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Dynamic Analysis of Fluid Distribution in the Gastrointestinal Tract in Rats: Positron Emission Tomography Imaging after Oral Administration of Nonabsorbable Marker, [18F]Deoxyfluoropoly(ethylene glycol) Tadayuki Takashima,*,† Tomotaka Shingaki,†,‡ Yumiko Katayama,† Emi Hayashinaka,† Yasuhiro Wada,† Makoto Kataoka,§ Daiki Ozaki,† Hisashi Doi,† Masaaki Suzuki,† Sho Ishida,∥ Kentaro Hatanaka,∥ Yuichi Sugiyama,⊥ Shuji Akai,∥ Naoto Oku,∥ Shinji Yamashita,§ and Yasuyoshi Watanabe† †

RIKEN Center for Molecular Imaging Science, 6-7-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan ADME Research Inc., 1-12-8 Senba-higashi, Minoh, Osaka 562-0035, Japan § Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan ∥ Graduate School of Pharmaceutical Sciences, University of Shizuoka, Yada Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan ⊥ Sugiyama Laboratory, RIKEN Innovation Center, RIKEN Research Cluster for Innovation, Yokohama Bio Industry Center, 1-6, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ‡

ABSTRACT: To develop potent drugs for oral use, information on their pharmacokinetic (PK) properties after oral administration is of great importance. We have recently reported the utility of positron emission tomography (PET) for the analysis of gastrointestinal (GI) absorption of radiolabeled compounds. In this study, PET image analysis was performed in rats using a novel PET probe, [18F]deoxyfluoropoly(ethylene glycol)s, with an average molecular weight of 2 kDa ([18F]FPEG), as a nonabsorbable marker to elaborate the GI physiology in more detail, such as segmental transition of the administered water, and fluid volume and distribution in the intestine. After oral administration of [18F]FPEG solution to rats, a 90 min PET scan with continuous blood sampling was performed, and then the disposition of radioactivity in each part of GI tract was investigated. From blood PK analysis, it was confirmed that the bioavailability of [18F]FPEG was quite low in rats. PET image analysis showed that the radioactivity after oral administration of [18F]FPEG solution rapidly passed through the stomach, spread into the proximal small intestine, and then transited toward the distal region of the small intestine without decreasing the radioactivity during GI transition. Radiometabolite analysis revealed that the radioactivity in intestinal mucosal tissues, blood, and urine was mainly derived from unchanged [18F]FPEG. It was also found that the volume of interest (VOI) after oral administration of the radiotracer enables an understanding of the time-dependent manner of effective fluid volume changes in the stomach and the small intestine. In addition, the rate constant of the intestinal transition of radioactivity in each intestinal segment was calculated by kinetic model analysis, which revealed that PET analysis enables us to determine the GI transit from the same individuals and that it is applicable to determine site-specific intestinal absorption. In conclusion, we demonstrated the high potency of PET imaging technique to elucidate the distribution of orally administered solution in the GI tract in vivo. KEYWORDS: oral absorption, positron emission tomography (PET), deoxyfluoropoly(ethylene glycol)s (FPEG), gastrointestinal transit, effective fluid volume

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INTRODUCTION

even though plasma pharmacokinetic (PK) analysis is feasible using data obtained from in vitro/in situ experiments. One reason for this is that the PK data from plasma profiles can provide only information on overall GI absorption and systemic elimination from the body, while each individual process of

Gastrointestinal (GI) absorption of orally administered drugs consists of many processes, which include disintegration of the formulation, dissolution of drugs into the GI fluid, transition through the GI tract, permeation across the GI mucosa, and intestinal metabolism. It is possible to determine the drug solubility, dissolution profile, and effective permeability of the intestinal membrane from in vitro or in situ experiments.1−3 However, accurate prediction and characterization of the drug absorption kinetics after oral administration are still difficult, © 2013 American Chemical Society

Received: Revised: Accepted: Published: 2261

August 24, 2012 April 4, 2013 April 19, 2013 April 19, 2013 dx.doi.org/10.1021/mp300469m | Mol. Pharmaceutics 2013, 10, 2261−2269

Molecular Pharmaceutics

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drug absorption cannot be separately evaluated using PK data. Therefore, methods that enable quantitative determination of the drug concentration in the GI tract and full elucidation of the drug absorption behavior in vivo are desirable. The GI transit parameter of orally administered drug has been obtained by time profiles of the blood concentration of marker drugs such as acetaminophen, sulfasalazine, and polyethylene glycol 4000 by an alternative technique.4−6 These parameters determined by the blood concentration−time profiles of the marker drug, like the gastric emptying rate and intestinal transit rate or residence time, are usually calculated from the time when the significant concentration of the drug was detected in the blood. Those parameters are profoundly affected by other factors, such as the rate of dissolution, absorption of drugs, and the detection limit of the blood concentration. Therefore, it would be difficult to determine the actual GI transit parameter from the blood PK analysis. To overcome these limitations, molecular imaging technology has been introduced to analyze GI absorption behavior, such as gamma-scintigraphy,7−9 magnetic resonance imaging (MRI),10 and positron emission tomography (PET).11−13 PET image analysis in particular allows us to assess the PK properties of drugs labeled with positron-emitting radionuclides in detail by externally monitoring the tissue distribution of the administered drugs at very high sensitivity.14 Drugs labeled with high specificity are commonly used in PET imaging, so that the mass of the total drug associated with a PET tracer is low enough to fulfill the definition of a microdose.15 PK parameters in drug absorption, distribution, and excretion assessed by microdosing PET studies and also the consideration of the dose issues assessed in preclinical stages can help in the planning of later phases of drug development.16,17 We have recently reported the utility of PET for the analysis of drug absorption in the GI tract in rats and humans using 2-[18F]fluoro-2-deoxy9,10 18 D-glucose ([ F]FDG) and [11C]telmisartan,11 which focused on visualizing and kinetically analyzing the gastric emptying and drug absorption from the entire small intestine. To evaluate intestinal absorption behavior using PET image analysis further, more detailed investigations are required through modeling of the absorption profiles and determination of the pharmacokinetic parameters related to the volume of intestinal fluid, which contributes to drug dissolution in the GI lumen. In the present study, a novel radiotracer, 18F-labeled deoxyfluoropoly(ethylene glycol)s, with an average mass of 2 kDa ([18F]FPEG), an analogue of polyethylene glycol (PEG), was used. PEG is well-known for having low toxicity and immunogenicity and is used as a nonabsorbable marker in the evaluation of intestinal absorption and secretion because of its lack of intestinal enzymatic degradation, bacterial metabolism, water-binding capacity, and so on. Previously, Akai et al.18 successfully achieved the labeling synthesis of long-chain PEGs using [18F]FPEGs (2 or 10 kDa) by nucleophilic substitution of the hydroxyl group of the PEGs with a [18F]fluoride ion and demonstrated the noninvasive pharmacokinetic analysis of the radiotracer by a planar positron imaging system after intravenous (IV) bolus administration to rats for the first time. The use of PET image analysis with orally administered [18F]FPEG solution enables the separate evaluation of each process of segmental GI behavior in vivo, particularly GI transition of the solution, and also clarifies the parameters related to the fluid volume presenting in the GI lumen.

Article

MATERIALS AND METHODS

Materials. Poly(ethylene glycol) (2 kDa) was kindly provided by NOF Corporation (Tokyo, Japan). All other chemicals were commercial products of reagent grade. All other materials were used without further purification. Scheme 1. 18F Labeling of PEG

18

F Labeling of PEG (2 kDa) (Scheme 1). On the basis of a previous 18F-labeling protocol of PEG reported by Akai et al.,18 the desired [18F]FPEG with a mass of 2 kDa was synthesized by nucleophilic [18F]fluorination of toluene sulfonyl-substituted poly(ethylene glycol), as a precursor prepared from Omethylpoly(ethylene glycol) (2 kDa) and p-toluene sulfonyl chloride under basic conditions, with tetra-n-butylammonium [18F]fluoride converted from tetra-n-butylammonium hydrogen carbonate and [18F]fluoride anion produced by cyclotron. Then, the reaction mixture was diluted with 0.3 mL of H2O and injected into semipreparative high-performance liquid chromatography (HPLC). The purification conditions were as follows: COSMOSIL 5C18-MS-II 10 × 250 mm (Nacalai Tesque, Inc., Kyoto, Japan), linear gradient elution: 25−40% of CH3CN in H2O from 0 to 15 min, flow rate: 5 mL/min, column temperature: RT, and retention time of [18F]FPEG: 15−17 min. The entire 18F-labeling operation and the purification of the desired [18F]FPEG were controlled remotely using a RIKEN original radiolabeling system. The isolated radioactivity and the specific radioactivity of [18F]FPEG at the end of synthesis were 1.2−2.3 GBq and 5.4 GBq/μmol, respectively (n = 2). The decay-corrected radiochemical yield was 9−17% based on the use of approximately 20 GBq of the [18F]fluoride anion. The chemical purity by HPLC analysis with a corona charged aerosol detector was 77% (up to 97% when strictly purifying by semipreparative HPLC), and the radiochemical purity by radio-HPLC with a γ-ray detector was 99%. Animals. Male Sprague−Dawley rats weighing 170−200 g (6−7 weeks old, n = 3 for each set of experiments) were fasted for 24 h prior to the end of the experiment and given free access to water prior to the following experiments. Rats were anesthetized using 1.5% isoflurane (Mylan, Tokyo, Japan) with 2 L/min of air for following surgical operations. The femoral vein was cannulated (MRE-040, 0.040 in. O.D. × 0.025 in. I.D., BIOSEB, Vitrolles, France). The abdomen was cut open along to the median line, and then the bile duct was exposed from the back side of the duodenum. The cannula (PE-10, 0.024 in. O.D. × 0.011 in. I.D., BD, NJ, USA) was inserted into the common bile duct to collect bile juice. All experimental protocols were approved by the Ethics Committee on Animal Care and Use of the Center for Molecular Imaging Science in RIKEN and were performed in accordance with the Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985). PET Scanning. All PET scans were performed using a microPET Focus220 scanner (Siemens, Knoxville, TN) designed for laboratory animals. The femoral vein of rats was 2262

dx.doi.org/10.1021/mp300469m | Mol. Pharmaceutics 2013, 10, 2261−2269


Article

standard 2D filtered back projection (FBP) using a Ramp filter with a cutoff at the Nyquist frequency. Regions of interest (ROIs) representing the stomach and the intestines after oral administration of the radiotracer were identified using a previously described procedure.12 In this PET analysis, the small intestine was further divided into three segments: duodenum (Duo; 10 cm forward from the pylorus), ileum (Ile; 10 cm back from the ileocecal junction), and jejunum (Jej; the remainder of the intestine). From the result of the biodistribution of isolated tissues measured using a gammacounter, the radioactivity levels in cecum and colon were quite low, and most radioactivity was located in Ile, even at 120 min postadministration. The ROIs of duodenum determined by PET images were obtained in the initial period because the images of the duodenum were visually identifiable on the basis of its anatomical features, in that the duodenum bends from the pylorus to the ligament of Treitz. The ROIs of the ileum region were determined by PET images taken at the end of the experiments because the biodistribution studies showed that most of the radioactivity was located at the end of the small intestine (10 cm back from the ileocecal junction) at 90 min postdose. The remainder of the intestine, after excluding the duodenal and ileal regions, was defined as the jejunum. A time profile of the radioactivity in each tissue was constructed by normalizing decay-corrected time−radioactivity measurements to the injected dose (%-dose) of [18F]FPEG. Determination of Pharmacokinetic Parameters. The GI tract was divided into four segments: stomach, duodenum, jejunum (proximal intestine), and ileum (distal intestine). The mean residence time (MRT) in each segment was calculated using the following equation:

cannulated with a polyurethane tube under anesthesia with a mixture of 1.5% isoflurane before the day of the PET study. On the day of the experiment, rats were fixed with a retainer for small animals for imaging in a conscious condition (Molecular Imaging Laboratory Inc., Osaka, Japan) for the following study. Before emission scans, rats were placed in the center of the field of view of a microPET camera, and a transmission scan with rotating 68Ge−68Ga point source was performed for 25 min to reproduce the abdomen positioning and attenuation correction. The concentration of radioactivity in [18F]FPEG solution for oral administration was set to 40−53 MBq/mL by adding an appropriate volume of saline. At the start of the emission scan, [18F]FPEG solution was administered orally in doses of 100− 160 MBq/kg. In addition, PET studies with [18F]FPEG after IV bolus administration were performed to investigate the distribution of the radioactivity. An emission scan was performed in 3D list-mode for 30 min and sorted into 57 dynamic sinograms according to the following sequence: 20 × 15 s, 25 × 60 s, and 12 × 300 s. The blood was sampled via the cannulated femoral vein at 5, 10, 20, 30, 45, 60, and 90 min after administration. Blood radioactivity was measured using a 1470 WIZARD Automatic Gamma Counter (PerkinElmer, Waltham, MA). The volume of blood sampled at each time point was within 150 μL, and the total blood volume sampled from one rat did not exceed 1.6 mL, about 10% of the total circulating blood volume. The radioactivity measured in each sample was decay-corrected to the administration time and is expressed as %-dose/tissue or %-dose/mL blood, normalized to the injected radioactivity. Radiometabolite Analysis. Radiometabolites in the intestinal contents, scraped intestinal epithelia from lumen, blood, urine, liver, and bile of a separate group of rats, were analyzed using radiometric HPLC. The whole intestine was extracted from a midline abdominal incision, and the intestinal contents in the luminal side were collected by slow infusion of an appropriate volume of saline followed by air 100 min after rats had received oral administration of [18F]FPEG. Then, the intestine was washed with saline and cut open, and intestinal epithelia were scraped off from the mucosal layer with a slide glass. A blood sample was collected 90 min after each rat received an IV injection of [18F]FPEG; bile was taken over a period of 60−90 min postinjection from the bile duct via the cannula, and urine was sampled from urinary bladder at 100 min postinjection. Samples were deproteinated using acetonitrile. After centrifugation, the supernatants were evaporated, reconstituted with HPLC mobile phase, and then analyzed for radioactive components using an HPLC system (Shimadzu Corporation, Kyoto, Japan) with a coupled NaI(TI) positron detector UGSCA30 (Universal Giken, Kanagawa, Japan) to measure the intact radiotracer and its metabolites. Chromatographic separation was carried out using a COSMOSIL C18 MS-II column (4.6 (i.d.) × 100 mm; Nacalai Tesque, Kyoto, Japan). The flow was 2.0 mL/min at initial conditions of 10 mM ammonium acetate (pH 7.4, solvent A) and acetonitrile (solvent B) (A/B = 95/5, v/v). Analytes were eluted using the following gradient conditions: 0−1 min: 5% solvent B in solvent A; 1−6 min: 5−90% solvent B in solvent A; and 6−8 min: 90% solvent B. Following elution, the column was returned to 5% solvent B in solvent A over 5 min until the time when the next sample was injected into the HPLC system. Analysis of PET Imaging Data. PET images were reconstructed using microPET Manager 2.4.1.1 (Siemens) by

MRT = AUMC/AUC

where AUMC is the area under the first moment curve and AUC is the area under the curve of each segment. On the basis of the principal equation described by Kimura et al.,8 GI transit constants in each segment were determined by fitting using the following equations: Xsto = X 0,sto × exp( −kel,sto × t )

(1)

Xduo = [X 0,duo × k in,duo/(k in,duo − kel,duo)] × {exp[−kel,duo × (t − tlag1)] − exp[−k in,duo × (t − tlag1)]}

(2)

X jej = [X 0,jej × k in,jej/(k in,jej − kel,jej)] × {exp[−kel,jej × (t − tlag2)] − exp[−k in,jej × (t − tlag2)]}

(3)

X ile = [X 0,ile × k in,ile/(k in,ile − kel,ile)] × {exp[−kel,ile × (t − tlag3)] − exp[−k in,ile × (t − tlag3)]}

(4)

where X0,sto, X0,duo, X0,jej, and X0,ile are the estimated fractions of administered radioactivity at the time of administration in the stomach, duodenum, jejunum, and ileum, respectively. kel,sto is the elimination rate constant in the stomach, and kin,duo is the rate constant for gastric emptying. kel,duo is the constant for 2263



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Figure 1. Coronal maximum-intensity-projection PET images of abdominal region serially obtained after oral administration (A) or intravenous bolus administration (B) of [18F]FPEG to rats. The abbreviations are as follows: anterior (A), posterior (P), left (L), and right (R).

elimination from the duodenum, and kin,jej is the rate constant for intestinal transit from the duodenum to the jejunum. kel,jej and kin,ile are rate constants for elimination from the jejunum and intestinal transit from the jejunum to the ileum, respectively. kel,ile is the rate constant for elimination from the ileum. However, as kel,ile showed a negative value in all animals tested, this value was considered as 0 in this case. tlag1, tlag2, and tlag3 represent the lag times in the duodenum, jejunum, and ileum, respectively. The damped Gauss−Newton algorithm was used with the MULTI program for nonlinear least-squares data fitting.19

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RESULTS Tissue Distribution and Blood Concentration Profile of Radioactivity Following Oral and Intravenous Administration of [18F]FPEG to Rats. The maximumintensity-projection PET images of radioactivity in the abdominal region over time following oral or intravenous (IV) administration of [18F]FPEG to rats are shown in Figure 1. After oral administration of [18F]FPEG solution, the radioactivity was found in the stomach and emptied into the intestine from 0.6 min postadministration. Subsequently, the radioactivity in the stomach gradually disappeared within 10 min and then periodically migrated along the intestinal tract until 90 min (Figure 1A). In contrast, after IV bolus administration of [18F]FPEG solution, the radioactivity initially localized in the blood vessels, was then distributed in the kidneys and liver within 1 min, and finally moved to the urinary bladder via the urinary duct. Even after IV injection, radioactivity was seen, but to a lesser degree, in the intestine via biliary excretion (Figure 1B), which coincided with previous data on the tissue distribution of [18F]FPEG determined using a planar positron imaging system.18 In addition, the radioactivity was also found in bone region within 1 min after IV bolus administration. Radioactivity in the kidneys and urinary bladder was not observed in PET images of orally administered [18F]FPEG. Blood radioactivity−time profiles after oral or IV bolus administration of [18F]FPEG are shown in Figure 2. PK

Figure 2. Time−radioactivity curves in the blood after oral and intravenous administration of [18F]FPEG to rats. Time profiles were determined by blood sampling over 90 min following oral administration (open symbols) or intravenous bolus administration (closed symbols) of [18F]FPEG. Each symbol with a bar represents the mean ± SD (n = 3 rats).

parameters were calculated from its time course and are summarized in Table 1. After IV bolus administration of [18F]FPEG, the radioactivity in the blood at 2 min was 1.6 ± 0.3% of the dose/mL and then decreased rapidly until 90 min. On the other hand, the radioactivity in the blood after oral administration of [18F]FPEG reached its maximum within 10 min but was very low (0.014 ± 0.008% of dose/mL). The calculated bioavailability of [18F]FPEG was 3.6 ± 2.5%. Time profiles of the radioactivity in the stomach and the intestine after oral administration of [18F]FPEG solution are shown in Figure 3. At 5 min after administration, approximately 95% of the radioactivity administered to the stomach had disappeared, and the radioactivity in the small intestine sharply increased and reached close to a plateau but slightly increased until the end of the scan. Estimation of the Effective Fluid Volume in the Gastrointestinal Lumen. The effective fluid volume relating to the drug distribution in the GI tract was calculated after oral administration of [18F]FPEG solution to rats (Figure 4). VOIs 2264



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Assessment of Radiometabolite Components in Biological Samples. Figure 5A shows a radiochromatogram of the intestinal luminal contents and scraped intestinal epithelia sampled at 100 min after oral administration of [18F]FPEG solution to rats. The radioactivity was predominantly associated with unchanged [18F]FPEG in these samples. Radioactivity in blood after oral administration of [18F]FPEG was below the detection limit for radiometric HPLC. In addition, HPLC analysis revealed that almost 100% of the radioactivity in blood, urine, and bile sampled at 90 min represented unchanged [18F]FPEG after IV bolus administration of [18F]FPEG (Figure 5B). These results indicate that [18F]FPEG did not undergo any metabolism or degradation in the intestine but was rapidly eliminated from the systemic circulation mainly via renal excretion. Kinetic Analysis of the Transit in Each Gastrointestinal Segment Using Compartment Model. A GI transitabsorption model was constructed to analyze time profiles of the radioactivity in each GI segment after the oral administration of [18F]FPEG to rats (Figure 6). The GI tract was divided into four segments, the stomach, the duodenum, the jejunum (proximal intestine), and the ileum (distal intestine), and time profiles of the radioactivity in each segment were fitted to the model (eqs 1−4) to obtain rate constants for GI transit. The fitting curves for each segment were in good agreement with the observed data in each individual (Figure 7). The corresponding GI transit parameters are summarized in Table 2. The half-life of elimination from the stomach was 1.1 ± 0.2 min, and the rate constant of elimination from the stomach (kel,sto) was calculated as 0.64 ± 0.11 min−1 from eq 1, which was comparable to kin,duo, the rate constant (0.68 ± 0.77 min−1) determined from eq 2. The rate constants related to the intestinal transit from the duodenum to the jejunum (kel,duo and kin,jej) were calculated as 0.32 ± 0.17 min−1 and 0.51 ± 0.26 min−1, from eqs 2 and 3, respectively. In addition, the transit rates determined from the jejunum to the ileum (kel,jej and kin,ile) were 0.014 ± 0.009 min−1 and 0.014 ± 0.004 min−1, from eqs 3 and 4, respectively. The MRTs of radioactivity in the stomach, the duodenum, and the jejunum were calculated as 1.6 ± 0.4 min, 11 ± 2 min, and 78 ± 51 min, respectively. MRT in the ileum could not be calculated because the elimination phase of radioactivity was not observed during the scan.

Table 1. Pharmacokinetic Parameters from Time Profiles of Blood Radioactivity after Intravenous and Oral Administrations of [18F]FPEG in Ratsa administration route pharmacokinetic parameter

IV bolus

oral

AUCblood,0−90min (%dose × min/mL) AUCblood,0‑infinity (%dose × min/mL) C0 (% of dose/mL) Cmax (% of dose/mL) Tmax (min) elimination T1/2 (min) CLtotal (mL/min/kg) Vd (mL/kg) bioavailability (%)

28 ± 7 35 ± 17 2.4 ± 0.4 N.C. N.C. 41 ± 26 17 ± 5 872 ± 249 N.C.

1.0 ± 0.6 2.6 ± 1.9 N.C. 0.0029 ± 0.0019 15 ± 10 113 ± 41 N.C. N.C. 3.5 ± 2.2

Each value represents the mean ± S.D. (n = 3−4). N.C.: Not calculated. a

Figure 3. Time−radioactivity curves determined by PET images of the stomach and the intestine after oral administration of [18F]FPEG to rats. The inset of each graph shows the detailed radioactivity profiles in the early time period of the stomach and the intestine. Each symbol with a bar represents the mean ± SD (n = 3 rats).

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DISCUSSION PET has great advantages in that the PK of drugs can be noninvasively determined on the basis of the drug concentration in tissue at very high sensitivity with good spatiotemporal resolution. Our previous PET studies using [18F]FDG, highly absorbable PET probe in the GI tract, demonstrated the utility of PET for visualizing and kinetically analyzing gastric emptying and drug absorption in the entire small intestine.11,12 In the present study, PET studies with [18F]FPEG were designed for further kinetic examination of the transit of GI fluid as well as its effective volume in the GI tract after oral administration of water containing a radiotracer. The pharmacokinetic analysis of blood radioactivity revealed that the bioavailability of [18F]FPEG was quite low (3.6 ± 2.5%), and the PET images showed that radioactivity was observed exclusively in the GI tracts after oral administration of [18F]FPEG. Furthermore, the radioactivity in the intestine was predominantly associated with unchanged [18F]FPEG, indicating that neither intestinal enzymatic degradation nor bacterial

Figure 4. Time course of the effective fluid volume in the gastric and intestinal lumen. The effective fluid volume related to the regional distribution of radioactivity in the stomach and the intestine was estimated by the volume of interest (VOI) determined by PET image analysis after oral administration of [18F]FPEG to rats. VOIs of each region were identified as the volume of fluid in which the radiotracer was dissolved.

of each region of the GI tract in the PET scan were identified as the effective volume of the gastric or intestinal luminal fluid in which [18F]FPEG was dissolved. The volume of the fluid in the stomach showed its maximum value at 0.1 min (first frame of the scan) as 2.4 ± 0.1 mL and decreased rapidly thereafter. The volume in the intestinal region peaked at 4.6 ± 0.8 mL at 4.6 min postdose and then declined until 15 min and kept a constant level of around 3.0 mL until the end of the scan. 2265



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Figure 5. Representative HPLC chromatograms of biological samples extracted from rats after oral or intravenous administration of [18F]FPEG. (A) Radiochromatograms of the extracts of intestinal contents (left) and intestinal epithelia scraped from the intestinal lumen (right) at 100 min after oral administration of [18F]FPEG. (B) Radiochromatograms of blood at 90 min (left), urine extracts sampled from urinary bladder at 100 min (center), and bile extracts sampled from the cannula inserted into the bile duct during 60−90 min (right) after IV bolus injection of [18F]FPEG to rats.

Figure 6. Pharmacokinetic model for analyzing the GI transit and the intestinal absorption after oral administration of [18F]FPEG to rats. GI transit constants in each segment were determined by fitting using eqs 1−4. Since radioactivity was not observed in the colon during the PET scan, the elimination rate constants of the ileum (kel,ile) and the rate constant of the intestinal transit into the colon (kin,co) were not determined in this study.

Figure 7. Time−radioactivity curves in each GI segment after oral administration of [18F]FPEG to rats. The segmental GI transit-absorption model was investigated, and the figures show the observed data and their fitting curves of three individual rats (a, b, and c) after oral administration of [18F]FPEG. Symbols are as follows: stomach (□), duodenum (■), jejunum (○), and ileum (●). Calculated time courses of the percent of dosed total radioactivity in each GI segment are expressed by solid lines.

Table 2. Parameters on GI Transit after Oral Administration of [18F]FPEG to Rats GI transit rate constant (min−1) eq 1 mean ± SD

eq 2

eq 3

eq 4

kel,sto

kin,duo

kel,duo

kin,jej

kel.jej

kin,ile

0.64 ± 0.11

0.68 ± 0.77

0.32 ± 0.17

0.51 ± 0.26

0.014 ± 0.009

0.014 ± 0.004

in the liver was negligible. The radioactivity was seen in bone region within 1 min but not after 2 min of IV bolus administration (Figure 1B). There was no obvious accumulation in any bone region in the abdominal PET images after

metabolism in the intestine participated after oral administration. The fact that the radioactivity in the blood, urine, and bile obtained after IV bolus administration also represented unchanged [18F]FPEG suggested that the first-pass metabolism 2266



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parameter. The ingested water volume (250 mL) is sometimes used in humans to calculate the drug concentration in the GI tract;22 however, it differs from the actual intestinal fluid volume owing to the secretion of fluids, such as saliva, gastric fluid, bile, and pancreatic fluid, and the absorption of water. In rats, water content in the GI tract was reported to be 3.2 ± 1.8 mL in fasted conditions by McConnell et al.23 However in their method, a large number of rats had to be sacrificed, and the dynamic analysis of the volume from individual animals is impossible.23 For these reasons, a novel method to estimate the effective fluid volume in the GI tract was desired, which should be noninvasive and independent of the drug and the subject/ patient. The VOIs in the stomach decreased rapidly in accordance with decreasing radioactivity. The volume of VOIs in the stomach determined in the present study was 2.4 ± 0.1 mL at 0.1 min postadministration, which was much higher than the volume of the administered solution (0.5 mL). It is assumed that there is some fluid in the gastric membrane when the animal is given free access to water; therefore, the radioactivity would immediately spread into the surface of the gastric mucosa or epithelium. Because of the spherical cavity shape of stomach, radioactivity of the surface of the gastric membrane may easily increase the apparent volume of VOIs by spillover effect at initial period. On the other hand, the volume of VOIs in the intestine determined in the present study showed that these consecutive volume changes were observed with the radiotracer moving toward the distal region. In the initial period, the volume of VOIs in the intestine peaked at around 4.6 mL. This transient increase of the volume in the intestine may have been caused by the rapid inflow of [18F]FPEG solution from the stomach in the early time period. After the transient increase, the volume was kept constant at around 3.0 mL, which was comparable to the water content in GI tract observed by McConnell et al.23 As a consequence, this study revealed that the use of VOIs helps to understand the time-dependent changes in relative fluid volume in the stomach and, moreover, dynamic changes in the effective volume in the small intestine after oral administration. Considering the distribution of [18F]FPEG after IV bolus injection, the radioactivity was rapidly excreted into the urine, which is thought to have occurred via glomerular filtration. On the other hand, to a lesser but still measurable extent, radioactivity was seen in the intestine owing to biliary excretion (Figure 1B). Roma et al. reported that, unlike sucrose, PEG-900 excretion is not associated with canalicular water movements but may be related to a vesicular transport process followed by a bile acid-stimulated discharge of secretory vesicles into bile through the lysosomal compartment.24 The use of a PET study with [18F]FPEG may also help to elucidate the detailed mechanisms of hepatobiliary transport of PEG. Recently, PEGs have been widely employed in pharmaceutical applications. The covalent attachment of PEGs to a drug or a therapeutic protein, called PEGylation, contributed to the avoidance of side effects and also to reduction of the frequency of administration, such as PEGylated Escherichia coli Lasparaginase (ONCASPAR) for the treatment of acute lymphoblastic leukemia25 and PEGylated antivascular endothelial growth factor RNA aptamer for the treatment of age-related macular degeneration (MACUGEN).26,27 PEGylation technology has also been applied to low-molecular-weight drugs.28,29 Moreover, PEGylation is also useful for achieving a longer circulation of drugs or drug carriers in the bloodstream by preventing uptake by the reticuloendothelial system in the

oral administration (Figure 1), indicating that the presence of free 18F− was negligible. These results indicate that [18F]FPEG can be used as a nonabsorbable and nonmetabolized marker for evaluation of the physiological parameters of the GI tract. Dynamic PET image analysis of the abdominal region in rats showed that orally administered solution containing [18F]FPEG rapidly passed through the stomach. After the radioactivity spread into the proximal small intestine, from the duodenum to the jejunum, it dynamically moved toward the ileum region without a change in its intensity (Figures 1 and 3). The detailed VOI definition in the GI region enabled kinetic analysis of the GI fluid transit in four segments, namely, the stomach, the duodenum, the jejunum, and the ileum. The kinetic analysis of the transit in the stomach and the intestine revealed that the elimination of [18F]FPEG in the stomach was predominantly derived from the gastric emptying due to kel,sto comparable to kin,duo. The half-life of the elimination in the stomach was 1.1 ± 0.2 min, which was faster than that observed in our previous PET study with [18F]FDG (3−4 min) in rats12 or in other studies with unabsorbed marker, [99mTc]DTPA, using gammascintigraphy.8,9 It is considered that the rapid gastric emptying of [18F]FPEG may be caused by its higher water-binding capacity and its weak interaction with the gastric mucosa or epithelium compared with other compounds. Moreover, kel,duo and kel,jej were comparable to kin,jej and kin,ile, respectively, which also shows that the elimination from the duodenum and the jejunum was derived from the intestinal transit, with intestinal absorption making less of a contribution. Therefore, the transit of radioactivity after oral administration of [18F]FPEG solution may represent the transit of GI fluid itself, which is a fundamental parameter of GI physiology and of importance when considering the transit of orally administered drugs as liquid or solid formulations. The order of intestinal transit constants was as follows: kel,sto (kin,duo) > kel,duo (kin,jej) > kel,jej (kin,ile), indicating that the transit rate was higher in the proximal region of the small intestine than the distal one. MRTs in the duodenum and the jejunum were calculated as 11 ± 2 min and 78 ± 51 min, respectively. MRT in the ileum could not be calculated because elimination of the radioactivity was not observed during the scan, but it is considered that MRT in the ileum would be much longer than that in the jejunum. Lennernas et al. reported that the small intestinal transit time of 14C-PEG4000 solution was more than 3 h and the transit rate was higher in the proximal part of the small intestine than in the distal part,20 which is comparable to our results. The GI transit rate constants obtained from this study were also comparable to those reported from gammascintigraphic analysis using [99mTc]DTPA (0.37 ± 0.22 min−1 for the stomach and 0.016 ± 0.006 min−1 for the jejunum). Gamma-scintigraphic analysis has been introduced for evaluation of the GI transit in experimental animals and clinical practice8,9,21 and is considered to help in elucidating the cause of variable absorption kinetics regulated by GI transit. In addition to this, PET image analysis allows the analysis of both the compartmental absorption and the GI transit based on the radioactivity of the administered drug itself and is applicable to the absorption model considering both segmental transit and drug absorption. We also demonstrated that the PET analysis enables the estimation of parameters relating to the volume of fluid presenting in the GI lumen in vivo, in which a considerable amount of drug dissolved. The volume of VOIs defined from PET images of each GI segment was calculated to estimate this 2267



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spleen and the liver, which facilitates access to a tumor via the enhanced permeability and retention (EPR) effect. Therefore, extensive application of PET image analysis using [18F]FPEG, particularly PET studies in humans, is expected not only to lead to the development of methods to evaluate the GI behavior of orally administered drugs, but also to provide basic information concerning the pharmacokinetics of [18F]FPEG for the development of PEGylated medicines. An extended singledose toxicity study of FPEG in rats was completed, which is mandatory for clinical trials with a single microdose, and no toxic effects and behaviors were observed in any of the assays used up to a dose 1000-fold higher than the estimated dose in human PET study (data not shown), showing extensive applicability of [18F]FPEG for PET study in humans. In conclusion, we have demonstrated that PET imaging analysis is applicable for separate evaluation of GI transit and absorption and also enables investigation of the parameters related to the volume of fluid presenting in the GI lumen in vivo. The present study on rats shows the feasibility of PET to investigate the pharmacokinetics in GI absorption behavior in humans in detail.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-78-304-7124. Fax: +81-78-304-7126. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was carried out as part of the Research Project for the “Establishment of Evolutional Drug Development with the Use of Microdose Clinical Trial” sponsored by the New Energy and Industrial Technology Development Organization (NEDO). Part of this work was also supported by a Grantin-Aid for Young Scientists (B) on KAKENHI (23790214) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese government (to T.T.). We thank Mr. Masahiro Kurahashi of Sumitomo Heavy Industry Accelerator Service Ltd. for operation of the cyclotron, Aya Mawatari for supporting the labeling synthesis of the radiotracer, and Tsuneo Yano for supervision of this study. We also express our appreciation to NOF Corporation for providing us with polyethylene glycols.

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REFERENCES

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