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NEUROPHARMACOLOGY

Pharmacokinetic Analysis of the Blood-Brain Barrier Transport of ¹²⁵I-Amyloid Protein 40 in Wild-Type and Alzheimer's Disease Transgenic Mice (APP,PS1) and Its Implications for Amyloid Plaque Formation

Molecular Neurobiology Laboratory, Departments of Neurology, Neuroscience, and Biochemistry/Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota

Received December 8, 2004; accepted February 25, 2005.

	Abstract

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Amyloid plaques are formed in the extracellular space of Alzheimer's disease (AD) brain due to the accumulation of amyloid (A) proteins such as A40. The relationship between A40 pharmacokinetics and its accumulation within and clearance from the brain in both wild-type (WT) and AD transgenic mice (APP,PS1) was studied to understand the mechanism of amyloid plaque formation and the potential use of A40 as a probe to target and detect amyloid plaques. In both WT and APP,PS1 mice, the ¹²⁵I-A40 tracer exhibited biexponential disposition in plasma with very short first and second phase half-lives. The ¹²⁵I-A40 was significantly metabolized in the liver kidney > spleen. Coadministration of exogenous A40 inhibited the plasma clearance and the uptake of ¹²⁵I-A40 at the blood-brain barrier (BBB) in WT animals but did not affect its elimination from the brain. The ¹²⁵I-A40 was shown to be metabolized within and effluxed from the brain parenchyma. The rate of efflux from APP,PS1 brain slices was substantially lower compared with WT brain slices. Since the A40 receptor at the BBB can be easily saturated, the blood-to-brain transport of A40 is less likely to be a primary contributor to the amyloid plaque formation in APP,PS1 mice. The decreased elimination of A40 from the brain is most likely responsible for the amyloid plaque formation in the brain of APP,PS1 mice. Furthermore, inadequate targeting of A40 to amyloid plaques, despite its high BBB permeability, is due to the saturability of A40 transporter at the BBB and its metabolism and efflux from the brain.

In recent years, substantial effort has focused on the development of a pre-mortem diagnosis of AD, which involves detection of the plaques using various imaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography. MRI used in conjunction with a contrast agent can resolve individual plaques and has the capability of differentiating plaques from other interfering structures such as blood vessels, myelinated fibers, and intracranial structures (Poduslo et al., 2002). Of the MRI contrast agents that are currently being developed for imaging amyloid plaques, the most notable is the amyloid protein itself, mostly A40 (Wengenack et al., 2000a; Lee et al., 2002; Poduslo et al., 2002, 2004; Wadghiri et al., 2003). ¹²⁵I-A40 was reported to have high binding affinity to the amyloid plaques in human and double transgenic AD mouse brain slices in vitro and high in vivo permeability at the BBB; however, the plaque targeting ability of ¹²⁵I-A40 after i.v. injection in AD transgenic mice was low (Wengenack et al., 2000a). Hence, methods such as modifying the protein so that it can be actively transported across the BBB have been used to increase the targeting of A40 after i.v. injection (Wengenack et al., 2000a; Poduslo et al., 2002, 2004). The inadequate targeting of A40 to amyloid plaques despite its high permeability at the BBB could be due in part to 1) rapid elimination of A40 from the systemic circulation, which leads to a reduction in its concentration at the BBB; 2) inadequate transcytosis across the capillary endothelium; 3) competing high level of efflux from the brain parenchyma; or 4) metabolism in the brain or uptake by various cells, which can deplete A40 concentrations from the extracellular space.

Some of these questions are addressed in the present study using mechanism-based pharmacokinetic experiments using a tracer (¹²⁵I-A40) in both wild-type (WT) and AD transgenic mice (APP,PS1). Our investigation also addresses questions regarding the relationship between endogenous A40 and the kinetics of A accumulation and clearance from the brain. Such information is not only helpful in elucidating the disease pathology but also in optimizing the delivery of diagnostic probes derived from A (Poduslo et al., 2004).

	Materials and Methods

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Animals. The double transgenic mice were bred in our mouse colony at Mayo. Hemizygous transgenic mice (mouse strain C57B6/SJL; i.d. no. Tg2576) expressing mutant human amyloid precursor protein (APP₆₉₅) (Hsiao et al., 1996) were mated with a second strain of hemizygous transgenic mice (mouse strain Swiss-Webster/B6D2; i.d. no. M146L6.2) expressing mutant human presenilin 1 (PS1) (Holcomb et al., 1998). These double transgenic mice have been shown to exhibit an accelerated phenotype with amyloid deposits and behavioral deficits by 12 weeks of age (Holcomb et al., 1998; Wengenack et al., 2000b). WT mice (B6/SJL) were obtained from The Jackson Laboratory (Bar Harbor, ME) at 6 to 8 weeks of age and were the same background strain as the transgenic mice. The animals were housed in a virus-free, indoor, light- and temperature-controlled barrier environment and were provided ad libitum access to food and water. All procedures with animals were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Mayo Institutional Animal Care and Use Committee.

Synthesis of A40. Human A40 was synthesized by the Mayo Protein Core Facility (Rochester, MN) on an ABI 433A peptide synthesizer (Applied Biosystems, Foster City, CA), using standard solid phase methods and procedures. Each peptide was purified by reverse phase high-performance liquid chromatography on a Jupiter C18 column (250 x 21.2 mm, 15µ; Phenomenex, Torrance, CA) in 0.1% trifluoroacetic acid/water with a 50-min gradient from 10 to 70% acetonitrile/0.1% trifluoroacetic acid. The integrity of the protein was verified by electrospray ionization mass analysis on a PerkinElmer Sciex API 165 mass spectrometer (Applied Biosystems, Foster City, CA). Protein concentration was determined using a bicinchoninic acid protein assay reagent kit (Pierce Chemical, Rockford, IL) and bovine serum albumin (BSA) standard.

Radioiodination of Proteins. Carrier-free Na¹²⁵I and Na¹³¹I were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Human A40 (500 µg) and BSA (500 µg) were labeled with ¹²⁵I and ¹³¹I, respectively, using the chloramine-T procedure as described previously (Poduslo et al., 1994). Free radioactive iodine was separated from the radiolabeled protein by dialysis against 0.01 M phosphate-buffered saline at pH 7.4 (Sigma-Aldrich, St. Louis, MO). Purity of the radiolabeled proteins was determined by trichloroacetic acid (TCA) precipitation. The radiolabeled protein was determined to be acceptable if the TCA precipitable counts were greater than 95% of the total counts. The final radioactivity associated with ¹²⁵I labeled A40 was determined to be 4 mCi/mg protein.

A40 Pharmacokinetic Studies. Before the beginning of experiment each mouse was weighed (WT = 18–21 g; APP,PS1 = 20–23 g), and the femoral vein and artery were catheterized under general anesthesia (isoflurane = 1.5% and oxygen = 4 l/min). The ¹²⁵I-A40 (100 µCi; 100 µl) was administered intravenously in the femoral vein. Blood was sampled (20 µl) from the femoral artery at various intervals. At the end of the experiment, an aliquot of ¹³¹I-BSA (100 µCi; 100 µl) was injected to serve as a measure of residual plasma volume (V_p). One minute after the ¹³¹I-BSA injection, the final blood sample was collected, and the animal was sacrificed. The blood samples, diluted to a volume of 100 µl using normal saline, were centrifuged, and the supernatant was obtained. After TCA precipitation, the samples were assayed for ¹²⁵I and ¹³¹I radioactivity in a two-channel gamma counter (Cobra II; Amersham Biosciences Inc., Piscataway, NJ). The measured activity was corrected for the background and crossover of ¹³¹I activity into the ¹²⁵I channel.

The plasma pharmacokinetics of ¹²⁵I-A40 was determined by collecting serial blood samples (20 µl) from the femoral artery over a period of 15 min at time points of 0.25, 1, 3, 5, 10, and 15 min. The accumulation of ¹²⁵I-A40 in the peripheral organs such as liver, kidney, and spleen was determined by perfusing the animals with PBS at the end of the experiment. The linearity of ¹²⁵I-A40 disposition was determined by repeating the experiment by coadministering 1 or 2 mg of cold A40 with 100 µCi of ¹²⁵I-A40.

The brain uptake studies of ¹²⁵I-A40 were conducted by collecting serial blood samples (20 µl) from the femoral artery over a period of 15 min at time points 0.25, 1, 3, 5, 10, and 15 min. At the end of the experiment, the brain of the animal was removed from the cranial cavity; dissected into the anatomical regions, cortex, caudate putamen, hippocampus, thalamus, brain stem, and cerebellum; and assayed for ¹²⁵I and ¹³¹I radioactivity. The brain regions were lyophilized, and dry weights were determined with a microbalance and converted to wet weights using wet weight/dry weight ratios determined previously. The saturability of ¹²⁵I-A40 transport at the BBB was determined by coadministering 0.5, 1, or 2 mg of cold A40 with 100 µCi of ¹²⁵I-A40.

To determine the influence of high circulating levels of A40 on the elimination of ¹²⁵I-A40 from the brain, 100 µCi of ¹²⁵I-A40 was administered to the animal intravenously followed by four i.v. bolus injections of 0.5 mg of cold A40 at 15-(T_max of ¹²⁵I-A40 in the brain), 30-, 45-, and 60-min intervals. Blood samples were collected over a period of 60 min at time points 0.25, 1, 3, 5, 10, 15, 30, 45, and 60 min. At the end of 90 min, ¹³¹I-BSA was administered; the animal was sacrificed a minute later to obtain the brain regions, which were assayed for ¹²⁵I and ¹³¹I radioactivity as described above.

Metabolism of ¹²⁵I-A40. To determine the metabolism of ¹²⁵I-A40 in the plasma of WT or AD transgenic animals, 0.1 µCi of ¹²⁵I-A40 was added to 350 µl of plasma and incubated at 37°C. Aliquots from the mixture (20 µl) were taken at regular intervals up to 60 min, and the amount of intact ¹²⁵I-A40 was determined by TCA precipitation. The metabolism of ¹²⁵I-A40 in the presence of brain, liver, kidney, and spleen slices was determined by obtaining the organs from WT and AD transgenic mice after perfusion with PBS. The organs were weighed, cut into 1-mm-thick slices using a tissue slicer (Stoelting Co., Wood Dale, IL), and placed in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) prewarmed to 37°C. ¹²⁵I-A40 (0.1 µCi) was added to the medium containing tissue slices and maintained at 37°C under 5% CO₂ for the entire length of the experiment. Aliquots (20 µl) of the medium were obtained at regular intervals and assayed for the intact protein using TCA precipitation method.

Efflux of ¹²⁵I-A40 from Brain Slices. WT and APP,PS1 transgenic mice were killed by decapitation under general anesthesia. The brains were rapidly removed, washed with PBS, and cut into 1-mm-thick cortical slices, containing hippocampus, using the tissue slicer. After equilibrating in oxygenated (95% O₂/5% CO₂) Krebs-Ringer bicarbonate buffer (KRB) for 15 min at 37°C, each slice was incubated in 1 ml of donor solution (0.6 µCi of ¹²⁵I-A40 in 1 ml of KRB) at 37°C for 30 min. The loaded brain slices were washed with KRB, and the efflux rate of ¹²⁵I-A40 from each brain slice was determined by incubating it in 5 ml of receiver medium (KRB or KRB + 1 mM 2,4-dinitrophenol) at 37°C. The receiver medium was replaced every 30 min to maintain sink conditions. One brain slice was sampled at each time interval and assayed for ¹²⁵I radioactivity.

Data Analysis. The A40 plasma concentration profile after a single i.v. bolus dose of ¹²⁵I-A40 was best described by a biexponential disposition function:

(1)

where C(t) = ¹²⁵I-A40 microcuries per milliliter of plasma, A and B are the intercepts, and and are the slopes of the biexponential curve. Pharmacokinetic parameters were estimated by nonlinear curve fitting using Gauss-Newton (Levenberg and Hartley) algorithm and iterative reweighting (WinNonlin Professional, version 4.1; Pharsight, Mountain view, CA). Secondary parameters such as the maximum plasma concentration (C_max), the first and second phase half-lives [t_1/2() and t_1/2(), respectively], the plasma clearance (Cl), the steady-state volume of distribution (V_ss), and area under the plasma concentration curve (AUC) were also calculated using WinNonlin. The mean values of controls and treatments were compared by Student's t test using GraphPad Prism version 3.03 (GraphPad Software Inc., San Diego, CA).

The residual brain region plasma volume (V_p, microliters per gram) and the cerebrovascular permeability-surface area product (PS) values were calculated as described previously by Poduslo (1993).

(2)

where q_p is the ¹³¹I-BSA content (cpm) of tissue, C_v is the ¹³¹I-BSA concentration (cpm per milliliter) in plasma, W is the dry weight (grams) of the brain region, and R is the wet weight/dry weight ratio for mice of a defined age group. From the total ¹²⁵I-A40 content (q_T) (cpm) of the brain region, the amount of ¹²⁵I-A40 that enters the brain region extravascular space (q) (cpm per gram) is calculated as follows:

(3)

where C_a is the final ¹²⁵I-A40 concentration (cpm per milliliter) in plasma. The PS (milliliters per gram per second) at the BBB is calculated as follows:

(4)

where t is the circulation time, q(t) is the extravascular ¹²⁵I activity in the brain region at time t, and ^t₀C_pdt is the plasma concentration time integral of ¹²⁵I-A40.

The rate of ¹²⁵I-A40 efflux (k) from the brain slices in vitro was determined by curve-fitting (one-phase exponential decay model) the amount of radioactivity retained in the brain slices versus time using GraphPad Prism version 3.03 as follows:

(5)

where Y_o is the amount of radioactivity (cpm) in the brain slice at 0 min, k is the decay rate constant, and t is the time in minutes. The adequacy of fit was determined by F-test.

The PS values of ¹²⁵I-A40 were plotted versus the log concentration (log C) of A40, and the inhibitor concentration 50% (IC₅₀) of A40 in various brain regions were calculated by fitting one-site competitive binding equation to the data using GraphPad Prism version 3.03:

(6)

where PS_max is the maximum PS value, which was obtained at the lowest A40 concentration. PS_min is the minimum PS value obtained at the highest A40 concentration.

	Results

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Recent reports have suggested that A40 exhibits bidirectional transport between the peripheral circulation and brain via the BBB (Shibata et al., 2000; Deane et al., 2003). According to Zlokovic (2004), such a bidirectional transport regulates the A40 equilibrium between central nervous system and peripheral circulation and contributes to the formation of amyloid plaques in the brain parenchyma. If peripherally circulating A40 can so directly impact AD pathogenesis, then it becomes important to study the plasma pharmacokinetics of ¹²⁵I-A40 in WT and AD mice. Differences in peripheral distribution and elimination between the two strains could have a profound influence on plasma and brain steady-state A concentrations, as well as on the time course of brain ¹²⁵I-A40 concentration after i.v. administration. The information obtained from such a comparative study could help optimize A40 delivery (or its derivatives) as a diagnostic probe and elucidate the physiological parameters responsible for plaque formation in AD mice.

¹²⁵I-A40 Plasma Pharmacokinetics and Metabolism. After i.v. administration, the ¹²⁵I-A40 concentration in the plasma of WT as well as APP,PS1 mice declined rapidly exhibiting a biexponential disposition with short first (t_1/2,) and second phase (t_1/2,) half-lives (Fig. 1A; Table 1). The plasma pharmacokinetic profile of ¹²⁵I-A40 in 8-week-old APP,PS1 mice (no amyloid plaque formation), was significantly different from that of the 8-week-old WT mice. However, this difference was not statistically significant when the animals were 24 weeks old (Fig. 1B; Table 1), an age with substantial amyloid burden (Wengenack et al., 2000b). Although no significant differences in the plasma pharmacokinetic parameters were observed between 8- and 24-week-old WT mice, significantly lower clearance and higher AUC was observed in 24-week-old APP,PS1 mice compared with that of 8-week-old mice (Table 1). These differences are most likely due to the increased A40 levels with age in APP,PS1 mice that could saturate peripheral elimination.

View larger version (17K): [in this window] [in a new window]   Fig. 1. A, plasma pharmacokinetics of 125I-A40 in WT and APP,PS1 mice at 8 weeks. Data are mean ± S.D. (n = 3); lines indicate the fit of the two-compartment pharmacokinetic model to the plasma concentrationtime data. B, plasma pharmacokinetics of 125I-A40 in WT (n = 6) and APP,PS1 (n = 3) mice at 24 weeks. Data are mean ± S.D; lines indicate the fit of the two-compartment pharmacokinetic model to the plasma concentration-time data.

View this table: [in this window] [in a new window]   TABLE 1 Plasma pharmacokinetic parameters of 125I-A40 in WT and APP,PS1 mice Data are mean ± S.D.

A substantial amount of ¹²⁵I-A40 was found in the liver, kidney, and spleen of both WT and APP,PS1 animals perfused with PBS at the termination of the experiment. The accumulation of ¹²⁵I-A40 was higher in the kidney than in the liver or spleen. Moreover, no significant differences in the accumulation of ¹²⁵I-A40 in these organs was observed between WT and APP,PS1 animals. To investigate the role of peripheral metabolism on the rapid elimination of ¹²⁵I-A40 from the systemic circulation, in vitro metabolism studies in the presence of plasma, liver, kidney, and spleen obtained from WT and APP,PS1 mice were conducted. Although the ¹²⁵I-A40 metabolism in the plasma was slightly higher than the degradation in Dulbecco's modified Eagle's medium at 37°C, less than 10% of the initial amount of ¹²⁵I-A40 was degraded in 60 min (Fig. 2). The metabolism of ¹²⁵I-A40 in the liver slices, however, was so rapid that A40 was degraded substantially before the initial sample (t = 0 min) could even be obtained, and proceeded to near completion by 60 min (Fig. 2). The metabolism in the APP,PS1 mouse peripheral tissues was not significantly different from that of WT mouse tissues. However, the ¹²⁵I-A40 metabolism in the brain slices of APP,PS1 mice is significantly lower compared with that in wild-type brain slices (Fig. 2).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Degradation of 125I-A40 in various tissues obtained from 24-week-old WT and APP,PS1 animals. Data are mean ± S.D. (n = 3).

To determine whether the elimination of ¹²⁵I-A40 from the peripheral circulation is saturable, the plasma kinetics of ¹²⁵I-A40 was studied by coadministering various amounts of unlabeled A40 (0.125–4 mg). When 0.125 mg of A40 was coadministered with 100 µCi of ¹²⁵I-A40, no significant changes in the plasma pharmacokinetic parameters were observed (Table 2). Upon coadministration of 1, 2, or 4 mg of A40, the pharmacokinetic parameters of clearance and V_ss of ¹²⁵I-A40 decreased significantly, whereas the AUC increased significantly (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Saturability of various plasma pharmacokinetic parameters in 24-week-old wild-type mice Data are mean ± S.D. Control experiments were performed by administering a 100-µCi bolus dose of 125I-A40 intravenously. Statistical significance was indicated by *p < 0.05 and **p < 0.01 using one-way analysis of variance.

¹²⁵I-A40 Brain Uptake. Upon i.v. administration in 24-week-old WT mice, ¹²⁵I-A40 showed rapid brain uptake (T_max of 15 min) (Fig. 3). ¹²⁵I-A40 pharmacokinetic profile in the brain was substantially different from that of the plasma. Elimination of ¹²⁵I-A40 from the brain was not as rapid as it was from the plasma.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Extravascular brain uptake and plasma pharmacokinetic profile of 125I-A40 in 24-week-old WT mice. Symbols represent observed data; line indicates the fit of the two-compartment pharmacokinetic model to the plasma concentration-time data.

View this table: [in this window] [in a new window]   TABLE 3 Effect of various amounts of A40 on the PS and Vp values of 125I-A40 at the BBB of wild-type mice (24 weeks) Data are mean ± S.D. According to two-way analysis of variance followed by Bonferroni post tests, PS values obtained after the coadministration of various amounts of A40 are significantly different (p < 0.05) from that of 125I-A40, whereas Vp values obtained after the coadministration of various amounts of A40 are not significantly different from that of 125I-A40 values, except those denoted by * (p > 0.05).

View larger version (26K): [in this window] [in a new window]   Fig. 4. A, effect of various doses of unlabeled A40 coadministered intravenously with 125I-A40 (100 µCi) on the permeability of 125I-A40 at the blood-brain barrier in 24-week-old WT mice. Each result represents the mean ± S.D. for three experiments. The differences between the control (125I-A40) and treatments (125I-A40 + various amounts of unlabeled A40) was determined to be statistically significant (p < 0.05) by two-way analysis of variance followed by Bonferroni post tests. B, inhibition of 125I-A40 permeability at the blood-brain barrier by various amounts of unlabeled A40 (cold) in 24-week-old WT mice. Data are mean ± S.D. (n = 3); curves represent predictions of the one-site competitive binding model.

The PS values of ¹²⁵I-A40 at the BBB in various brain regions of 8- and 24-week-old APP,PS1 transgenic mice were significantly lower than that in age matched WT mice (Table 4). There was no significant difference in the V_p values of ¹²⁵I-A40 in various brain regions except in hippocampus and brain stem (Table 4). These findings differ from that of our previous report where no differences in the PS values of ¹²⁵I-A40 between AD transgenic and normal mice were observed (Poduslo et al., 2001). In the previous study, we used ¹³¹I-A40 as vascular space marker, but in this study ¹³¹I-BSA was used to estimate the V_p values of ¹²⁵I-A40 in both WT and AD transgenic mice. Using the same protein labeled with a different isotope helps correct for potential artifacts such as nonspecific adherence to vessel walls and allows for an accurate estimation of residual brain plasma volume (V_p). However, further studies conducted in our laboratory demonstrated that rapid elimination of proteins such as A40 (unpublished data), whose plasma concentrations are reduced by 50% in 45 s, can lead to an underestimation of PS values. Moreover, differences in the circulating levels of endogenous A40 in WT and AD transgenic mice further confounds the determination of these parameters (Poduslo et al., 2001). Another reason for the differences between the two studies is the manner in which the blood samples were processed. In the former study, the TCA precipitation to quantify the intact protein was conducted on whole blood, whereas in the current study, the blood was diluted and centrifuged to remove cells, and the TCA precipitation was conducted on the diluted plasma.

View this table: [in this window] [in a new window]   TABLE 4 PS and Vp of A40 at the BBB in WT and APP,PS1 transgenic mice Data are mean ± S.D. Statistical significance was indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 using Student's t test.

The APP,PS1 mice at the age of 6 months develop distinct plaques, mostly in the cortex and hippocampus (Wengenack et al., 2000). They also carry significantly higher levels of A40 (13.9 pmol/ml) in the peripheral circulation compared with wild-type mice (1.07 pmol/ml) (Poduslo et al., 2001). If one of the primary elimination pathways of A40 from the brain involves efflux into peripheral circulation across the BBB via low-density lipoprotein receptor-related protein 1 located on the abluminal surface (Shibata et al., 2000), the higher levels of A40 in the peripheral circulation of the transgenic mice could reduce A40 efflux by saturating the efflux transporter. To investigate this hypothesis, a pharmacokinetic study based on the absorption and elimination profile of ¹²⁵I-A40 in the extravascular brain tissue (Fig. 3) was designed. A bolus dose of ¹²⁵I-A40 was administered to WT mice intravenously starting at the T_max of ¹²⁵I-A40 in the brain (15 min); four i.v. bolus doses of unlabeled A40, each 0.5 mg, were injected at 15-min intervals up to 60 min; and the brain levels of ¹²⁵I-A40 were then measured at the end of 90 min. Control experiments were performed on a similar set of three WT animals by injecting saline instead of unlabeled A40. The assumptions behind this experiment were as follows: 1) most of the uptake of ¹²⁵I-A40 into endothelial cells or brain parenchyma takes place during the absorption phase (0–15 min); and 2) if the efflux of the absorbed ¹²⁵I-A40 from brain is inhibited by higher concentrations of A40 (resulting from multiple bolus doses) on the luminal side, higher amounts of ¹²⁵I-A40 remain in the brain compared with the control.

The amount of ¹²⁵I-A40 in various brain regions of mice injected with unlabeled A40 (treatment) was not significantly different from the amount present in the brain regions of mice injected with saline (control) (Table 5). No significant differences were found in the V_p values as well. These results suggest that the higher concentration of A40 in the peripheral circulation does not significantly influence the brain efflux of ¹²⁵I-A40.

View this table: [in this window] [in a new window]   TABLE 5 Comparison of extravascular content and Vp values of 125I-A40 resulting from multiple dosing experiments in 24-week-old WT mice According to Student's t test, there is no statistically significant (N.S., p > 0.05) difference between the control and the treatment.

Efflux of ¹²⁵I-A40 from Brain Slices. In the past, investigators have demonstrated that intracerebrally injected ¹²⁵I-A40 was effluxed into CSF and blood (Ghersi-Egea et al., 1996; Shibata et al., 2000). Although the mechanism of efflux is still not clear, it is reasonable to assume that the transport across the brain parenchyma plays a significant role in the efflux of ¹²⁵I-A40 from brain. Hence, the efflux rate of ¹²⁵I-A40 from the brain slices of WT and APP,PS1 animals was determined in vitro. The efflux of ¹²⁵I-A40 from the brain slices of WT (in the presence and absence of 2,4-DNP) and APP,PS1 transgenic animals (24 weeks) was significantly different from each other (Fig. 5) (F-test, p < 0.0001). The efflux rate of ¹²⁵I-A40 from the WT mouse brain slices was 0.06 ± 0.01 min^-1 and was significantly higher than that of brain slices obtained from APP,PS1 transgenic animals (0.02 ± 0.009 min^-1) (mean ± S.E.M.). In the presence of a metabolic inhibitor like 2,4-DNP, which interferes with the energy metabolism, the efflux of ¹²⁵I-A40 from the WT mouse brain slices was reduced to 0.02 ± 0.003 min^-1 (mean ± S.E.M.).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Efflux of 125I-A40 from the 24-week-old WT (n = 6) and APP,PS1 (n = 3) brain slices in vitro; effect of 2,4-dinitrophenol on the efflux of 125I-A40 from WT brain slices. Data are mean ± S.D.; curves represent predictions of the one-phase exponential decay model.

	Discussion

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The objective of this work was to elucidate the role of absorption, distribution, metabolism, and elimination characteristics of A40 in the plasma and brain of APP,PS1 mice. This objective was accomplished with the resulting conclusions: 1) accumulation of A40 in the systemic circulation of APP,PS1 mice is not due to impaired plasma clearance; 2) neither A40 influx nor efflux across the BBB plays a pivotal role in the accumulation of A40 in the brain; and 3) accumulation of A40 in the brains of APP,PS1 animals is due to ineffective efflux in the brain parenchyma and/or reduced metabolism.

Our experiments demonstrated no major differences in the plasma pharmacokinetics or peripheral metabolism of ¹²⁵I-A40 between WT and APP,PS1 animals. The high level of circulating A40 in APP,PS1 animals could saturate the transporter at the BBB and hence contribute insignificantly to the amyloid plaque formation in the brain. Also, these kinetic experiments provided no direct evidence for the existence of A40 efflux transport at the BBB. On the other hand, ¹²⁵I-A40 efflux studies conducted in the brain slices, used as an vitro model for brain parenchymal transport demonstrated substantially higher efflux rate from WT brain slices compared with that of APP,PS1 brain slices. Moreover, the rate of ¹²⁵I-A40 degradation in the presence of APP,PS1 brain slices is substantially lower compared with that of WT brain slices. The conclusions from our experimental studies that lead to these key findings are further elaborated below.

After i.v. administration, our studies have demonstrated that the tracer ¹²⁵I-A40 was eliminated rapidly from the plasma. Such rapid clearance of A40 from the systemic circulation could adversely affect its permeability at the BBB and limit its utility as a diagnostic probe. The distribution and elimination half-lives of ¹²⁵I-A40 in WT mice are lower than those reported in nonhuman primate model of cerebral -amyloidosis (Mackic et al., 2002). Similar results are expected with AD transgenic mice used in this study, because they have higher plasma A40 levels than the WT animals (Poduslo et al., 2001). The higher plasma levels of A40 could saturate the receptors and/or enzymes involved with the distribution and metabolism of A40, resulting in higher distribution and elimination half-lives. Surprisingly, ¹²⁵I-A40 clearance and AUC were significantly higher in APP,PS1 mice (8 weeks) compared with that in 8-week-old WT mice. However, at 24 weeks of age, when the peripherally circulating A40 in APP,PS1 mice is 12 times higher than in WT mice (Poduslo et al., 2001), the ¹²⁵I-A40 plasma profiles were similar.

To investigate the effect of higher endogenous plasma A40 concentrations on the elimination of A40 from the plasma, ¹²⁵I-A40 was coadministered with various amounts of unlabeled A40 into the systemic circulation of WT mice. In the presence of high plasma concentrations of A40, the clearance and V_ss of ¹²⁵I-A40 decreased significantly, which suggest that ¹²⁵I-A40 exhibits nonlinear disposition in the systemic circulation. In vitro metabolism studies conducted using WT and APP,PS1 mouse tissue slices indicated that ¹²⁵I-A40 is substantially metabolized in liver and kidney. However, no significant difference in the extent of metabolism between WT and APP,PS1 animals was observed. These results demonstrate that the lower clearance of A40 in 24-week-old APP,PS1 mice compared with that in 8-week-old animals is due to the higher circulating A40 levels saturating the peripheral elimination. Despite the absence of any differences in the metabolism of A40 in the peripheral tissues such as plasma, liver, kidney, and spleen of APP,PS1 and WT mice, the increase in the plasma levels of A40 with age in APP,PS1 mice indicates that A40 is replenished at a rate faster than it is removed from the peripheral circulation. Excess A40 in the peripheral circulation could be due to overproduction by the peripheral tissues and/or probable continual efflux from the brain.

For A40 in the peripheral circulation to directly contribute to the plaque formation in the brain parenchyma, it has to be transported across the BBB. Several investigators have reported previously that A40 is actively transported at the BBB (Zlokovic et al., 1993; Poduslo et al., 1997, 1999). Brain pharmacokinetic profile obtained in the present study demonstrated that ¹²⁵I-A40 is absorbed at the cerebral vasculature very rapidly without any lag time, which is expected if the transport is receptor mediated. A decrease in the PS value of ¹²⁵I-A40 in the presence of various amounts of unlabeled A40 provides further evidence that ¹²⁵I-A40 exhibits receptor-mediated transport at the BBB.

Deane et al. (2003) claimed that RAGE mediates A transport across the BBB and accumulation in brain. They reported that RAGE is up-regulated in cerebral vasculature of patients with Alzheimer's disease and in AD mouse model and hypothesized that inhibition of RAGE at the BBB may limit accumulation of A in the brain. If the A40 transport across the BBB is mediated primarily by RAGE, we would expect to see higher ¹²⁵I-A40 PS values in APP,PS1 mice compared with WT mice consistent with the RAGE up-regulation. On the contrary, the current study demonstrates that the PS value of ¹²⁵I-A40 is significantly lower in APP,PS1 mice compared with WT mice most likely due to the saturation of uptake receptors at the BBB with increasing levels of A40 in the plasma.

Although researchers in the past have reported that ¹²⁵I-A40 have reasonable permeability at the BBB in APP,PS1 mice and nonhuman primates (Poduslo et al., 1999; Mackic et al., 2002), our laboratory and others have realized that the transport of A40 into brain parenchyma is poor (Wengenack et al., 2000a; Lee et al., 2002; Poduslo et al., 2002; Wadghiri et al., 2003). The PS value represents the rate at which a protein is transferred from the blood to the endothelial cell, but it offers no information on the amount of protein delivered to the brain parenchyma. Hence, care must be taken not to overinterpret this parameter. Wengenack et al. (2000a) reported that no detectable signal due to ¹²⁵I-A40 was observed on the plaques when 250 µg of ¹²⁵I-A40 was administered intravenously. Based on the pharmacokinetic parameters determined in this study, such a dose can produce plasma concentrations, at least 50 times greater than the physiological concentrations usually observed in APP,PS1 mice. On a similar note, Selkoe and colleagues (Craft et al., 2002), based on mathematical simulations, have reported that a 100-fold reduction in plasma to brain transport of A can only reduce the brain A burden by less than 0.2%. These studies indicate, therefore, that plasma-to-brain transport of A is not an important factor for plaque generation in the brain parenchyma of APP,PS1 transgenic mice. One may hypothesize, however, that very small amounts of A40 transported across the BBB over a long period can accumulate in high enough quantities to form plaques. This is kinetically feasible if the clearance mechanism of A40 from the brains of AD mice is impaired.

The clearance of A40 from the brain is known to be mediated by enzymes such as insulin degrading enzyme that metabolize the protein (Qiu et al., 1998) and the receptors (Shibata et al., 2000; Lam et al., 2001) that are involved in the efflux of the protein from central nervous system to plasma. Our in vitro efflux studies conducted in brain slices of 24-week-old normal and APP,PS1 transgenic mice demonstrated a significantly higher rate of ¹²⁵I-A40 efflux in the normal mice brain slices compared with the AD mice. The efflux rate decreased in the presence of 2,4-DNP (metabolic inhibitor), suggesting that ¹²⁵I-A40 efflux across the brain slices of normal mice is carrier-mediated. The lower rate of ¹²⁵I-A40 efflux across the brain slices of APP,PS1 mice could be due to 1) binding of ¹²⁵I-A40 to amyloid plaques, 2) impaired metabolism of ¹²⁵I-A40, or 3) inefficient efflux transport. More experiments are needed to elucidate the extent each of these factors contributes to the elimination kinetics of A40 from the brain.

It has been reported that A40 concentration in peripheral circulation and brain exist in dynamic equilibrium, with the A present in the peripheral circulation being transported to brain parenchyma to compensate for the reduction in CSF A levels due to plaque formation (DeMattos et al., 2002). Similarly, A from brain is transported to peripheral circulation if plasma A levels are depleted due to the presence of antibodies that could sequester A (DeMattos et al., 2002). Based on these studies, higher plasma concentrations of A40 in APP,PS1 animals could reduce the clearance of A40 from the brain. However, our experiments demonstrated that the clearance of ¹²⁵I-A40 from the brain is not affected by the high levels of peripheral A40, suggesting once again that the influx of A40 at the luminal surface of the cerebrovascular endothelium is not significant enough to impact the capacity of the efflux transporter supposedly located on the abluminal surface.

Our studies demonstrate that the higher level of A40 in the peripheral circulation in APP,PS1 mice compared with WT mice is not due to differences in the plasma, hepatic, or renal metabolism of the protein. The higher plasma concentration of exogenously added A40 in WT animals inhibited the uptake of ¹²⁵I-A40 at the BBB, but it did not affect its elimination from the brain. The decreased permeability of ¹²⁵I-A40 at the BBB in APP,PS1 animals compared with WT animals therefore is a direct result of higher A40 levels in the plasma. Since the A40 receptor at the BBB can be easily saturated, the blood-to-brain transport of A40 is less likely to be a primary contributor to the amyloid plaque formation in older (6-month) APP,PS1 animals, as claimed by other investigators. The ¹²⁵I-A40 was shown to be metabolized and effluxed in the brain parenchyma. The rate of ¹²⁵I-A40 efflux in APP,PS1 brain slices was substantially lower compared with WT brain slices. Also, the metabolism of ¹²⁵I-A40 was significantly lower in APP,PS1 brain slices compared with WT brain slices. Therefore, the decreased efflux of A40 from the brain and its possible decreased metabolism are reasonable explanations for the A accumulation and subsequent amyloid plaque formation in the brain of APP,PS1 transgenic mice. This study also demonstrates that inadequate targeting of A40 to amyloid plaques despite its high BBB permeability is due to the saturability of A40 transporter at the BBB. This saturability coupled with metabolism and efflux of A40 in the brain parenchyma significantly affects the plaque targeting of A40. The knowledge gained from these studies is being used in the development of new A derivatives with improved BBB permeability and plaque targeting.

	Acknowledgements

 
We thank Dr. Karen Duff for the PS1 transgenic mouse line and Dawn Gregor for excellent technical assistance.

	Footnotes

 
This work is supported by National Institute on Aging Grant AG22034 (to J.F.P.) and the Mayo Foundation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.081901.

ABBREVIATIONS: AD, Alzheimer's disease; A, amyloid ; CSF, cerebrospinal fluid; BBB, blood-brain barrier; RAGE, receptor for advanced glycation end products; MRI, magnetic resonance imaging; APP, amyloid precursor protein; PS1, presenilin 1; WT, wild-type; BSA, bovine serum albumin; TCA, trichloroacetic acid; PBS, phosphate-buffered saline; KRB, Krebs-Ringer bicarbonate; AUC, area under the plasma concentration curve; PS, cerebrovascular permeability coefficient-surface area product; 2,4-DNP, 2,4-dinitrophenol.

Address correspondence to: Dr. Joseph F. Poduslo, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. E-mail: poduslo.joseph{at}mayo.edu

	References

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