesis 2.3-fold in submaxillary gland, but decreased it in kidney. The data show that endogenous kallikrein synthesis differs markedly in various tissues, and that interventions which are known to change kallikrein content or excretion also change the relative rate of enzyme synthesis.     

In vitro metabolic stabilities and metabolism of 2'-O-(methoxyethyl) partially modified phosphorothioate antisense oligonucleotides in preincubated rat or human whole liver homogenates

In vitro metabolic stability testing of phosphorothioate 2'-O-methoxyethyl (2'-MOE) partially modified antisense oligonucleotides (ASOs) is not routinely performed to help screen discovery compounds (eg, predict in vivo half-lives), as no suitable in vitro test system currently exists. The aims of this work were to develop, optimize, and evaluate an in vitro whole liver homogenate (rat or human) test system. The test system was used to evaluate in vitro metabolic stabilities (intrinsic clearance) of selected ASOs, with results compared to reported in vivo half-lives, and generated metabolites also identified. Test system optimization involved preincubating whole liver homogenates at 37°C for ≥24 hours, which increased in vitro ASO metabolism rate. From calculated in vitro intrinsic clearance (CL(int)) values in preincubated rat or human whole liver homogenates, metabolic stabilities of fully phosphorothioated 2'-MOE ASOs (ISIS 104838 and ISIS 301012) were, as expected, greater (ie, lower CL(int)) than a 2'-MOE ASO containing a single phosphodiester substitution (ISIS 104838PO10). However, comparable-to-lower in vitro metabolic stability for ISIS 301012 was seen compared to ISIS 104838, in contrast to reported ～2-fold longer in vivo tissue elimination half-lives for ISIS 301012. Identified in vitro metabolic products of ISIS 301012 were consistent with previously reported in vivo observations.     

The metabolism of potassium dodecyl [35S]-sulphate in the rat

The metabolic fate of potassium dodecyl [(35)S]sulphate was studied in rats. Intraperitoneal and oral administration of the ester into free-ranging animals were followed by the excretion of the bulk of the radioactivity in the urine within 12hr., approximately 17% being eliminated as inorganic [(35)S]sulphate. Similar results were obtained in experiments in which potassium dodecyl [(35)S]sulphate was injected intravenously into anaesthetized rats with bile-duct and ureter cannulae. Analysis of urinary radioactivity revealed the presence of a new ester sulphate (metabolite A). This metabolite was isolated, purified and subsequently identified as the sulphate ester of 4-hydroxybutyric acid by paper, thin-layer and gas chromatography, by paper electrophoresis and by comparison of its properties with those of authentic butyric acid 4-sulphate. The identity of the metabolite was confirmed by isotope-dilution experiments. When either purified metabolite A or authentic potassium butyric acid 4[(35)S]-sulphate was administered to free-ranging rats the bulk of the radioactivity was eliminated unchanged in the urine within 12hr., approx. 20% of the dose appearing as inorganic [(35)S]sulphate. Whole-body radioautography and isolated-liver-perfusion experiments implicated the liver as the major site of metabolism of potassium dodecyl [(35)S]sulphate. It is suggested that butyric acid 4-sulphate probably arises by omega-oxidation of dodecyl sulphate to a fatty acid-like compound, which is then degraded by beta-oxidation.     

Synthesis of 11C-labelled (R)-OHDMI and CFMME and their evaluation as candidate radioligands for imaging central norepinephrine transporters with PET

(R)-1-(10,11-Dihydro-dibenzo[b,f]azepin-5-yl)-3-methylamino-propan-2-ol ((R)-OHDMI) and (S,S)-1-cyclopentyl-2-(5-fluoro-2-methoxy-phenyl)-1-morpholin-2-yl-ethanol (CFMME) were synthesized and found to be potent inhibitors of norepinephrine reuptake. Each was labelled efficiently in its methyl group with carbon-11 (t(1/2)=20.4 min) as a prospective radioligand for imaging brain norepinephrine transporters (NET) with positron emission tomography (PET). The uptake and distribution of radioactivity in brain following intravenous injection of each radioligand into cynomolgus monkey was examined in vivo with PET. After injection of (R)-[(11)C]OHDMI, the maximal whole brain uptake of radioactivity was very low (1.1% of injected dose; I.D.). For occipital cortex, thalamus, lower brainstem, mesencephalon and cerebellum, radioactivity ratios to striatum at 93 min after radioligand injection were 1.35, 1.35, 1.2, 1.2 and 1.0, respectively. After injection of [(11)C]CFMME, radioactivity readily entered brain (3.5% I.D.). Ratios of radioactivity to cerebellum at 93 min for thalamus, occipital cortex, region of locus coeruleus, mesencephalon and striatum were 1.35, 1.3, 1.3, 1.2 and 1.2, respectively. Radioactive metabolites in plasma were measured by radio-HPLC. (R)-[(11)C]OHDMI represented 75% of plasma radioactivity at 4 min after injection and 6% at 30 min. After injection of [(11)C]CFMME, 84% of the radioactivity in plasma represented parent at 4 min and 20% at 30 min. Since the two new hydroxylated radioligands provide only modest regional differentiation in brain uptake and form potentially troublesome lipophilic radioactive metabolites, they are concluded to be inferior to existing radioligands, such as (S,S)-[(11)C]MeNER, (S,S)-[(18)F]FMeNER-D(2) and (S,S)-[(18)F]FRB-D(4), for the study of brain NETs with PET in vivo.     

Antisense knockdown of PKC-alpha using LNA-oligos

Full-length and 4 nucleotides truncated Locked Nucleic Acid (LNA) modifications of ISIS 3521 were compared for antisense properties in a cellular assay. ISIS 3521 is a 20-mer phosphorothioate designed to hybridise to human protein kinase C-alpha (PKC-alpha) mRNA and is currently submitted to clinical trials against cancer. We report that LNA can potentate this antisense oligo and retain the antisense potential with shorter oligos.     

Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts

Epoxyeicosatrienoic acids (EETs), the eicosanoid biomediators synthesized from arachidonic acid by cytochrome P450 epoxygenases, are inactivated in many tissues by conversion to dihydroxyeicosatrienoic acids (DHETs). However, we find that human skin fibroblasts convert EETs mostly to chain-shortened epoxy-fatty acids and produce only small amounts of DHETs. Comparative studies with [5,6,8,9,11,12,14,15-(3)H]11,12-EET ([(3)H]11,12-EET) and [1-(14)C]11,12-EET demonstrated that chain-shortened metabolites are formed by removal of carbons from the carboxyl end of the EET. These metabolites accumulated primarily in the medium, but small amounts also were incorporated into the cell lipids. The most abundant 11, 12-EET product was 7,8-epoxyhexadecadienoic acid (7,8-epoxy-16:2), and two of the others that were identified are 9, 10-epoxyoctadecadienoic acid (9,10-epoxy-18:2) and 5, 6-epoxytetradecaenoic acid (5,6-epoxy-14:1). The main epoxy-fatty acid produced from 14,15-EET was 10,11-epoxyhexadecadienoic acid (10, 11-epoxy-16:2). [(3)H]8,9-EET was converted to a single metabolite with the chromatographic properties of a 16-carbon epoxy-fatty acid, but we were not able to identify this compound. Large amounts of the chain-shortened 11,12-EET metabolites were produced by long-chain acyl CoA dehydrogenase-deficient fibroblasts but not by Zellweger syndrome and acyl CoA oxidase-deficient fibroblasts. We conclude that the chain-shortened epoxy-fatty acids are produced primarily by peroxisomal beta-oxidation. This may serve as an alternate mechanism for EET inactivation and removal from the tissues. However, it is possible that the epoxy-fatty acid products may have metabolic or functional effects and that the purpose of the beta-oxidation pathway is to generate these products.     

A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain

Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomography. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [(14)C]7m6CP was prepared by the reaction of 6-chloropurine with [(14)C]CH(3)I as a model of [(11)C]CH(3)I. The radiochemical yield and purity of [(14)C]7m6CP were 10-20% and greater than 99%, respectively. High brain uptake (0.8% ID/g) at 1 min was observed, followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [(14)C]7m6CP into rats. Analysis of metabolites confirmed that the presence of [(14)C]7m6CP was hardly observed, and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [(14)C]7m6CP, and the first-order efflux rate constant of the conjugate, estimated from the slope, was 0.0253 min(-1). These results showed that (1) [(14)C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [(14)C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.     

Maternal and fetal distribution of a phosphorothioate oligonucleotide in rats after intravenous infusion

BACKGROUND: Fetal uptake of an antisense oligonucleotide was evaluated after intravenous (i.v.) dosing of ISIS 2105, a 20-base phosphorothioate oligonucleotide, in timed-pregnant Sprague-Dawley rats. METHODS: To maximize the potential for fetal exposure, ISIS 2105 was administered as a 3-hr infusion at 6.6 mg/kg/hr with a total dose of 20 mg/kg, or as a continuous 7-day infusion at 0.35 mg/kg/hr with a total dose of 59 mg/kg. This dosing regime is higher than a patient would be expected to receive in the clinical use of oligonucleotides. Infusions were delivered through a jugular vein cannula by syringe pump on gestation day (GD) 19 (3-hr exposure) or by osmotic pumps implanted subcutaneously (s.c.) starting on GD 12 (7-day exposures). RESULTS: After a 3-hr infusion, maternal and fetal plasma concentrations of ISIS 2105 were >100 microg/ml and <0.07 microg/ml, respectively with a maternal fetal ratio of >1,000. Maternal regions of the placenta had twice the oligonucleotide concentration compared to fetal regions of the placenta (6 microg/g vs. 3 microg/g). After this acute exposure the concentrations in fetal kidney and liver were approximately 140- and 500-fold less than the maternal kidney and liver respectively. After 7-day infusion maternal plasma concentrations were 0.82 microg/ml and fetal concentrations were <0.22 microg/ml. By capillary gel electrophoresis (CGE) only the fetal liver consistently had quantifiable oligonucleotide concentrations (range=1.01-4.95 microg/g) compared to a mean concentration of 50.11+/-1.71 microg/g in the maternal liver a maternal to fetal ratio of approximately 10:50 after 7 days of infusion. CONCLUSIONS: There was a low level of transfer from dam to fetus, consistent with a slow equilibrium but the permeability of placenta to this 6 kDa polyanionic compound seemed to be limited even at supraclinical doses.