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NEUROPHARMACOLOGY

Physiological and Biophysical Factors That Influence Alzheimer's Disease Amyloid Plaque Targeting of Native and Putrescine Modified Human Amyloid 40

Molecular Neurobiology Laboratory, Departments of Neurology, Neuroscience, and Biochemistry/Molecular Biology (K.K.K., G.L.C., S.S.H., E.J.G., T.M.W., J.F.P.) and Department of Biochemistry/Molecular Biology (M.R.-A.), Mayo Clinic College of Medicine, Rochester, Minnesota

Received for publication September 15, 2005
Accepted February 27, 2006.

	Abstract

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Amyloid 40 (A40) and its derivatives are being developed as probes for the ante-mortem diagnosis of Alzheimer's disease. Putrescine-A40 (PUT-A40) showed better plaque targeting than the native A40, which was not solely explained by the differences in their blood-brain-barrier (BBB) permeabilities. The objective of this study was to elucidate the physiological and biophysical factors influencing the differential targeting of A40 and PUT-A40. Despite better plaque-targeting ability ¹²⁵I-PUT-A40 was more rapidly cleared from the systemic circulation than amyloid 40 labeled with ¹²⁵I (¹²⁵I-A40) after i.v. administration in mice. The BBB permeability of both compounds was inhibited by circulating peripheral A40 levels. ¹²⁵I-A40 but not ¹²⁵I-PUT-A40 was actively taken up by the mouse brain slices in vitro. Only fluorescein-A40, not fluorescein-PUT-A40, was localized in the brain parenchymal cells in vitro. The metabolism of ¹²⁵I-A40 in the brain slices was twice as great as ¹²⁵I-PUT-A40. ¹²⁵I-A40 efflux from the brain slices was saturable and found to be 5 times greater than that of ¹²⁵I-PUT-A40. Thioflavin-T fibrillogenesis assay demonstrated that PUT-A40 has a greater propensity to form insoluble fibrils compared with A40, most likely due to the ability of PUT-A40 to form sheet structure more readily than A40. These results demonstrate that the inadequate plaque targeting of A40 is due to cellular uptake, metabolism, and efflux from the brain parenchyma. Despite better plaque targeting of PUTA40, its propensity to form fibrils may render it less suitable for human use and thus allow increased focus on the development of novel derivatives of A with improved characteristics.

Currently, no method exists to image "individual" amyloid plaques in humans for a definitive and early diagnosis of AD. Radiolabeled molecular probes that bind to -amyloid plaques have recently been demonstrated both in vitro and in animal studies (Skovronsky et al., 2000; Wengenack et al., 2000a; Agdeppa et al., 2001; Bacskai et al., 2001). The most notable of these probes is the Pittsburgh compound-B, which allowed the researchers to visualize amyloid plaques by bulk tissue enhancement after positron emission tomography imaging for the first time in AD patients (Klunk et al., 2004). Although scintigraphic imaging of -amyloid plaques is promising, certain difficulties may be envisioned for clinical application. The most obvious is poor spatial resolution. The spatial resolution of clinically available tomographic scintigraphic techniques (positron emission tomography or single-photon emission computed tomography) is several times poorer than that of standard three-dimensional magnetic resonance imaging (MRI).

The ability of human A40 and putrescine-A40 (PUTA40) to selectively target amyloid plaques in AD transgenic mouse (APP,PS1) brain has provided an opportunity to use them as molecular probes to image amyloid plaques with MRI. ¹²⁵I-A40 was reported to have high in vitro binding affinity to the amyloid plaques in human and APP,PS1 mouse brain slices and high in vivo permeability at the blood-brain barrier (BBB) (Zlokovic et al., 1993; Maness et al., 1994; Poduslo et al., 1997, 1999; Mackic et al., 2002); however, the plaque-targeting ability of ¹²⁵I-A40 after i.v. injection in AD transgenic mice was low (Wengenack et al., 2000a). PUT-A40 was shown to have 2-fold higher BBB permeability than unmodified A40 and was able to target and label amyloid plaques in APP,PS1 animals after i.v. administration. When covalently linked to gadolinium diethylenetriaminepentaacetic acid, which provides contrast for MRI imaging, Gd-PUT-A40 was able to provide enough contrast to image amyloid plaques in APP,PS1 mice, whereas Gd-A40 failed to provide the required contrast (Poduslo et al., 2002). The differences in the efficacy of these compounds to label plaques ex vivo cannot be completely explained by the differences in their permeability values at the BBB.

Permeability at the BBB, endothelial transcytosis, as well as diffusion within and efflux from the brain parenchyma are important factors that determine the amount of A-probe available to target plaques. The proportion of A-probe in the extracellular space of the brain parenchyma versus intracellular uptake is another important consideration that will determine the successful targeting of amyloid plaques. Adequate knowledge of the transport kinetics of human A protein and its derivatives at the BBB and in the brain will allow us to design smart molecular probes capable of labeling plaques in Alzheimer's patients as well as provide further information regarding the dynamics of A transport in the progression of AD.

The objective of the current study was to investigate various factors affecting the in vivo uptake of A40 and PUTA40 at the BBB, accumulation within and efflux from the brain tissue in vitro, and binding to the amyloid plaques, as a means to explain the better targeting ability of PUT-A40 over native A40.

	Materials and Methods

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Synthesis of Human A40
A40 was synthesized on an ABI 433 peptide synthesizer (Applied Biosystems, Foster City, CA) using 2-(H-benzotriazol-1-yl)1,1,3,3-tetramethyl uranium hexafluorophate activation and the manufacturer's suggested synthesis protocols. The starting resin was Val-NovaSyn TGA resin (Calbiochem-Novabiochem, San Diego, CA). The peptides were cleaved from the solid support using 5% crystalline phenol, 5% water, 2.5% triisopropylsilane, and 87.5% trifluoroacetic acid for 2 h at room temperature and were purified by reverse-phase high-performance liquid chromatography using a heated 250- x 21.2-mm C18 Jupiter column (Phenomenex, Torrance, CA). The weights of the peptides were confirmed by electrospray ionization mass spectrometry (Sciex API 165; Applied Biosystems/MDS Sciex, Foster City, CA).

Synthesis of Fluorescein-Labeled A40 (F-A40)
After the final deprotection of the N-terminal N-fluoren-9-ylmethyloxycarbonyl group, the peptide resin was washed with 12% diisopropylethylamine/dichloromethane (DCM). N-Hydroxysuccinimide-fluorescein (0.2 mM; Pierce, Rockford, IL) was dissolved in 6 ml of dimethylformamide and added to the resin saturated with 12% diisopropylethylamine/DCM. The resin slurry was mixed overnight at room temperature, followed by several washes with dimethylformamide and DCM. The success of the fluorescein addition was confirmed by a negative ninhydrin reaction.

Putrescine Modification of A40 and F-A40
Putrescine modification of synthetic human A40 and F-A40 was performed by covalent linkage of the polyamine to carboxylic acid groups using carbodiimide at pH of 6.7 as described previously (Poduslo and Curran, 1996a,b).

Radioiodination of Proteins
Five hundred micrograms of human A40 or PUT-A40 was labeled with carrier-free Na¹²⁵I, whereas the bovine serum albumin (BSA) (500 µg) was labeled with carrier-free Na¹³¹I using the chloramine-T procedure as described previously (Poduslo et al., 2001). Free radioactive iodine was separated from the radiolabeled protein by dialysis against 0.01 M phosphate-buffered saline at pH 7.4 (Sigma-Aldrich Co., St. Louis, MO). Purity of the radiolabeled proteins was determined by trichloroacetic acid (TCA) precipitation. The radiolabeled protein was considered to be acceptable if the precipitable radioactive counts were greater than 95% of the total counts. The specific activity of the proteins thus obtained was determined as 2.0 ± 0.1 µCi/µg. No significant difference was observed between the specific activities of ¹²⁵I-A40 and ¹²⁵I-PUT-A40.

Animals
Wild-type (WT) mice (B6/SJL strain) were obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were housed in a virus-free barrier facility under a 12-h light/dark cycle, with ad libitum access to food and water. All the experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using protocols approved by the Mayo Institutional Animal Care and Use Committee.

PUT-A40 Pharmacokinetics Studies
Before the beginning of the experiment, the femoral vein and the femoral artery of each mouse were catheterized under general anesthesia (Poduslo et al., 2001). An i.v. bolus dose of ¹²⁵I-PUT-A40 (100 µCi in 100 µl) was administered through the femoral vein. Blood samples (20 µl) were obtained from the femoral artery at 0.25-, 1-, 3-, 5-, 10-, and 15-min intervals. At the end of the experiment, an i.v. bolus dose of ¹³¹I-BSA (100 µCi in 100 µl) was administered 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 were diluted to a volume of 100 µl using normal saline, centrifuged, and the supernatant was obtained. After TCA precipitation, the supernatant was assayed for ¹²⁵I and ¹³¹I radioactivity in a two-channel gamma counter (Cobra II; PerkinElmer Life and Analytical Sciences, Boston, MA). The measured activity was corrected for the background and crossover of ¹³¹I activity into the ¹²⁵I channel. At the end of the experiment, the brain of the animal was removed from the cranial cavity, dissected into cortex, caudate putamen, hippocampus, thalamus, brain stem, and cerebellum, and assayed for ¹²⁵I and ¹³¹I radioactivity. The effect of A40 on the ¹²⁵I-PUT-A40 uptake at the blood brain barrier was determined by both coadministering and sequential administration of 1 mg of unlabeled A40 with 100 µCi of ¹²⁵I-PUT-A40.

Metabolism of ¹²⁵I-PUT-A40
The metabolism of ¹²⁵I-PUT-A40 in the plasma of WT mice was determined by adding 0.1 µCi of ¹²⁵I-PUT-A40 to 350 µl of plasma. A 20-µl sample was taken from the mixture at regular intervals up to 60 min, and the amount of intact ¹²⁵I-PUT-A40 in the sample was determined by TCA precipitation. The metabolism of ¹²⁵I-PUT-A40 in the presence of brain, liver, kidney, and spleen slices was determined using the organs obtained from WT mice after perfusion with PBS. The organs were weighed, chopped into 1-mm-thick slices using a tissue slicer (Stoelting Co., Wood Dale, IL), and placed in oxygenated (95% O₂ and 5% CO₂) KRB until the start of the experiment. The tissue slices were then transferred to 5 ml of KRB containing 0.1 µCi of ¹²⁵I-PUT-A40 and incubated at 37°C under 5% CO₂ for the entire length of the experiment. Samples (20 µl) of the medium were obtained at regular intervals and assayed for the intact protein by TCA precipitation.

Brain Slices
After sacrificing with an overdose of sodium pentobarbital (200 mg/kg i.p.), the animals were decapitated, and the brains were carefully removed from the cranial cavity. Each brain was cut coronally into 1-mm-thick slices containing cortex and hippocampus. The slices were placed in oxygenated (95% O₂ and 5% CO₂) KRB until the start of the experiment.

Brain Slice Uptake of ¹²⁵I-A40 and ¹²⁵I-PUT-A40
Effect of Time. After the equilibration in KRB, each brain slice was incubated in 1 ml of donor solution containing ¹²⁵I-A40 (0.8 µCi/ml KRB) at 37°C. The brain slices were sampled at different time points (0, 10, 15, 30, 45, and 60 min), rinsed with KRB, and assayed for ¹²⁵I radioactivity in a two-channel gamma counter (Cobra II; PerkinElmer Life and Analytical Sciences).

Effect of Donor Concentration. After the equilibration, each brain slice was incubated at 37°C in a 1-ml donor solution containing different concentrations of ¹²⁵I-A40 (0.05-28.4 µCi/ml KRB). After 15 min, the brain slices were removed from the donor solutions, rinsed with KRB, and assayed for radioactivity using a dual-channel gamma counter.

Effects of Metabolic Inhibitors. After the equilibration, brain slices was preincubated with the metabolic inhibitors ouabain (1 mM) or 2,4-dinitrophenol (2,4-DNP) (1 mM) for 30 min. Each brain slice was then transferred to 1 ml of donor solution (preincubate + 0.6 µCi/1 ml of ¹²⁵I-A40 or PUT-A40) maintained at 37°C. After 15 min, the brain slices were removed, rinsed with KRB, and assayed for radioactivity in a two-channel gamma counter.

Cellular Accumulation of F-A40 and F-PUT-A40 in the Brain Parenchyma. Each brain slice was incubated in 1 ml of donor solution containing F-A40 or F-PUT-A40 (40 µg/ml KRB) at 37°C. After 15 min, the slice was removed from the donor solution, placed on a coverslip moistened with KRB, and imaged under a Zeiss LSM 410 (Carl Zeiss, Thornwood, NY) laser confocal microscope.

¹²⁵I-A40 and ¹²⁵I-PUT-A40 Efflux from Brain Slices
WT mice brain slices were equilibrated in KRB for 15 min at 37°C. Each slice was then incubated in a 1-ml donor solution containing various concentrations of ¹²⁵I-A40 or ¹²⁵I-PUT-A40 at 37°C for 30 min, washed with KRB, and incubated in 5 ml of KRB (receiving medium). The brain slices were harvested at various time points: 0, 10, 15, and 30 min, rinsed with KRB, and assayed for ¹²⁵I radioactivity. The initial rates of efflux (<15 min) of these compounds from the brain slices were calculated at various donor concentrations. The rates of efflux were normalized by the donor concentrations and plotted against log donor concentrations.

A40 and PUT-A40 Fibril Formation
Thioflavin T (THT) is a fluorescent dye that exhibits substantial enhancement in the fluorescence intensity upon binding to amyloid fibrils. Due to an excellent correlation between the THT fluorescence and the amount of amyloid fibrils, THT fibrillogenesis assay is widely used to quantify the amyloid fibril formation. In the current study, A40 and PUT-A40 fibril formation kinetics were followed using THT fibrillogenesis assay, which is detailed as follows: A40 and PUT-A40 were incubated at 37°C in TRIS-EDTA buffer (50 mM Tris, 5 mM EDTA, and 150 mM NaCl) under continuous shaking (200 rpm) for 24 h. A 60-µl aliquot of the reaction mixture was obtained at 0, 1, 2, 4, 6, 9, 12, 24, 48, 90, and 120 h, mixed with 1.2 ml of 5 µM THT in TRIS-EDTA and assayed for fluorescence (excitation , 450 nm; emission , 480 nm). The fluorescence intensity of each compound was normalized with the maximal fluorescence value of the compound and plotted against time.

Circular Dichroism Spectroscopy
The secondary structures of A40 and PUT-A40 were determined using circular dichroism (CD) spectroscopy. A 20 µM solution of each protein was prepared in PBS, pH 7.2, filtered using a 0.22-µm syringe filter, and transferred to a 0.2-cm path length quartz cuvette. The CD spectra of A40 and PUT-A40 were obtained from 260 to 200 nm at 4°C, scanning every 1 nm with an averaging time of 5 s, on an AVIV CD spectrometer model 215 (Aviv Instruments, Newington, NH).

Data Analysis
The ¹²⁵I-PUT-A40 plasma concentration profile after the single i.v. bolus dose of ¹²⁵I-A40 was best described by a biexponential disposition function C(t) = Ae^-t + Be^-t, where C(t) = ¹²⁵I-PUTA40 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 (Win-Nonlin Professional, version 4.1; Pharsight, Mountain View, CA). Secondary parameters such as the C_max (maximum plasma concentration), the first [t_1/2()] and the second [t_1/2()] phase half-lives, 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 4.0 (GraphPad Software, San Diego, CA).

Cerebrovascular permeability coefficient-surface area product (PS) and V_p measurements were calculated as described previously by Poduslo (1993). The residual brain region V_p (microliters per gram) is calculated as:

(1)

where q_p is the radioactivity (counts per minute) of ¹³¹I-BSA present in the tissue. C_v is the ¹³¹I-BSA concentration (counts per minute 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) (counts per minute) of the brain region, the amount of ¹²⁵I-A40 that enters the brain region extravascular space (q) (counts per minute per gram) is calculated as:

(2)

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

(3)

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 initial rates of efflux of ¹²⁵I-A40 and ¹²⁵I-PUT-A40 from the WT brain slices were normalized by the donor concentrations and plotted against log donor concentrations (log C). The IC₅₀ of ¹²⁵IA40 efflux from the brain slices in vitro was determined by fitting the following equation to the data using GraphPad Prism version 3.03 (GraphPad Software).

(4)

where NE_max is the maximum normalized efflux value, which was obtained at the lowest ¹²⁵I-A40 concentration. NE_min is the minimum NE value obtained at the highest ¹²⁵I-A40 concentration.

	Results

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The in vivo targeting abilities of molecular probes may be compromised because of rapid elimination from the systemic circulation, competitive inhibition by high circulating A levels in APP,PS1 mice, proteolytic degradation in the brain parenchyma, uptake and degradation by neurons, and efflux from the brain. In this study, we systematically studied and compared the influence of each of the above factors on the amyloid plaque targeting of A40 and PUT-A40.

¹²⁵I-PUT-A40 Plasma Pharmacokinetics
Upon i.v. administration, the ¹²⁵I-PUT-A40 concentrations in the plasma of WT mice declined rapidly, exhibiting a biexponential disposition with short and half-lives [t_1/2() and t_1/2(), respectively] (Fig. 1; Table 1). The C_max, t_1/2(), and AUC were significantly lower, whereas the Cl and V_ss were significantly higher for ¹²⁵I-PUT-A40 compared with previously reported values for ¹²⁵I-A40 (Kandimalla et al., 2005). These differences are most likely due to rapid clearance normally observed for cationized proteins (Dellian et al., 2000) or significantly higher metabolism of ¹²⁵I-PUT-A40 in the peripheral tissues compared with that of ¹²⁵I-A40.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Plasma pharmacokinetics of 125I-A40 [n = 12; from Kandimalla et al. (2005)] and 125I-PUT-A40 in 24-week-old WT mice (n = 3). 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 Comparison of the plasma pharmacokinetic parameters of 125I-A40 and 125I-PUT-A40 in 24-week-old WT mice Mean ± S.D. (n = 3); statistical significance was indicated by *, p < 0.05 and **, p < 0.01 using Student's t test; N.S., p > 0.05.

¹²⁵I-PUT-A40 Metabolism in the Peripheral Tissues
To investigate the contribution of peripheral tissue metabolism on the rapid elimination of ¹²⁵I-PUT-A40 from the systemic circulation, in vitro degradation of ¹²⁵I-PUT-A40 in the presence of WT mice tissue slices (liver, kidney, and spleen) as well as plasma was studied. Although ¹²⁵I-PUTA40 degradation in the WT plasma was slightly higher than the degradation in Dulbecco's modified Eagle's medium, not more than 10% of the initial amount of ¹²⁵I-PUT-A40 was degraded in 60 min (Fig. 2). The degradation of ¹²⁵I-PUTA40 in the presence of liver slices was similar to that of ¹²⁵I-A40, with substantial degradation in 60 min. The extent of ¹²⁵I-PUT-A40 degradation in the kidney and spleen was significantly lower compared with that of ¹²⁵I-A40 (Fig. 2). These results demonstrate that despite rapid clearance from the peripheral circulation compared with ¹²⁵I-A40, the metabolism of ¹²⁵I-PUT-A40 in the peripheral organs of elimination was significantly lower than that of ¹²⁵I-A40.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Degradation of 125IA40 and 125I-PUT-A40 in various tissues obtained from 24-week-old WT mice. Data are mean ± S.D. (n = 5); 125I-A40 data are from Kandimalla et al. (2005).

¹²⁵I-A40 Brain Uptake

View larger version (21K): [in this window] [in a new window]   Fig. 3. Effect of 1 mg (231 nM) of unlabeled A40 coadministered i.v. with 125I-PUT-A40 (100 µCi) on the PS and brain vascular volume (Vp) of 125I-PUT-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 PS values of control (125I-PUT-A40) and treatment (125IPUT-A40 + A40) was determined to be statistically significant (p < 0.05) by two-way analysis of variance followed by Bonferroni post tests.

Brain Slice Uptake Studies
After permeating the BBB, ¹²⁵I-A40 or ¹²⁵I-PUT-A40 must diffuse across the brain parenchyma to reach the plaque sites. The relative diffusivities of ¹²⁵I-A40 and ¹²⁵IPUT-A40 across the brain parenchyma may be directly related to their plaque-targeting capabilities. Hence, the uptake of ¹²⁵I-A40 and ¹²⁵I-PUT-A40 into WT mouse brain slices was determined in vitro.

Effect of Time. The uptake of ¹²⁵I-A40 into WT mouse brain slices was linear till 15 min and reached a plateau from 15 to 60 min (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effect of time on the uptake of 125I-A40 into 24-week-old WT brain slices in vitro. Data are mean ± S.D. (n = 5).

View larger version (13K): [in this window] [in a new window]   Fig. 5. Effect of donor concentration on the uptake of 125I-A40 into 24-week-old WT brain slices in vitro. Data are mean ± S.D. (n = 5). Curves represent predictions of Michaelis-Menten-type model fitted to the data.

Localization of F-A40 and PUT-A40 (F-PUT-A40) in the Brain Parenchyma.

View larger version (40K): [in this window] [in a new window]   Fig. 6. Accumulation of fluorescein-labeled A40 and fluorescein-labeled PUT-A40 in the neurons and extracellular space of WT mouse brain slices in vitro, respectively (20x).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effect of metabolic inhibitors on the uptake of 125I-A40 and 125I-PUT-A40 in 24-week-old WT brain slices in vitro. Data are mean ± S.D. (n = 5); *, p < 0.05, Student's t test.

¹²⁵I-A40 and ¹²⁵I-PUT-A40 Efflux from Brain Slices

View larger version (16K): [in this window] [in a new window]   Fig. 8. Effect of donor concentration on the efflux rates of 125I-A40 and 125I-PUT-A40 from 24-week-old WT brain slices in vitro. Initial rates of efflux from the brain slices were normalized by the donor concentrations and plotted against log donor concentrations. Curve represents prediction of one-site competitive binding model using GraphPad Prism version 3.03.

Biophysical Characterization of A40 and PUT-A40
CD Spectra. The CD spectra of A40 and PUT-A40 illustrated in Fig. 9 demonstrate that A40 has a random coil structure, whereas PUT-A40 assumes a -sheet structure in phosphate-buffered saline, pH 7.2, at 4°C.

View larger version (14K): [in this window] [in a new window]   Fig. 9. CD spectra of 125I-A40 and 125I-PUT-A40 in PBS, pH 7.2, at 4°C.

View larger version (13K): [in this window] [in a new window]   Fig. 10. Kinetics of 125I-A40 and 125I-PUT-A40 fibril formation. THT binds to the -sheet structure of the fibrils and emits fluorescence. The intensity of the fluorescence provides a direct measure of the extent of fibril formation.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
¹²⁵I-A40 and ¹²⁵I-PUT-A40 have demonstrated a differential ability to target amyloid plaques in APP,PS1 transgenic mice after i.v. injection. As a result, they are used in this study as model compounds to investigate various physiological factors (plasma clearance, BBB permeability, parenchymal diffusion and metabolism, and efflux from the central nervous system) and biophysical factors (effect of charge, ability to form fibrils, and affinity to plaques) that affect plaque targeting.

It is generally believed that lower plasma clearance resulting in high sustained plasma levels of the probe will enhance its BBB permeability. Our earlier studies demonstrated that ¹²⁵I-A40 is rapidly eliminated from the systemic circulation as a result of significant renal clearance and hepatic metabolism (Kandimalla et al., 2005). Despite better plaque-targeting ability, ¹²⁵I-PUT-A40 is more rapidly cleared from the systemic circulation than ¹²⁵I-A40 after i.v. administration. Unlike ¹²⁵I-A40, the rapid clearance of ¹²⁵I-PUT-A40 did not correlate with its metabolism in the peripheral tissues such as liver, kidney, and spleen. Although the metabolism of ¹²⁵I-PUT-A40 in the liver slices was only slightly lower compared with ¹²⁵I-A40, its metabolism in kidney slices was significantly lower than ¹²⁵I-A40. Because the molecular size of ¹²⁵I-PUT-A40 or ¹²⁵I-A40 is small, these molecules can be eliminated by the kidney without the need for initial catabolism in the liver. Moreover, the microvascular permeability of cationized proteins like ¹²⁵I-PUT-A40 was found to be substantially higher than the native proteins (Dellian et al., 2000), resulting in their increased renal clearance. Although higher microvascular permeability can increase the distribution of ¹²⁵I-PUT-A40 to brain (Poduslo and Curran, 1996b), increase in the renal clearance and rapid distribution to peripheral tissues could diminish the net effect. Hence, it is unlikely that the better plaque-targeting ability of ¹²⁵IPUT-A40 compared with ¹²⁵I-A40 is due to favorable plasma pharmacokinetics.

Like the BBB permeability of ¹²⁵I-A40, which decreased significantly in the presence of unlabeled A40 (0.125-2 mg) in a dose-dependent manner (Kandimalla et al., 2005), the BBB permeability of ¹²⁵I-PUT-A40 decreased when coadministered with 1 mg of A40. Even though the PS value of ¹²⁵I-PUT-A40 is higher than that of ¹²⁵I-A40 in WT mice, the PS value of ¹²⁵I-PUT-A40 coadministered with 1 mg of A40 was similar to the PS value of ¹²⁵I-A40 coadministered with 1 mg of A40. These results indicate that the BBB permeability of ¹²⁵I-PUT-A40 is also saturable. Therefore, endogenous A40 levels in the peripheral circulation of APP,PS1 animals, which were reported to increase linearly with age, could affect the BBB permeability of ¹²⁵I-PUT-A40 to a similar extent as ¹²⁵I-A40. Based on this information, it is likely that a modest 1.5- to 2.0-fold higher PS value of ¹²⁵IPUT-A40 compared with ¹²⁵I-A40 (Wengenack et al., 2000a) may, at the best, have a limited contribution to the differences in their plaque-targeting abilities.

Our earlier work demonstrated that the accumulation of ¹²⁵I-A40 in the extracellular space of APP,PS1 mouse brain is primarily influenced by the reduced clearance of A40, mediated by metabolism and/or efflux, from the brain parenchyma (Kandimalla et al., 2005). Mouse brain slices containing cortex and hippocampus were used in this study as in vitro model to investigate the role of parenchymal metabolism and uptake on the differential plaque-targeting ability of ¹²⁵I-A40 and ¹²⁵I-PUT-A40. This in vitro model has been used by several researchers to study the metabolism (Newman et al., 1990) and diffusion of ions (Newman et al., 1995) and small (Gredell et al., 2004) and large molecules (Patlak et al., 1998) in the brain parenchyma. Uptake of ¹²⁵I-A40 into WT mouse brain slices followed Michaelis-Menten kinetics, which suggested that the uptake is saturable. The inhibition of ¹²⁵I-A40 uptake in WT mouse brain slices by metabolic inhibitors such as 2,4-DNP and ouabain suggested that it is energy-dependent and most likely carrier-mediated. Preferential localization of F-A40 in the brain parenchymal cells, most likely neurons, after a short incubation time (5 min) further confirms the presence of carrier-mediated transport of A40.

In contrast, the metabolic inhibitors such as 2,4-DNP did not affect the uptake of ¹²⁵I-PUT-A40 significantly, thereby suggesting that the uptake of ¹²⁵I-PUT-A40 in WT mouse brain slices is most likely by passive diffusion. Localization of F-PUT-A40 in the extracellular space of the brain parenchyma but not in the cells provides further evidence that there is no receptor-mediated uptake of PUT-A40. The cellular uptake of A40 in the brain parenchyma makes it unavailable for targeting the plaques located in the extracellular space. In contrast, PUT-A40 is not taken up by the cells, remains in the extracellular space, and is available for binding to plaques, a desirable feature for imaging amyloid plaques.

In vitro fibril binding studies demonstrated that both ¹²⁵IA40 and ¹²⁵I-PUT-A40 bind to A40 fibrils with similar affinity (data not shown). However, the differences in the extracellular concentrations of the probe can alter plaque binding significantly. The extracellular concentration of ¹²⁵IA40 is not only reduced because of cellular uptake but also by the efflux and parenchymal metabolism. The degradation of ¹²⁵I-PUT-A40 in the WT mouse brain slices was significantly lower than the previously reported degradation of ¹²⁵I-A40 (Kandimalla et al., 2005). The efflux of ¹²⁵I-A40 across WT mouse brain slices was found to be saturable and inhibited by 2,4-DNP (Kandimalla et al., 2005), thereby suggesting that it is energy-dependent and carrier-mediated. The efflux of ¹²⁵I-PUT-A40, on the other hand, was much slower compared with that of ¹²⁵I-A40 and was not dependent upon the donor concentration, suggesting that it occurs via passive diffusion.

Despite its success in providing contrast enhancement of plaques after i.v. injection during MRI of APP,PS1 mouse brains ex vivo, the utility of PUT-Gd-A40 for diagnostic use in animal models and patients is limited because carbodiimide-mediated modification of A40 with putrescine is associated with problems inherent with the protein itself, such as crosslinking, aggregate and/or fibril formation, and insolubility. In addition, PUT-A40 forms fibrils more readily than A40. The ease with which a molecule can form fibrils is considered as a direct measure of its likelihood to provide a seed for plaque formation.

Hence, a new probe, Gd[N-4ab/Q-4ab]A30, was produced as a putative MRI contrast enhancement agent by first synthesizing a glutamyl-4-aminobutane or asparagyl-4-aminobutane, which were then incorporated into the synthesis of the protein using standard solid-phase methods (Poduslo et al., 2004). The complete chemical synthesis of this probe eliminates peptide crosslinking, aggregate and fibril formation, and insolubility that affected the carbodiimide-mediated modification of A40 with putrescine. Apart from having the chemical purity, this probe is devoid of the neurotoxic domain found in A40 and is not amyloidogenic like A40. In addition, Gd[N-4ab/Q-4ab]A30 has good BBB permeability and labels neuritic plaques in vitro with affinity comparable with PUT-A40. However, attempts to image amyloid plaques in APP,PS1 animals in vivo using this agent yielded only modest results. Studies are being conducted to enhance the in vivo efficacy of this probe through appropriate structural modifications to influence various physiological factors, parenchymal metabolism and neuronal uptake, that were determined in the present study to adversely affect the plaque-targeting ability.

In summary, these studies demonstrated that both ¹²⁵IA40 and ¹²⁵I-PUT-A40 are rapidly eliminated from the systemic circulation after i.v. administration in WT animals. However, the peripheral pharmacokinetics of the probes did not significantly influence their plaque-targeting capabilities. The higher concentration of endogenous A40 in the peripheral circulation decreases the permeability of both compounds at the BBB. The metabolism of ¹²⁵I-PUT-A40 is substantially lower in the brain slices compared with that of ¹²⁵I-A40. This indicates that ¹²⁵I-PUT-A40 is metabolically stable and may remain intact in the brain parenchyma with higher concentrations being available for plaque targeting than ¹²⁵I-A40. In addition, the rate of ¹²⁵I-PUT-A40 efflux in WT brain slices was substantially lower compared with that of ¹²⁵I-A40. Hence, ¹²⁵I-PUT-A40 tends to remain in brain parenchyma longer than ¹²⁵I-A40. This study also demonstrates that the fluorescein-labeled A40, but not ¹²⁵I-PUT-A40, is taken up by cells, which makes the former unavailable for targeting the plaques located in the extracellular space. By comparing the distribution and metabolism of ¹²⁵I-A40 and ¹²⁵I-PUT-A40 after i.v. administration, it can be concluded that the inadequate targeting of ¹²⁵I-A40 to amyloid plaques despite its carrier-mediated uptake at the BBB could be due to cellular uptake, metabolism, and efflux of A40 in the brain parenchyma. The knowledge gained from these studies will be very useful in the development of new A derivatives with improved BBB permeability and plaque targeting.

	Acknowledgements

 
We thank Dan McCormick and Jane A. Petersen for extending technical expertise in synthesizing A40, Karen Duff for the PS1 transgenic mouse line, Dawn Gregor for excellent technical assistance, and Jennifer Scott for excellent secretarial assistance.

	Footnotes

 
This work was supported by National Institutes of Health Grant R01 AG22034 and by National Institutes of Health/National Center for Research Resources/Research Centers in Minority Institutions Grant G12RR03020.

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

doi:10.1124/jpet.105.095711.

ABBREVIATIONS: A, amyloid ; AD, Alzheimer's disease; MRI, magnetic resonance imaging; A40, unlabeled amyloid 40; PUT-A40, putrescine-A40; ¹²⁵I-A40, amyloid 40 labeled with ¹²⁵I; APP, amyloid precursor protein; PS1, presenilin 1; BBB, blood-brain barrier; F-A40, fluorescein-labeled A40; DCM, dichloromethane; BSA, bovine serum albumin; TCA, trichloroacetic acid; WT, wild type; V_p, residual brain region plasma volume; KRB, Krebs-Ringer bicarbonate; 2,4-DNP, 2,4-dinitrophenol; THT, thioflavin T; CD, circular dichroism; Cl, clearance; V_ss, steady-state volume of distribution; AUC, area under the plasma concentration curve; PS, cerebrovascular permeability coefficient-surface area product.

¹ Current affiliation: College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL.

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

	References

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