Quantitative Whole-body Autoradiography Research Articles

Quantitative whole-body autoradiography, LC–MS/MS and MALDI for drug-distribution studies in biological samples: the ultimate matrix trilogy

The drug-development process requires an understanding of the ADME properties of the novel therapeutic agent. Determination of drug concentrations and identity in excreta (urine and feces) examines the products of these processes. Similar measurements made on plasma, while accurately determining exposure, show only what is being transported around the body. Both activities fail to confirm the nature of components at the pharmacologically relevant matrix - the tissue. Attention is therefore being directed towards methods that can be employed to address this lack in our current methodologies, to provide better quality data on which risk assessments can be made, so that pharmacological models can be refined, and drug safety improved. In this article, we will look at the current methods used to obtain tissue drug and drug metabolite concentrations, and their potential use in drug discovery.

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.

Evaluation of inhibitory effect of recreational drugs on dopaminergic terminal neuron by PET and whole-body autoradiography.

There is little investigation for the functional roles of peripheral dopamine. [18F]FDOPA has been used in cancer imaging (i.e., neuroendocrine and tumors pancreatic tumors) and neuroimaging (i.e., Parkinson's disease and Huntington's disease). Here, we accessed side effects of recreational drugs such as ketamine, cocaine, and methamphetamine on dopamine neurons in peripheral organs by using positron emission tomography (PET) imaging and quantitative whole-body autoradiography (QWBAR) with [18F]FDOPA. The images were applied for the measurement of specific binding ratios (SBRs) of striatum with the cerebellum as the reference region. Clear striatal [18F]FDOPA-derived radioactivity was observed. Moderate level of radiotracer accumulation was presented in the mucosal layers of the stomach and small intestine. The medulla layers of kidney had higher radioactivity than that of the cortex. Blocking images markedly eliminated the specific binding of [18F]FDOPA in the striatum and in peripheral organs such as stomachs, intestines, and kidney. Ketamine showed the highest inhibitory effect on striatal [18F]FDOPA-derived radioactivity followed by cocaine and methamphetamine. The current results demonstrated a useful crossing-validating tool that enhances the capability of [18F]FDOPA for further investigations of the alteration of dopaminergic neurons in the brain disorder or cancer diseases in peripheral tissues.

Generating an In Vitro–In Vivo Correlation for Metabolism and Liver Enrichment of a Hepatitis C Virus Drug, Faldaprevir, Using a Rat Hepatocyte Model (HepatoPac)

Hepatocytes provide an integrated model to study drug metabolism and disposition. As a result of a loss of polarity or a significant decrease in the expression of enzymes and transporters, suspended and sandwich-cultured hepatocytes have limitations in determining hepatocellular drug concentrations. Underprediction of the extent of glucuronidation is also a concern for these hepatocyte models. Faldaprevir is a hepatitis C virus protease inhibitor in late-stage development that has demonstrated significant liver enrichment in in vivo rat models based on quantitative whole-body autoradiography (QWBA) and liver-to-plasma area under-the-curve ratio. In bile duct cannulated rats, the primary biliary metabolite was a glucuronide. Owing to ethical concerns, it is difficult to assess liver enrichment in humans, and a lack of in vitro and in vivo correlation of glucuronidation has been reported. The current study was conducted to verify whether a hepatocyte model, rat HepatoPac, could overcome some of these limitations and provide validity for follow-up studies with human HepatoPac. With rat HepatoPac, liver enrichment values averaged 34-fold and were consistent with rat QWBA (26.8-fold) and in vivo data (42-fold). In contrast, liver enrichment in suspended hepatocytes was only 2.8-fold. Furthermore, the extent of faldaprevir glucuronidation in HepatoPac studies was in agreement with in vivo results, with glucuronidation as the major pathway (96%). Suspended rat hepatocytes did not generate the glucuronide or two key hydroxylated metabolites that were observed in vivo. Overall, our studies suggest that HepatoPac is a promising in vitro model to predict in vivo liver enrichment and metabolism, especially for glucuronidation, and has demonstrated superiority over suspended hepatocytes.

Tu1830 Predictors of Slow Healing of Reflux Esophagitis With Potent Acid Suppression

Introduction: Proton pump inhibitors (PPIs) are now widely used for the treatment of acidrelated diseases, such as peptic ulcer and gastroesophageal reflux disease (GERD). PPIs which are weak-base prodrugs are activated in an acidic condition and only inhibit H+/K+ATPase that has already been activated. Therefore, the effect of PPIs on acid-related diseases is slow onset and their maximum efficacy can be achieved after several days of dosing, leading to unmet medical needs such as nocturnal acid breakthrough and unsatisfied symptom relief. Methods: We investigated the pharmacological profiles of YH4808, a novel potent, highly selective K+-competitive acid blocker (P-CAB), in terms of hog gastric H+/K+-ATPase activity, gastric acid secretion, and acute gastroesophageal lesions in animal models, in comparison with esomeprazole. In addition, a quantitative whole body autoradiography (QWBA) study was performed with [14C]YH4808 (10 mg/kg) in rats to characterize tissue distribution. Results: YH4808 inhibited H+/K+-ATPase activity in a concentration-dependent manner, with an IC50 value of 3.4 nM which is more potent than that of esomeprazole. The inhibitory mechanism of YH4808 on H+/K+-ATPase was a competitive antagonism to the K+-binding site of H+/K+-ATPase, and it was also a reversible inhibition. In a selectivity assay, YH4808 showed 3,000-fold greater selectivity against Na+/K+-ATPase. In pylorus-ligated rats, oral administration of YH4808 inhibited basal acid secretion in a dose-dependent manner with the ID50 value of 1.1 mg/kg. Anti-secretory activity of YH4808 against histamine-stimulated acid secretion in rats after IV injection (ID50=0.1 mg/kg) was stronger than that of esomeprazole (ID50=0.9 mg/kg). In Heidenhain pouch dogs, IV administration of YH4808 inhibited histamine-stimulated acid secretion in a dose-dependent manner with almost complete inhibition at the dose of 0.6 mg/kg. In a reflux esophagitis model, intraduodenal administration of YH4808 prevented esophageal lesions by 30, 82 and 91% at a dose of 1, 3, and 10 mg/kg, respectively while esomeprazole reduced the lesion by 47% at 10 mg/kg. A QWBA study in rats showed YH4808 accumulated and was retained in the gastric tissue for more than 24h after oral administration. Conclusion: YH4808, a novel selective P-CAB exerted a potent, longer-lasting anti-secretory efficacy through high accumulation in the gastric tissue, which translated into the more effective inhibition of reflux esophagitis than esomeprazole. These findings indicate that YH4808 can provide significant clinical benefits to patients with acid-related diseases.

Tu1827 Yh4808, a Potent, Highly Selective K+-Competitive Acid Blocker, Suppressed Rat Acute Reflux Esophagitis More Effectively Than Esomeprazole

Introduction: Proton pump inhibitors (PPIs) are now widely used for the treatment of acidrelated diseases, such as peptic ulcer and gastroesophageal reflux disease (GERD). PPIs which are weak-base prodrugs are activated in an acidic condition and only inhibit H+/K+ATPase that has already been activated. Therefore, the effect of PPIs on acid-related diseases is slow onset and their maximum efficacy can be achieved after several days of dosing, leading to unmet medical needs such as nocturnal acid breakthrough and unsatisfied symptom relief. Methods: We investigated the pharmacological profiles of YH4808, a novel potent, highly selective K+-competitive acid blocker (P-CAB), in terms of hog gastric H+/K+-ATPase activity, gastric acid secretion, and acute gastroesophageal lesions in animal models, in comparison with esomeprazole. In addition, a quantitative whole body autoradiography (QWBA) study was performed with [14C]YH4808 (10 mg/kg) in rats to characterize tissue distribution. Results: YH4808 inhibited H+/K+-ATPase activity in a concentration-dependent manner, with an IC50 value of 3.4 nM which is more potent than that of esomeprazole. The inhibitory mechanism of YH4808 on H+/K+-ATPase was a competitive antagonism to the K+-binding site of H+/K+-ATPase, and it was also a reversible inhibition. In a selectivity assay, YH4808 showed 3,000-fold greater selectivity against Na+/K+-ATPase. In pylorus-ligated rats, oral administration of YH4808 inhibited basal acid secretion in a dose-dependent manner with the ID50 value of 1.1 mg/kg. Anti-secretory activity of YH4808 against histamine-stimulated acid secretion in rats after IV injection (ID50=0.1 mg/kg) was stronger than that of esomeprazole (ID50=0.9 mg/kg). In Heidenhain pouch dogs, IV administration of YH4808 inhibited histamine-stimulated acid secretion in a dose-dependent manner with almost complete inhibition at the dose of 0.6 mg/kg. In a reflux esophagitis model, intraduodenal administration of YH4808 prevented esophageal lesions by 30, 82 and 91% at a dose of 1, 3, and 10 mg/kg, respectively while esomeprazole reduced the lesion by 47% at 10 mg/kg. A QWBA study in rats showed YH4808 accumulated and was retained in the gastric tissue for more than 24h after oral administration. Conclusion: YH4808, a novel selective P-CAB exerted a potent, longer-lasting anti-secretory efficacy through high accumulation in the gastric tissue, which translated into the more effective inhibition of reflux esophagitis than esomeprazole. These findings indicate that YH4808 can provide significant clinical benefits to patients with acid-related diseases.

Tu1828 Impact of Regurgitation on Health-Related Quality of Life (HRQoL) in Gastroesophageal Reflux Disease (GERD)

Introduction: Proton pump inhibitors (PPIs) are now widely used for the treatment of acidrelated diseases, such as peptic ulcer and gastroesophageal reflux disease (GERD). PPIs which are weak-base prodrugs are activated in an acidic condition and only inhibit H+/K+ATPase that has already been activated. Therefore, the effect of PPIs on acid-related diseases is slow onset and their maximum efficacy can be achieved after several days of dosing, leading to unmet medical needs such as nocturnal acid breakthrough and unsatisfied symptom relief. Methods: We investigated the pharmacological profiles of YH4808, a novel potent, highly selective K+-competitive acid blocker (P-CAB), in terms of hog gastric H+/K+-ATPase activity, gastric acid secretion, and acute gastroesophageal lesions in animal models, in comparison with esomeprazole. In addition, a quantitative whole body autoradiography (QWBA) study was performed with [14C]YH4808 (10 mg/kg) in rats to characterize tissue distribution. Results: YH4808 inhibited H+/K+-ATPase activity in a concentration-dependent manner, with an IC50 value of 3.4 nM which is more potent than that of esomeprazole. The inhibitory mechanism of YH4808 on H+/K+-ATPase was a competitive antagonism to the K+-binding site of H+/K+-ATPase, and it was also a reversible inhibition. In a selectivity assay, YH4808 showed 3,000-fold greater selectivity against Na+/K+-ATPase. In pylorus-ligated rats, oral administration of YH4808 inhibited basal acid secretion in a dose-dependent manner with the ID50 value of 1.1 mg/kg. Anti-secretory activity of YH4808 against histamine-stimulated acid secretion in rats after IV injection (ID50=0.1 mg/kg) was stronger than that of esomeprazole (ID50=0.9 mg/kg). In Heidenhain pouch dogs, IV administration of YH4808 inhibited histamine-stimulated acid secretion in a dose-dependent manner with almost complete inhibition at the dose of 0.6 mg/kg. In a reflux esophagitis model, intraduodenal administration of YH4808 prevented esophageal lesions by 30, 82 and 91% at a dose of 1, 3, and 10 mg/kg, respectively while esomeprazole reduced the lesion by 47% at 10 mg/kg. A QWBA study in rats showed YH4808 accumulated and was retained in the gastric tissue for more than 24h after oral administration. Conclusion: YH4808, a novel selective P-CAB exerted a potent, longer-lasting anti-secretory efficacy through high accumulation in the gastric tissue, which translated into the more effective inhibition of reflux esophagitis than esomeprazole. These findings indicate that YH4808 can provide significant clinical benefits to patients with acid-related diseases.

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.

ADME characterization in rats revealed immediate secretion of AZD7903 into the stomach after IV dosing

1. The distribution of AZD7903 and/or its metabolites was studied in rats following a single p.o. or i.v. dose using quantitative whole-body autoradiography (QWBA). At 5 min after i.v. administration of the 14C compound, high levels of radioactivity were observed in the fundus of the stomach compared to blood and plasma and the rest of the stomach, indicating an active secretion of 14C material into the stomach.2. Also, excretion and pharmacokinetics were studied following p.o. and i.v. dosage in rats. The radioactivity was mainly excreted via feces, and even in i.v. administered bile-duct cannulated (BDC) animals a significant part of radioactivity (26% in males and 57% in females) was recovered in the feces in the form of parent compound and two minor metabolic products.3. The compound was well absorbed (F% 98 in males and 76 in females), and showed low clearance in plasma (0.14 L/h/kg in males and 0.03 L/h/kg in females) and low-intermediate Vdss (0.7 L/kg). Clear differences in metabolic pathways (qualitative) and rates (quantitative) and consequently in PK parameters between sexes were observed.4. In summary, the results indicate AZD7903 being substrate for a transporter protein and support the hypothesis that the differences in disposition between sexes are due to differences in metabolic pathways and rates.

Tissue Accumulation Kinetics of Ciclesonide‐active Metabolite and Budesonide in Mice

Inhaled corticosteroids (ICS) are mainstay treatment of asthma and chronic obstructive pulmonary disease. However, highly lipophilic ICS accumulate in systemic tissues, which may lead to adverse systemic effects. The accumulation of a new, highly lipophilic ICS, ciclesonide and its active metabolite (des-CIC) has not yet been reported. Here, we have compared tissue accumulation of des-CIC and an ICS of a moderate lipophilicity, budesonide (BUD), after 14days of once-daily treatment in mice. Single, three or 14 daily doses of [(3) H]-des-CIC or [(3) H]-BUD were administered subcutaneously to male CD1 albino mice, which were killed at 4hr, 24hr or 5days after the last dose. Distribution of tissue concentration of radioactivity was studied by quantitative whole-body autoradiography. Pattern of radioactivity distribution across most tissues was similar for both corticosteroids after a single as well as after repeated dosing. However, tissue concentration of radioactivity differed between des-CIC and BUD. After a single dose, concentrations of radioactivity for both corticosteroids were low for most tissues but increased over 14days of daily dosing. The tissue radioactivity of des-CIC at 24hr and 5days after the 14th dose was 2-3 times higher than that of BUD in majority of tissues. Tissue accumulation, assessed as concentration of tissue radioactivity 5days after the 14th versus 3rd dose, showed an average ratio of 5.2 for des-CIC and 2.7 for BUD (p<0.0001). In conclusion, des-CIC accumulated significantly more than BUD. Systemic accumulation may lead to increased risk of adverse systemic side effects during long-term therapy.

Peginesatide Clearance, Distribution, Metabolism, and Excretion in Monkeys following Intravenous Administration

Peginesatide, a polyethylene glycol (PEG)ylated peptide-based erythropoiesis-stimulating agent, stimulates the erythropoietin receptor dimer that governs erythropoiesis. Studies were designed to determine the erythropoietic response, pharmacokinetics (PK), tissue distribution, metabolism, and excretion of peginesatide in nonhuman primates following a single i.v. dose. The PK profile of peginesatide (0.1-5 mg/kg) is characterized by low, dose-dependent plasma clearance; small volume of distribution; and long half-life. The peginesatide PK profile following a single i.v. dose is consistent with the sustained erythropoiesis. Biodistribution quantitative whole-body autoradiography demonstrated high peginesatide levels in bone marrow (i.e., primary hematopoietic site) as well as other known hematopoietic sites persisting through at least 3 weeks at 2.1 mg/kg. Microautoradiography analysis at 48 hours postdose revealed uniform and high distribution of radioactivity in the bone marrow and splenic red pulp with less extensive distribution in the renal cortex (glomeruli, associated ducts, interstitial cells). Radioactivity in the kidney was most prominent in the outer medullary and papillary interstitium. At 2 weeks after dosing, cumulative radioactivity recovery in the urine and feces was 60 and 7% of the administered dose, respectively, with most of the radioactivity associated with the parent molecule. In conclusion, the PK characteristics are consistent with a PEGylated peptide of a 45-kDa molecular mass, specifically low volume of distribution and long half-life. Drug was localized principally to hematopoietic sites, and nonspecific tissue retention was not observed. The nonhuman primate data indicate that peginesatide is metabolically stable and primarily excreted in the urine.

Evaluation of Metabolism and Disposition of GDC-0152 in Rats Using 14C Labeling Strategy at Two Different Positions: A Novel Formation of Hippuric Acid from 4-Phenyl-5-Amino-1,2,3-Thiadiazole

The compound (S)-1-[(S)-2-cyclohexyl-2-([S]-2-[methylamino]propanamido)acetyl]-N-(4-phenyl-1,2,3-thiadiazol-5-yl)pyrrolidine-2-carboxamide (GDC-0152) is a peptidomimetic small molecule antagonist of inhibitor of apoptosis (IAP) proteins with antitumor activity. The mass balance, pharmacokinetics, tissue distribution and metabolism of GDC-0152 was investigated in rats following intravenous administration of 15 mg/kg of [(14)C]GDC-0152, labeled either at the terminal phenyl ring (A) or at the carbonyl of the 2-amino-2-cyclohexylacetyl moiety (B). In rats, 92.2%-95.1% of the radiolabeled GDC-0152 dose was recovered. Approximately 62.3% and 25.1% of A was excreted in urine and feces, respectively. By contrast, B was excreted almost equally in urine (27.2%), feces (32.2%), and expired air (27.5%). GDC-0152 underwent extensive metabolism, with less than 9% of the dose recovered as parent in excreta. Similarly, in plasma, GDC-0152 represented 16.7% and 7.5% of the area under the curve of the total radioactivity for A and B, respectively. The terminal half-life (t(1/2)) for total radioactivity was longer for B (21.2 hours) than for A (4.59 hours). GDC-0152 was highly metabolized via oxidation and amide hydrolysis, followed by subsequent sulfation and glucuronidation. The most abundant circulating metabolites were the amide hydrolyzed products, M26, M28, M30, M31, and M34, which ranged from 3.5% to 9.0% of total radioactivity. In quantitative whole-body autoradiography studies, the residence of radioactivity in tissues was longer for B than for A, which is consistent with the t(1/2) of the total radioactivity in circulation. A novel 4-phenyl-5-amino-1,2,3-thiadiazole (M28) oxidative cleavage resulted in the formation of hippuric acid (M24). This biotransformation was also observed in rat hepatocyte incubations with para-substituted M28 analogs. In addition, the formation of M24 was inhibited by 1-aminobenzotriazole, which points to the involvement of P450 enzymes.

Different Contribution of Hematopoietic and Somatic Cell Types Expressing FcRn to the Prolonged Half-Life of Recombinant Factor VIII Fc and Recombinant FIX Fc

AbstractAbstract 1103 Introduction:Recombinant fusion of a single molecule of either FVIII or FIX to the Fc domain of IgG1 (rFVIIIFc or rFIXFc, respectively) has been shown to decrease clearance compared to rFIX or rFVIII in an FcRn-dependent manner (Blood (2010) 115: 2057; Blood (2012) 119:3024). The long-lasting procoagulant activity of these two molecules relies on a natural pathway in somatic (endothelial) and hematopoietic (monocyte) cells that recirculate antibodies into the blood stream. In hemophilia A subjects, rFVIIIFc has demonstrated ∼1.6-fold longer half-life compared to recombinant full length FVIII (Advate®) (Blood (2012) 119:3031), while rFIXFc shows a ∼3-fold longer half-life than that reported for current rFIX products (Blood (2012) 119:666). In the current study we determined the biodistribution of rFVIIIFc in hemophilia A (HemA) mice and sought to identify the cell types and tissues that contribute to FcRn-mediated protection and recycling of rFVIIIFc or rFIXFc. Methods:The pharmacokinetics of rFVIIIFc or rFIXFc were compared to IgG1 in bone marrow chimeric mice of wild-type and FcRn-KO mice. The plasma FVIII activity was determined using a chromogenic assay, and the decay of antigen levels of rFIXFc or IgG1 in plasma post dosing was determined by ELISA. The biodistribution of iodinated rFVIIIFc in HemA mice was evaluated by Quantitative Whole Body Autoradiography (QWBA). Results:We have shown that the rFVIIIFc half-life is ∼2-fold longer in wild type mice than in FcRn-KO mice. However, bone marrow transplantation of FcRn-expressing hematopoietic cells into FcRn-deficient mice did not substantially increase the half-life of rFVIIIFc. Also, bone marrow transplantation of FcRn-deficient cells into wild-type mice did not decrease the half-life of rFVIIIFc, suggesting a substantional role of somatic FcRn. In contrast, while rFIXFc has a ∼3 fold longer half-life in FcRn wild-type mice than in knockout mice, both FcRn-deficient mice receiving wild-type bone marrow, as well as wild-type mice receiving FcRn-deficient bone marrow, displayed clearance levels of rFIXFc intermediate to that of wild-type and FcRn-KO mice. These studies suggest that rFVIIIFc is recycled via FcRn predominantly in somatic cells, while rFIXFc, similar to IgG1, is recycled approximately equally in both FcRn-expressing hematopoietic and somatic cells.Furthermore, QWBA using 125I-labelled rFVIIIFc in HemA mice show extensive localization of rFVIIIFc in the liver, suggesting that somatic cells in the liver may mediate rFVIIIFc recycling through Fc – FcRn interaction. In summary, these data suggest that similar to IgG1, rFVIIIFc and rFIXFc are recycled in both somatic and hematopoietic FcRn-expressing cells. However, the relative contributions of the various cell types are dependent on the particular Fc-fusion protein, suggesting additional regulation at the level of protein-specific interactions for cellular uptake. Characterization of the cell-type specific recycling pathways for rFVIIIFc and rFIXFc is in progress to support further delineation of the mechanism of action for these two novel therapeutics currently in registrational trials for treatment of severe Hemophilia A and B, respectively. Disclosures:van der Flier:BiogenIdec: Employment. Liu:BiogenIdec: Employment. Drager:BiogenIdec: Employment. Vallee:former BiogenIdec: Employment. Chen:BiogenIdec: Employment. Tan:BiogenIdec: Employment. Wharton:former BiogenIdec: Employment. Light:biogenidec: Employment. Jiang:biogenidec: Employment.

Development of a Carbon-14 Labeling Approach to Support Disposition Studies with a Pegylated Biologic

Although it is widely accepted that one can extend the pharmacokinetic half-life of a therapeutic protein by covalent conjugation with polyethylene glycol (PEG), the disposition properties of such biologics have not yet been fully evaluated. Therefore, a novel [¹⁴C]-labeling method was developed that can be applied to a biologic conjugated with PEG through a maleimide-cysteine reaction. The method was used to study the tissue and tumor distribution of a PEGylated Adnectin, a recombinant protein derived from the 10th type III domain of fibronectin, in nude mice bearing human xenograft tumors. The PEGylated Adnectin contained a 40-kDa branched PEG (P40B) that was labeled with [¹⁴C] at the linker region between the PEG and Adnectin, without compromising cellular activity and plasma half-life in mice. After a single intravenous or intraperitoneal dose (33 mg/kg, 1.7 μCi per mouse) of [¹⁴C]-P40B-Adnectin, quantitative whole-body autoradiography analysis revealed that the liver had the highest uptake of the radioactivity among nontumor tissues, followed by the kidneys and lung. The muscle and brain showed the least penetration of the radioactivity among all tissues examined. In addition, the [¹⁴C]-P40B-EI-tandem penetrated into the tumor tissue, although the extent of accumulation was largely dependent on tumor type. Therefore, it was possible to assess the tissue distribution of a PEGylated biologic after it had been [¹⁴C] labeled using the novel method described herein.

Novel physiologically based pharmacokinetic modeling of patupilone for human pharmacokinetic predictions

Patupilone (EPO906) is a novel potent microtubule stabilizer, which has been evaluated for cancer treatment. A novel physiologically based pharmacokinetics (PBPK) model was developed based on nonclinical data to predict the disposition of patupilone in cancer patients. After a single intravenous dose (1.2 mg/kg) in male Han-Wistar rats, the tissue distribution of (14)C-patupilone was investigated by quantitative whole-body autoradiography (QWBA). The blood radioactivity and patupilone concentration were determined by LC-MS/MS and liquid scintillation counting. A novel PBPK model was developed based on rat tissue concentration data to predict blood concentration-time profiles of patupilone in cancer patients. PBPK parameters derived from the rat were applied to a human PBPK model. Phase I clinical pharmacokinetic data in Caucasian and Japanese cancer patients at various doses ranging from 0.75 to 10 mg/m(2) were successfully described using the PBPK approach. Patupilone dispositions in lung, heart, muscle, spleen, liver, brain, adipose, and testes of rats were well described using the PBPK model developed assuming a perfusion rate-limited distribution between different compartments. For skin and bone marrow, concentration-time profiles were modeled assuming a permeability-limited distribution between different compartments. The simulated human pharmacokinetic profiles from the PBPK model showed good agreement with observed clinical pharmacokinetic data, where the model predicted AUC, t(1/2), V(ss), and CL values were within approximately twofold of the observed values for all dose groups. The distribution of patupilone in rats was well described by a PBPK model based on measured tissue distribution profiles generated by QWBA combined with metabolism data. The human PBPK model adequately predicted blood pharmacokinetics of patupilone in cancer patients. The PBPK model based upon preclinical tissue distribution data can aid in successful prediction of pharmacokinetics in humans.

Use of Radioactive Compounds and Autoradiography to Determine Drug Tissue Distribution

Radioactivity has been used in drug discovery and development for several decades because it offers researchers a highly sensitive way to quantitatively assess the absorption, distribution, metabolism, and/or excretion (ADME) of chemical entities by incorporating a radioactive isotope into the structure of the drug molecule. Regulatory agencies around the world require drug makers to characterize the ADME properties of prospective new drugs as one way to help ensure that patients are not exposed to dangerous drug and/or drug metabolite levels before they can be approved for human use. Radiolabeled compounds have consistently proved to be the most efficient tool for determining that information, even though attempts have been made to use nonradioactive techniques. The techniques of quantitative whole-body autoradiography (QWBA) and microautoradiography (MARG), which rely on the use of radiolabeled drugs, are two techniques that are routinely used to examine tissue distribution of drugs in discovery and development. These techniques provide drug researchers with quantitative tissue concentration data and a visual location of those concentrations in intact organs, tissues, and cells of laboratory animals. It is important for readers to realize that these techniques visualize total radioactivity, which can include the parent molecule along with its metabolites, and/or degradation products or impurities. This requires investigators to treat the quantitative data with caution unless the identity of the radioactivity is determined using some type of other bioanalytical techniques, such as mass spectroscopy and/or radio-HPLC, which can be easily performed on the tissue obtained from the animals used for QWBA and/or MARG. Nevertheless, these data are used in drug discovery and development to answer questions related to tissue penetration, fetal/placental transfer, tissue retention, routes of elimination, drug-drug interactions, enzyme induction/inhibition, formulation comparisons, in vivo compound solubility, differential metabolite distribution, interspecies comparisons, and to predict human exposure to parent drugs, metabolites, and radiation during clinical studies. This review will consider the strategic use of WBA, QWBA, and MARG in the pharmaceutical industry. Case studies and anecdotal information will also be presented; however, readers should realize that these are general examples and that some details have been omitted for brevity and/or because the data is proprietary and could not be presented at this time. Nevertheless, the images and discussions are provided to demonstrate how the techniques can and have been used to examine in situ tissue distribution of therapeutic compounds.

Absorption, Distribution and Excretion Pattern of Oral and Transdermal Donepezil Hydrochloride after Single and Repeated Administration to the Rat

Donepezil hydrochloride formulated in a transdermal patch for weekly administration is an alternative to the approved daily oral tablet. Transdermal delivery has a locally high concentration at the application site causing potential safety issues. To assess the skin exposure for safety evaluation, studies were performed comparing the donepezil transdermal patch with an oral donepezil dose previously tested in a carcinogenicity study, in which no test article-induced tumors were found. Quantitative Whole Body Autoradiography (QWBA) comparison in Long-Evans rats administered 14C-donepezil either as single oral dose (30 mg/kg) or a 24-hour transdermal patch (~60 mg/kg) revealed similar organ distribution, including skin. Pigmented tissue, namely, skin and eye showed sustained labeling over 7-10 days, suggesting melanin binding. Subsequently, a 14C-donepezil study in Long-Evans rats comparing seven daily oral doses (10-30 mg/kg) with a seven-day transdermal patch (~40 mg/kg) again revealed similar tissue distributions between the two delivery methods, however, skin not directly exposed to the transdermal patch had less label as compared to both unpigmented and pigmented skin after oral dosing. Similarly, eye concentrations generally had higher label after oral dosing as compared to the transdermal administration. Upon patch removal, the non-viable stratum corneum under the dose-site contained the majority of the label with the underlying skin layers returning to baseline levels within 5 and 7 days for unpigmented and pigmented skin, respectively, illustrating dermal clearance. Skin structures were comparably labeled after oral and transdermal administration, as evidenced by microautoradioagraphy.

Pharmacokinetics and tissue distribution of galantamine and galantamine-related radioactivity after single intravenous and oral administration in the rat.

The plasma kinetics and tissue distribution of galantamine hydrobromide [4aS-(4a alpha,6beta,8aR*)]-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro-[3a,3,2-ef] [2benzazepin-6-ol hydrobromide, CAS-1953-04-4], a reversible acetylcholinesterase inhibitor, were studied in male and female non-pregnant and pregnant SPF Wistar rats and in male Fisher x Copenhagen pigmented rats. Most studies were performed using 3H-labelled galantamine hydrobromide, measuring unchanged drug (UD) and non-volatile radioactivity (NVR) in plasma and tissues by high-performance liquid chromatography (HPLC), liquid scintillation counting and quantitative whole-body autoradiography (QWBA). Plasma levels after single intravenous administration of UD (1.25-2.5 mg/kg) declined bi- or triphasically, with an elimination half-life of 3.5 h in male, and 5.1 h in female rats. The plasma clearance (Cl) averaged 1.9 l/kg/h (male rats) and 0.9 l/kg/h (female rats), and the volume of distribution (VdSS) was about 5 l/kg for both male and female rats. Following oral administration (2.5-10 mg/kg), galantamine was rapidly absorbed in both sexes, with an absolute oral bioavailability of 77%. Distribution studies after oral administration of 3H-galantamine showed an almost immediate equilibrium between plasma and tissues, with highest tissue levels of NVR and UD in liver, kidney, salivary glands, adrenal glands and, for the female rat, spleen, and lowest in white fat. To most tissues and especially to brain, the distribution of UD was more pronounced than that of its metabolites. Tissue concentrations of UD and NVR declined at a similar rate as plasma, showing no undue retention. QWBA in the pigmented rat showed the same distribution and elimination pattern of NVR. Only in hair follicles and choroid some retention of NVR was seen, but the calculated half-life was less than one day. In the female pregnant SPF Wistar rat, maternal tissue distribution of NVR was similar to that of the non-pregnant rat. NVR tissue levels in the foetus were similar to those found in maternal blood during the whole experiment, indicating a rapid equilibrium without accumulation.

Absorption, distribution, metabolism and excretion of peginesatide, a novel erythropoiesis-stimulating agent, in rats

The pharmacokinetics (PK) (absorption, distribution, metabolism, excretion) of peginesatide, a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent (ESA), was evaluated in rats. The PK profile was evaluated at 0.1–5 mg·kg−1 IV using unlabeled or [14C]-labeled peginesatide. Mass balance, tissue distribution and metabolism were evaluated following IV administration of 5 mg·kg−1 [14C]-peginesatide, with tissue distribution also evaluated by quantitative whole-body autoradiography (QWBA) following an IV dose of 17 mg·kg−1 [14C]-peginesatide.Plasma clearance was slow and elimination was biphasic with unchanged peginesatide representing >90% of the total radioactivity of the total radioactive exposure. Slow uptake of the radiolabeled compound from the vascular compartment into the tissues was observed.Biodistribution to bone marrow and extramedullary hematopoietic sites, and to highly vascularized lymphatic and excretory tissues occurred.A predominant degradation event to occur in vivo was the loss of one PEG chain from the branched PEG moiety to generate mono-PEG.Renal excretion was the primary mechanism (41%) of elimination, with parent molecule (67%) the major moiety excreted.In conclusion, elimination of [14C]-peginesatide-derived radioactivity was extended, retention preferentially occurred at sites of erythropoiesis (bone marrow), and urinary excretion was the primary elimination route.

Investigation of Figopitant and Its Metabolites in Rat Tissue by Combining Whole-Body Autoradiography with Liquid Extraction Surface Analysis Mass Spectrometry

This article describes the combination of whole-body autoradiography with liquid extraction surface analysis (LESA) and mass spectrometry (MS) to study the distribution of the tachykinin neurokinin-1 antagonist figopitant and its metabolites in tissue sections of rats after intravenous administration of 5.0 mg/kg figopitant. An overview of autoradiography results is presented together with mass spectrometry identification and semiquantification of parent drug and its metabolites based on LESA-MS. The quality and accuracy of data generated by LESA-MS were assessed in comparison with classic tissue extraction, sample cleanup, and high-performance liquid chromatography analysis. The parent drug and the N-dealkylated metabolite M474(1) (BIIF 1148) in varying ratios were the predominant compounds in all tissues investigated. In addition, several metabolites formed by oxygenation, dealkylation, and a combination of oxygenation and dealkylation were identified. In summary, the LESA-MS technique was shown to be a powerful tool for identification and semiquantification of figopitant and its metabolites in different tissues and was complementary to quantitative whole-body autoradiography for studying the distribution.