ADME studies of [5-(3)H]-2'-O-methyluridine nucleoside in mice: a building block in siRNA therapeutics.

The chemical modification 2'-O-methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)-properties of tritium-labeled 2'-O-methyluridine, following a single intravenous dose to male CD-1 mice. The single intravenous administration of [5-(3)H]-2'-O-methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [(3)H]-2'-O-Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [(3)H]-AUC 0-48 h). Three [(3)H]-2'-O-methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine.

Biodistribution and metabolism studies of lipid nanoparticle-formulated internally [3H]-labeled siRNA in mice.

Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

Metabolism studies of unformulated internally [3H]-labeled short interfering RNAs in mice.

Absorption, distribution, metabolism, and excretion properties of two unformulated model short interfering RNA (siRNAs) were determined using a single internal [(3)H]-radiolabeling procedure, in which the full-length oligonucleotides were radiolabeled by Br/(3)H -exchange. Tissue distribution, excretion, and mass balance of radioactivity were investigated in male CD-1 mice after a single intravenous administration of the [(3)H]siRNAs, at a target dose level of 5 mg/kg. Quantitative whole-body autoradiography and liquid scintillation counting techniques were used to determine tissue distribution. Radiochromatogram profiles were determined in plasma, tissue extracts, and urine. Metabolites were separated by liquid chromatography and identified by radiodetection and high-resolution accurate mass spectrometry. In general, there was little difference in the distribution of total radiolabeled components after administration of the two unformulated [(3)H]siRNAs. The radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated at later time points (24 and 48 hours for [(3)H]MRP4 (multidrug resistance protein isoform 4) and [(3)H]SSB (Sjögren Syndrome antigen B) siRNA, respectively). After an initial rapid decrease, concentrations of total radiolabeled components in dried blood decreased at a much slower rate. A nearly complete mass balance was obtained for the [(3)H]SSB siRNA, and renal excretion was the main route of elimination (38%). The metabolism of the two model siRNAs was rapid and extensive. Five minutes after administration, no parent compound could be detected in plasma. Instead, radiolabeled nucleosides resulting from nuclease hydrolysis were observed. In the metabolism profiles obtained from various tissues, only radiolabeled nucleosides were found, suggesting that siRNAs are rapidly metabolized and that the distribution pattern of total radiolabeled components can be ascribed to small molecular weight metabolites.

Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies.

Precision-cut tissue slices (PCTS) are viable ex vivo explants of tissue with a reproducible, well defined thickness. They represent a mini-model of the organ under study and contain all cells of the tissue in their natural environment, leaving intercellular and cell-matrix interactions intact, and are therefore highly appropriate for studying multicellular processes. PCTS are mainly used to study the metabolism and toxicity of xenobiotics, but they are suitable for many other purposes. Here we describe the protocols to prepare and incubate rat and human liver and intestinal slices. Slices are prepared from fresh liver by making a cylindrical core using a drill with a hollow bit, from which slices are cut with a specially designed tissue slicer. Intestinal tissue is embedded in cylinders of agarose before slicing. Slices remain viable for 24 h (intestine) and up to 96 h (liver) when incubated in 6- or 12-well plates under 95% O(2)/5% CO(2) atmosphere.

Induction of metabolism and transport in human intestine: validation of precision-cut slices as a tool to study induction of drug metabolism in human intestine in vitro.

Induction of drug enzyme activity in the intestine can strongly determine plasma levels of drugs. It is therefore important to predict drug-drug interactions in human intestine in vitro. We evaluated the applicability of human intestinal precision-cut slices for induction studies in vitro. Morphological examination and intracellular ATP levels indicated tissue integrity up to 24 h of incubation, whereas in proximal jejunum slices, the metabolic rate toward most substrates remained at 40 to 50% of initial values. In colon slices, the cytochrome P450 conversions were below the detection limit, but conjugation rates remained relatively constant during incubation. The inducibility of drug-metabolizing enzymes and P-glycoprotein was evaluated using prototypical inducers for five induction pathways. beta-Naphthoflavone (aryl hydrocarbon receptor ligand) induced CYP1A1 (132-fold in colon and 362-fold in proximal jejunum) and UDP glucuronosyltransferase (UGT) 1A6 mRNA (9.8-fold in colon and 3.2-fold in proximal jejunum). In proximal jejunum, rifampicin (RIF) [pregnane X receptor (PXR) ligand] induced CYP3A4 (5.2-fold), CYP2B6 (2-fold), UGT1A6 (2.2-fold), and multidrug resistance-1 (MDR1)/ABCB1 mRNA (2.7-fold), whereas 6beta-hydroxytestosterone formation (CYP3A4) increased 2-fold. In colon, RIF induced UGT1A6 32-fold and MDR1 2.2-fold. Dexamethasone (glucocorticoid receptor and PXR ligand) induced CYP3A4 mRNA (3.5-fold) and activity (5-fold) in proximal jejunum. Phenobarbital (constitutive androstane receptor activator) induced CYP3A4 (4.1-fold, only in jejunum), CYP2B6 (4.9-fold in colon and 2.3-fold in proximal jejunum), and MDR1/ABCB1 mRNA and CYP3A4 activity (2-fold only proximal jejunum). Quercetin (nuclear factor-E2-related factor 2 activator) induced UGT1A6 mRNA (6.7-fold in colon and 2.2-fold in proximal jejunum). In conclusion, this study shows that human intestinal precision-cut slices are useful to study induction of drug-metabolizing enzymes and transporters in the human intestine.

In vitro methods to study intestinal drug metabolism.

Although the liver has long been thought to play the major role in drug metabolism, also the metabolic capacity of the intestine is more and more recognized. In vivo studies eventually pointed out not only the significance of first-pass metabolism by the intestinal wall for the bioavailability of several compounds, but also the relevance of transporters in this process. Only a few methods are available to study drug metabolism in vivo or in situ and with most of these methods it remains difficult to discriminate between the contribution of liver and extrahepatic tissues. To study intestinal drug metabolism in vitro, apart from subcellular fractions, several intact cell systems are nowadays available. This review discusses the available intestinal in vitro methods to study drug metabolism. The advantages and limitations of intact cell systems (isolated intestinal perfusion, everted sac, Ussing chamber preparations, biopsies, precision-cut slices, primary cells), subcellular fractions (S9 fractions, microsomes) and intestinal cell lines (caco-2, LS180 cells amongst others) are discussed. Their applicability to different species and to study phase I and II metabolism/transport and drug-drug interactions are summarized. Furthermore, causes of variation within and between methods are discussed and metabolic rates obtained with different methods are compared. Whereas subcellular fractions and cell lines are efficient methods to study mechanistic aspects of drug metabolism at the enzyme level, the isolated intestinal perfusion, everted sac and Ussing chamber appear particularly useful for studying drug metabolism of rapidly metabolised drugs and interactions with transporters. Biopsies, precision-cut slices and primary cells seem all appropriate to study induction and metabolism of slowly metabolised drugs.

Innovative methods to study human intestinal drug metabolism in vitro: precision-cut slices compared with ussing chamber preparations.

Predictive in vitro methods to investigate drug metabolism in the human intestine using intact tissue are of high importance. Therefore, we studied the metabolic activity of human small intestinal and colon slices and compared it with the metabolic activity of the same human intestinal segments using the Ussing chamber technique. The metabolic activity was evaluated using substrates to both phase I and phase II reactions: testosterone, 7-hydroxycoumarin (7HC), and a mixture of cytochrome P450 (P450) substrates (midazolam, diclofenac, coumarin, and bufuralol). In slices of human proximal jejunum, the metabolic activity of several P450-mediated and conjugation reactions remained constant up to4hof incubation. In the colon slices, conjugation rates were virtually equal to those in small intestine, whereas P450-mediated conversions occurred much slower. In both organs, morphological evaluation and ATP content implied tissue integrity within this period. P450 conversions using the Ussing chamber technique showed that the metabolic rate (sum of metabolites measured in apical, basolateral, and tissue compartments) was constant up to 3 h. For 7HC conjugations, the metabolic rate remained constant up to 4 h. The distribution of the metabolites in the compartments differed between the substrates. Overall, metabolic rates were surprisingly similar in both techniques and appear similar to or even higher than in liver. In conclusion, this study shows that both human intestinal precision-cut slices and Ussing chamber preparations provide useful tools for in vitro biotransformation studies.

Empirical validation of a rat in vitro organ slice model as a tool for in vivo clearance prediction.

Tissue slices have been shown to be a valuable tool to predict metabolism of novel drugs. However, besides the numerous advantages of their use for this purpose, some potential drawbacks also exist, including reported poor penetration of drugs into the inner cell layers of slices and loss of metabolic capacity during prolonged incubation, leading to underprediction of metabolic clearance. In the present study, we empirically identified (and quantified) sources of underprediction using rat tissue slices of lung, intestine, kidney, and liver and found that thin liver slices (+/-100 mum) metabolized model substrates (7-hydroxycoumarin, testosterone, warfarin, 7-ethoxycoumarin, midazolam, haloperidol, and quinidine) as rapidly as isolated hepatocytes. Furthermore, it was found that organ slices remain metabolically active for sufficient periods of incubation, enabling study of the kinetics of low clearance compounds. In addition, we determined the influence of albumin on the clearance prediction of six model substrates. For three of these substrates, the intrinsic clearance in the presence of albumin was approximately 3 times higher than that obtained from incubations without albumin, but corrected for unbound fraction. This resulted in a much more accurate prediction of in vivo whole body metabolic clearance for these compounds. Collectively, these results show that drawbacks of the use of slices for clearance prediction are largely surmountable. Provided that thin liver slices and physiological albumin concentration are used, whole body metabolic clearance is predicted with acceptable (2-fold) accuracy with organ slices. These results emphasize the applicability of organ slices in this field of research.

Characterization of rat small intestinal and colon precision-cut slices as an in vitro system for drug metabolism and induction studies.

The aim of this study was to characterize rat small intestinal and colon tissue slices as a tool to study intestinal metabolism and to investigate gradients of drug metabolism along the intestinal tract as well as drug-induced inhibition and induction of biotransformation. Tissue morphology and the intestinal mucus layer remained intact in small intestinal and colon slices during 3 h of incubation, while alkaline phosphatase was retained and the rate of metabolism of three model compounds (7-hydroxycoumarin, 7-ethoxycoumarin, and testosterone) appeared constant. Phase I and phase II metabolic gradients, decreasing from stomach toward colon were shown to be clearly different for the model compounds used. Furthermore, the observed slice activities were similar or even higher compared with the literature data concerning metabolism of in vitro intestinal systems. Preincubation with beta-naphthoflavone for 24 h induced the O-deethylation of 7-ethoxycoumarin from nearly undetectable to 140 pmol/min/mg protein in small intestine (fresh slices, 43 pmol/min/mg protein) and to 100 pmol/min/mg protein in colon slices (fresh slices, undetectable). Ketoconazole inhibited metabolism of testosterone by 40% and that of 7-ethoxycoumarin by 100%. In conclusion, we showed that the intestinal slice model is an excellent model to study drug metabolism in the intestine in vitro, since we found that the viability parameters remain constant and the measured enzyme activities are relevant, sensitive to inhibitors, and inducible. Therefore, it is a promising tool to study intestinal drug metabolism in human intestine in vitro in the future.

A new technique for preparing precision-cut slices from small intestine and colon for drug biotransformation studies.

INTRODUCTION: A new technique was developed to prepare precision-cut slices from small intestine and colon with the object of studying the biotransformation of drugs in these organs. METHODS: Rat intestinal slices were prepared in two different ways. In the first method, slices were punched out of the small intestine. In the second method, precision-cut slices were made from agarose-filled and -embedded intestines, using the Krumdieck tissue slicer. This method was also applied to colon tissue. Viability of the slices was determined by analysis of intracellular ATP and RNA levels and morphology. Drug metabolizing activity was studied using lidocaine, testosterone, and 7-ethoxycoumarin (7-EC) as phase I substrates, and 7-hydroxycoumarin (7-HC) as a phase II substrate. RESULTS: Precision-cut slices made from agarose-filled and -embedded intestine better preserved ATP levels than tissue that was punched out of the intestinal wall. After 24 h of incubation, morphology in precision cut-slices showed was quite well preserved while punched out tissue was almost completely autolytic after incubation. In addition, total RNA amount and quality was much better maintained in precision-cut slices, when compared to punched out tissue. Both intestinal slices and punched-out tissue showed high, and comparable, phase I and phase II biotransformation activities. DISCUSSION: It is concluded that preparing precision-cut 0.25 mm slices out of agarose-filled and -embedded intestine provides an improvement, compared with punched-out tissue, and that both intestinal and colon slices are useful preparations for in vitro biotransformation studies.