Preliminary studies of 99mTc-memantine derivatives for NMDA receptor imaging

Nuclear Medicine and Biology 39 (2012) 1034–1041

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Nuclear Medicine and Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n u c m e d b i o

Preliminary studies of

99m

Tc-memantine derivatives for NMDA receptor imaging

Xingqin Zhou ⁎, Jiankang Zhang, Chenglong Yan, Guoxian Cao, Rongjun Zhang, Gangming Cai, Mengjun Jiang, Songpei Wang Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China

a r t i c l e

i n f o

Article history: Received 14 December 2011 Received in revised form 10 February 2012 Accepted 28 February 2012 Keywords: NMDA receptor Competitive binding assay Biodistribution Nerve cell 99m Tc-memantine derivatives

a b s t r a c t Introduction: Novel technetium-labeled ligands, 99mTc-NCAM and 99mTc-NHAM were developed from the N-methyl-D-aspartate (NMDA) receptor agonist memantine as a lead compound by coupling with N2S2. This study evaluated the binding affinity and specificity of the ligands for the NMDA receptor. Methods: Ligand biodistribution and uptake specificity in the brain were investigated in mice. Binding affinity and specificity were determined by radioligand receptor binding assay. Three antagonists were used for competitive binding analysis. In addition, uptake of the complexes into SH-SY5Y nerve cells was evaluated. Results: The radiochemical purity of 99mTc-labeled ligands was more than 95%. Analysis of brain regional uptake showed higher concentration in the frontal lobe and specific uptake in the hippocampus. 99mTc-NCAM reached a higher target to nontarget ratio than 99mTc-NHAM. The results indicated that 99mTc-NCAM bound to a single site on the NMDA receptor with a Kd of 701.21 nmol/l and a Bmax of 62.47 nmol/mg. Specific inhibitors of the NMDA receptor, ketamine and dizocilpine, but not the dopamine D2 and 5HT1A receptor partial agonist aripiprazole, inhibited specific binding of 99mTc-NCAM to the NMDA receptor. Cell physiology experiments showed that NCAM can increase the viability of SH-SY5Y cells after glutamate-induced injury. Conclusions: The new radioligand 99mTc-NCAM has good affinity for and specific binding to the NMDA receptor, and easily crosses the blood–brain barrier; suggesting that it might be a potentially useful tracer for NMDA receptor expression. © 2012 Elsevier Inc. All rights reserved.

1. Introduction The N-methyl-D-aspartate (NMDA) receptor is the most abundant receptor of the glutamatergic system in the brain. It has roles in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. Disturbances of the brain glutamatergic neurotransmitter system are evident in many neuropsychiatric disorders [1]. Recent evidence indicated that excess activation of the NMDA receptor plays an important role in the onset and development of neurodegenerative diseases. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Alzheimer's disease (AD), schizophrenia, and epilepsy [2–5]. Imaging the NMDA receptor in the brain by positron emission tomography (PET) or single photon emission computed tomography (SPECT) would provide useful information on various neurological disorders [6]. By mapping changes in the density and quantity of NMDA receptors in specific brain regions, a tracer could play a significant role in understanding the relationship between the NMDA receptor and neurodegenerative diseases, as well as in the diagnosis and treatment of these disorders. However, imaging NMDA receptors in vivo has progressed slowly compared to dopamine receptor imaging. ⁎ Corresponding author. Tel.: +86 510 85514482 3523; fax: +86 510 85513113. E-mail address: [email protected] (X. Zhou). 0969-8051/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2012.02.008

Since the first SPECT image of NMDA receptors using (+)3-[ 123I] Iodo-MK-801 developed in 1997 [7], series of new NMDA receptor tracers have emerged after long-term trials. Shiue et al. [8] prepared [ 11C] ketamine to image NMDA receptors using PET. However, due to its rapid metabolism in the brain and similar uptake in the striatum and cerebellum, it was not an ideal radiotracer. Similarly, [ 23I]MK-801 was made to explore glutamatergic pathways in AD, but ultimately did not provide sufficient evidence to demonstrate their involvement [9]. The utility of (+)3-[123I]Iodo-MK-801 as a SPECT ligand for assessing modest alterations in NMDA receptor activity is ultimately limited by its lipophilicity and consequent high non-specific binding. Subsequently, a variety of probes such as [ 11C]CP101606, [ 11C] 3MPICA, and [ 123I]-IBZM were developed for NMDA receptor imaging [10–12]. Unfortunately, these tracers were ultimately limited by their low fat solubility and consequent high non-specific binding. [ (123)I] CNS 1261 is a novel SPECT NMDA receptor radiotracer which binds the intra-channel MK 801/PCP/ketamine site. It brought new hope for NMDA receptor imaging in neurodegenerative diseases [13–17]. A recent study reported an abnormal high-intense area in the right occipital lobe of patients with epilepsy using brain magnetic resonance imaging and this lesion was demonstrated as an area of hyperperfusion on [ 123I]-BZ-SPECT [18]. Further study of NMDA probes is needed. Many researchers are trying to develop improved SPECT and PET imaging agents for the

X. Zhou et al. / Nuclear Medicine and Biology 39 (2012) 1034–1041

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NMDA receptor. The anatomical location of the NMDA receptor not only makes it an important imaging target, but also complicates the development of suitable PET and SPECT radiotracers. Memantine is a partial NMDA receptor antagonist approved in the US and Europe for the treatment of moderate to severe AD. The molecular mechanism of memantine is by preferentially blocking excessive NMDA receptor activity without disrupting normal activity [19]. In this study, new NMDA receptor radioligands N-[2-(N-(2mercaptoethyl)) amino ethyl]-N-(2-mercaptoethyl)-3,5-dimethyl acetamide amantadine-technetium ( 99mTc-NCAM) and [1-[N-[N-(2mercaptoethyl)]-N-[2-[N-(2-mercaptoethyl) amino] ethyl] amino ethyl] amino-3,5-dimethyladmantane-technetium ( 99mTc-NHAM) (Fig. 1) were designed and synthesized with memantine as a lead compound with coupling using N2S2 [20]. The activities of the two radioligands were characterized using binding assays, biodistribution in mouse brains, and uptake into nerve cells. 2. Materials and methods Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AM-400 (400 MHz) spectrometer. Electrospray ionization mass spectra were obtained using a MAT 2.2 trap mass spectrometer (Varian, USA). Radioactivity was assayed using a Wizard 1470 γ-automatic counting device (Perkin Elmer, USA). TENSOR2Z FT-IR was from Bruker. The ZT-II cell collector (Zhejiang Shaoxing Satellite Machinery Co., China), AC22105 analytical balance (Sartorius Company, Germany) and J2-HS centrifuge (Beckman, USA) were used for competitive binding assays. Memantine was provided by Jiangsu Institute of Nuclear Medicine. Ketamine, dizocilpine, and aripiprazole were purchased from Sigma-Aldrich (St Louis, MO, USA). All the chemicals used were analytical grade. Rats (Sprague–Dawley) and mice (ICR) were purchased from Shanghai SLAC Laboratory Animal Center (Shanghai, China). The animal experiments in this study were approved by the Animal Care and Ethics Committee of Jiangsu Institute of Nuclear Medicine. 2.1. Syntheses The synthesis route for each product is depicted in Scheme 1. The details are as follows. 2.1.1. N-[2-((2-(S-(4-methoxybenzyl) mercapto) ethyl) amino) acetyl]S-(4-methoxy-benzyl)-2-aminoethanethiol (II) A mixture of 5.47 g (20 mmol) 4-methoxybenzyl-(2-chloroacetylaminoethyl) sulfide, 3.94 g (20 mmol) 2-(4-methoxybenzyl thio) ethylamine, and 4.2 ml dry triethylamine in anhydrous acetonitrile (100 ml) with potassium iodide as catalyst was refluxed for 22 h. After the reaction, solvents were evaporated slowly at room temperature, and the reaction mixture was extracted twice with CH2Cl2 (20 ml). The organic layers were combined and washed with H2O, dried over Na2SO4, filtered, and then evaporated to dryness. Pure product (II) was obtained by flash chromatography (ethanol:ethyl

Fig. 1. The structure of

99m

Tc-memantine derivatives.

Scheme 1. Synthesis of memantine derivatives.

acetate = 1:20, v/v) with 80% yield. 1HNMR (CDCl3 ), δ:1.82(s,1H); 2.58(m,4H); 2.74(m,2H); 3.21(s,2H); 3.42(t,2H); 3.64 ∼ 3.82(dd,10H); 6.82(d,4H); 7.22(m,4H); 7.58(s,1H). MS, m/z (%): 434(M+), 121 (CH3OC6H5CH2+). 2.1.2. Chloroacetyl-3,5-dimethyl amantadine (III) Chloroacetyl chloride (20 mmol) and 30 ml CH2Cl2 were mixed in a flask. Amantadine (3.58 g, 20 mmol) and triethylamine (4 ml, 30 mmol) in CH2Cl2 (50 ml) were added dropwise to the stirred mixture at −45 °C. The mixture was stirred for an additional 30 min at −45 °C and then stirred at room temperature for 1 h under flowing N2. After the reaction, 50 ml H2O was added. The organic layer was washed three times with H2O to neutralize, dried over Na2SO4, filtered, and evaporated to dryness. The light yellow solid was collected, and pure product (III) was obtained by flash chromatography (ethyl acetate:petroleum ether = 1:5, v/v) with 83% yield.

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X. Zhou et al. / Nuclear Medicine and Biology 39 (2012) 1034–1041

1

H NMR (CDCl3), δ:0.86(s,6H); 1.11–1.25(m,2H), 1.28–1.32(m,2H), 1.37–1.42(m,2H), 1.60–1.75(m,4H), 1.84–1.89(m,2H); 2.15– 2.19(m,1H); 3.92(s,2H); 6.23(s,1H). MS, m/z (%): 256(M+1). 2.1.3. N-[2-(S-(4-methoxybenzyl) thio) ethyl]-N-[(N-(2-(S-(4methoxybenzyl) thio) ethyl) amino) carbonyl methyl] -3,5-dimethyl acetamide amantadine (IV) Potassium carbonate (5 g, 36 mmol) followed by potassium iodide (0.6 g, 3.6 mmol) were added to a mixture of compound II (212 mg, 0.69 mmol) and compound III (0.98 g, 3.6 mmol) in anhydrous acetonitrile (100 ml). The resulting mixture was stirred under reflux for 12 h. After the reaction, the mixture was evaporated to dryness, and 100 ml H2O and 100 ml CH2Cl2 were added. The organic layer was washed three times with H2O to neutralize, dried over Na2SO4, filtered, and evaporated to dryness. The crude product obtained was 2.2 g. Pure product (IV) was obtained by flash chromatography (ethanol:ethyl acetate:petroleum ether = 0.4:8:4, v/v/v) with 67% yield. 1H NMR (CDCl3), δ:0.832(s,6H); 1.10–1.18(m,2H), 1.23–1.29(m,2H), 1.35– 1.37(d,J = 10HZ,2H), 1.62–1.67(t,4H), 1.827–1.831(d,J = 2HZ,2H); 2.11–2.13(m,1H); 2.51–2.53(t,2H); 2.55–2.58(t,2H); 2.69– 2.72(t,2H); 2.69 ppm–2.72(t,2H); 3.42–3.46(m,2H); 3.02(s,2H); 3.13(s,2H); 3.656(s,2H); 3.661(s,2H); 3.772(s,3H); 3.776(s,3H); 6.82–6.85(m,4H); 7.18–7.22(m,4H); 6.55(s,1H); 7.32–7.34(m,1H). MS (m/z): 654(M+1), 676(M+Na), 692(M+K), 708(M+NaS). 2.1.4. N-[2-(N-(2-mercaptoethyl)) amino ethyl]-N-(2-mercaptoethyl)3,5-dimethyl acetamide amantadine (V) V was prepared according to a previously described procedure [21]. Compound IV (0.327 g, 0.05 mmol) was dissolved in 3.3 ml trifluoroacetic acid (TFA), and 0.13 ml dried anisole and 318 g (1 mmol) mercury acetate were added. The mixture was stirred for 30 min at 0 °C under flowing N2. A viscous oily substance was obtained by condensation using a vacuum. A colorless solid was obtained by adding 5 ml dry ether to the oily product and ultrasonicating for 5 min. The collected solid was filtered and dried, and dissolved in 5 ml anhydrous ethanol followed by addition of dry H2S for 30 min. The mixture was centrifuged to remove the black mercuric sulfide. A white solid (compound V) was collected by evaporating at room temperature under N2. MS (m/z): 414 (M+1), 436 (M+Na). 2.1.5. 1-N-[N-(2-(S-(4-methoxybenzyl) thiol) ethyl)-N-[2-[N-(2-(S-(4methoxybenzyl) thiol) ethyl) amino] ethyl] amino ethyl] amino-3,5dimethyladmantane (VI) Compound IV (3.72 g, 5 mmol) was dissolved in 0 °C anhydrous tetrahydrofuran (50 ml) and the solution was drop-wise added to BH3·THF (50 ml, 1 mmol/ml) with stirring and the mixture refluxed for 24 h under a N2 stream. The liquid slowly changed to colorless. The mixture was cooled to 0 °C and drop-wise added to HCl (1 N) until gas generation stopped. A white solid was obtained after removing THF using vacuum. HCl (1 N, 100 ml) was added to the solid and the mixture reacted at 90 °C for 30 min. The solution was cooled to 0 °C and pH was adjusted to neutral using ammonia. The reaction mixture was extracted twice with CH2Cl2 (20 ml). The organic layers were combined and washed with H2O, dried over Na2SO4, filtered, and evaporated. Pure product (VI) was obtained by flash chromatography (methanol:CH2Cl2:triethylamine = 1:10:0.5, v/v/v) (yield, 63%). 1H NMR (CDCl3) δ:0.896(s,6H); 1.18(s,2H), 1.25–1.4 ppm(m,4H), 1.65– 1.79(m,4H), 2(s,2H); 2.2(m,1H); 2.49–2.51(m,2H), 2.81–2.96(m,6H), 3.03–3.15(m,8H); 3.69(m,4H); 3.74–2.72(m,6H); 6.83–6.88(m,4H), 7.22–7.29(m,4H); 9 ppm(1H, bs), 9.14(1H, bs). MS (m/z): 626(M+1). 2.1.6. [1 - [N-[N-(2-mercaptoethyl)]-N-[2-[N-(2-mercaptoethyl) amino] ethyl] amino ethyl] amino-3,5-dimethyladmantane (VII) Compound VII was prepared using the method described above for V. MS (m/z): 386(M+1).

2.2. Labeling and characterizing activities of the radioligands 2.2.1. Labeling of NHAM and NCAM 99m Tc-NHAM was prepared as follows: in a 1.5-ml centrifuge tube, 0.1 ml of sodium glucoheptonate (GH, 40 mg/ml), 0.05 ml of EDTA-2Na (10 mg/ml), 0.1 ml of NHAM (2 mg/ml, dissolved in 70% ethanol solution) were added; followed by 0.1 ml of [ 99mTc]NaTcO4 (37 MBq) and then immediately by 0.1 ml of fresh SnF2 (1 mg/ml in 0.1 M HCl) solution. The volume was adjusted to 1 ml using phosphate buffer (0.5 mol/l NaHPO4.12H2O, 0.17 mol/l KH2PO4, pH 6.5). The vial was heated in a boiling water bath for 45 min and allowed to cool to room temperature. Radiochemical purity (RCP) was determined by thinlayer chromatography using diethyl ether:methanol:ammonia = 50:20:5, (v/v) as the mobile phase. 99mTc-NHAM migrated to the top of the strip (Rf = 0.8–1.0), while [ 99mTc]-, 99mTc-colloid, 99mTcGH and radiochemical impurities remained near the origin (Rf = 0– 0.2). The radiochemical purity was N95%. 99mTc-NCAM was labeled in the same way. The radiochemical purity of 99mTc-NCAM was also N95%. After more than 6 h at room temperature, the products retained a radiochemical purity N90%. Reverse-phase high-performance liquid chromatography (HPLC) was done using a C18 column (4.6 × 250 mm, 5 mm). The samples were eluted using 0.01 mol/l TFA in water (eluent A) and 0.01 mol/l TFA in acetonitrile (eluent B) with an isocratic flow rate of 0.8 ml/min of A/B = 2/1 (v/v). 2.2.2. Lipid–water partition coefficient Partition coefficients were measured by shake-flask method [22]. 1-Octanol and phosphate-buffered saline (PBS, pH = 7.4) were cosaturated with each other before use. 1 ml PBS saturated with 1octanol and 1 ml 1-octanol saturated with PBS were added to a centrifuge tube containing 200 μl 99mTc-complex(0.37 MBq). The tube was capped and vigorously vortexed for 5 min at room temperature and then allowed to stand for 5 min. After reaching equilibrium, the tube was centrifuged at 2000 rpm (r = 6.0 cm) for 10 min. Aliquots were taken from each phase and radioactivity counted. The partition coefficient was calculated by dividing the radioactivity of the 1octanol layer with that of the water layer. This measurement was repeated five times. The results are listed in Table 1. 2.2.3. Plasma protein binding A mixture of 100 µl of the 99mTc-complex (3.7 MBq, 0.74 MBq, =0.148 MBq) in 200 μl plasma was incubated for 3 h at 37 °C. Following equilibrium, 1 ml 15% trichloroacetic acid was added to each tube, followed by vortexing and centrifuging at 2000 rpm (r = 6.0 cm) for 10 min. The supernatant was collected and the steps were repeated four times. The radioactivity of the supernatant and residue was counted using a gamma counter. Protein binding to plasma was calculated by the relationship: protein binding to plasma = [(residue counts)/(residue counts + supernatant counts)] × 100% (Table 1). 2.2.4. Rate of uptake into SH-SY5Y cells The experimental group was a mixture of 1 ml SH-SY5Y cell suspension (4.0 × 10 5 cells/ml) and 0.1 ml (0.185 MBq) [ 99mTc]NHAM Table 1 Plasma protein binding and PO/W values of

99m

Test items

Plasma protein binding (%) PO/W value Kd (mmol/l) Bmax (nmol/mg)

High (3.7 MBq) Middle (0.74 MBq) Low (0.148 MBq)

Tc-NCAM and

99m

Tc-NHAM.

99m

Tc-memantine derivatives

99m

Tc-NHAM

62.5 58.1 63.2 1.95 ± 0.05 584.32 ± 87.27 267.05 ± 22.06

99m

Tc-NCAM

79.6 81.3 83.5 1.79 ± 0.02 701.21 ± 24.15 62.47 ± 10.23

X. Zhou et al. / Nuclear Medicine and Biology 39 (2012) 1034–1041

and the negative control was 1 ml of cell suspension and 0.1 ml (0.185 MBq) [ 99mTc]NaTcO4. A blank control was made by adding 1 ml medium to 0.1 ml (0.185 MBq) [ 99mTc] NHAM. Similar experimental groups were created with 99mTc-NCAM. After mixing, the tubes were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 10, 30, 60, 90, 120, 180, and 360 min, respectively. After incubation tubes were centrifuged at 2000 rpm (r = 6.0 cm) for 5 min to remove supernatant and then washed three times using PBS. After washing, the residues were counted by γ-counter. All tubes were incubated with 0.5% fetal calf serum for more than 1 h to reduce nonspecific adsorption before use. Percent cellular uptake was calculated as (residue counts−blank counts)/(residue counts + supernatant counts) × 100%. The final results are expressed as mean ± standard deviation (SD) of six independent parallel experiments and plotted as

Fig. 2. The HPLC radiochromatogram of [99mTc]-,

99m

1037

a function of time as described previously [23,24]. During the whole procedure, the cell viability was more than 90%. The protective effect of the precursors (NCAM and NHAM) on SHHY5Y cells treated with glutamate (Glu, 5 mmol/l) was compared. Five groups were tested: a control group (4.0 × 10 5/ml SH-SY5Y cells + 200 μl HBSS); a negative control (20 μl [5 mmol/l] glutamate and 180 μl HBSS added to SH-SY5Y cells in); a positive control (SH-HY5Ycells + 160 μl HBSS + 20 μl glutamate [5 mmol/l] + 20 μl memantine [3 mmol/l]); and two experimental groups (SH-HY5Y cells + 160 μl HBSS + glutamate [5 mmol/l] + 20 μl NCAM or NHAM [3 mmol/l]). 2.2.5. Equilibrium dissociation constant Kd NMDA receptor protein was prepared from Sprague Dawley rats as described in reference [25]. Coomassie Brilliant Blue was used to

Tc-NCAM, and

99m

Tc-NHAM. a, [99mTc]; b,

99m

Tc-NCAM; c,

99m

Tc-NHAM.

1038

X. Zhou et al. / Nuclear Medicine and Biology 39 (2012) 1034–1041

Fig. 3. A. The rate of radioligand uptake rate by SH-HY5Y cells. B. Protective effect of NHAM and NCAM on SH-HY5Y cells treated with glutamate (Glu, 5 mmol/l). The concentrations of memantine, NHAM and NCAM were 3 mmol/l. The control group is untreated SH-HY5Y cells.

determine protein amount. Buffer C was prepared by adding 5% Tween-20 in 10 mmol/l HEPES (pH 7.4). Total binding (TB) was determined by adding 0.1 ml of different concentrations of 99mTcmemantine derivatives (5, 10, 25, 50, 100, 180, 360 nmol/l), 0.1 ml buffer C, 0.3 ml NMDA receptor protein, and the volume was adjusted to 0.6 ml by adding buffer C. The NSB was performed in the same way as above, except that 0.1 ml NCAM (or NHAM) was added. The blank was made by adding 0.5 ml buffer C to 0.1 ml of 99mTc-memantine derivatives. All tubes were incubated at 37 °C for 25 min in a water bath, and then placed on ice for 5 min to stop the reaction. A GF/B glass fiber filter paper was used to infiltrate 5% bovine serum albumin for over 2 h. A ZT-II cell collector was used for filtration. The products were washed with 3 ml buffer C four times. The samples were counted after removing the membranes. The equilibrium dissociation constant Kd was estimated using a saturation curve (Table 1). 2.2.6. Competition binding assay Three antagonists, ketamine, dizocilpine, and aripiprazole were used to assay competition binding for NMDA. 99mTc-memantine derivatives (0.1 ml, 1.85 μmol/l) and a range of 11 concentrations (6 × 10 −1 to 6 × 10 −11 mol/l) of antagonist were added to each tube, and then leached and counted. 2.2.7. Biodistribution studies in normal mice The biodistribution of 99mTc-memantine derivatives was studied in normal Kunming mice (25 ± 2 g) to evaluate pharmacokinetic properties. A volume of 0.2 ml of the purified radiotracer solution (∼10 MBq) was injected into the mice via the tail vein. The mice

(n = 5) were sacrificed at 5, 15, 30, 60, 120, 180, 240, and 360 min after injection. Organs and brain regions of interest were dissected, weighed, and counted for radioactivity. The percentage of injected dose per gram (%ID/g) was calculated by comparing its activating with appropriate standard of injected dose (ID) (Table 3). 2.2.8. Blood kinetic test 99m Tc-NCAM (0.2 ml, 10 MBq) was injected through the tail vein into six mice of (20 ± 1 g). At 2, 5, 10, 30, 60, 120, and180 min, 10 μl blood was taken from the tail vein by quantitative vessel bleeding. The samples were counted and the data were analyzed using 3p87 pharmacokinetic software to fit the appropriate compartment model. 3. Results 3.1. Radiochemistry New technetium-labeled memantine derivatives 99mTc-NCAM and 99mTc-NHAM were characterized by HPLC (Fig. 2). Under the selected chromatographic conditions, it was possible to clearly separate the peaks corresponding to the complexes 99mTc-NCAM (tR = 11.27 min) and 99mTc-NHAM (tR = min 7.9) from [ 99mTc] [TcO4]-(tR = 3.3 min).One important factor that determines whether a drug penetrates the blood–brain barrier is its lipid–water partition coefficient. Both 99mTc-NCAM and 99mTc-NHAM are lipophilic, but the lipid–water partition coefficient of 99mTc-NHAM is slightly larger than 99mTc-NCAM (Table 1). The polar atoms that act as hydrogen bond donors or acceptors play important roles in drug kinetics. The number of polar atoms, net charge and surface area often affect pharmacodynamics. The degree to which drugs bind plasma proteins is significantly affected by molecular weight, and the total surface area of hydrogen bond acceptors and donors. Stronger hydrogen binding with plasma protein results in greater proportion of plasma protein binding. In the two complexes, the hydrogen bond acceptor (oxygen atom) surface area of NCAM is greater than NHAM and the data indicated that NCAM has slightly higher plasma protein binding than NHAM.

Table 2 Binding affinities (Ki) of NMDA receptor antagonists. Ketamine (mol/l)

99m

Tc-NCAM 99m Tc-NHAM Fig. 4. Competition curves for ketamine, dizocilpine and aripiprazole with 99mTc-NCAM and 99mTc-NHAM binding to the NMDA receptor.

Dizocilpine (mol/l)

Ki

IC50

Ki

IC50

8.43 × 10-6 9.28 × 10-7

1.22 × 10-5 9.46 × 10-6

3.15 × 10-8 2.43 × 10-8

5.08 × 10-8 3.69 × 10-8

Values are means (n = 3) obtained after adding antagonist (6 × 10 −1 to 6 × 10−11 mol/l) and 99mTc-memantine derivatives (0.1 ml, 1.85 μmol/l) to NMDA protein.

X. Zhou et al. / Nuclear Medicine and Biology 39 (2012) 1034–1041 Table 3 In vivo organ distribution of Tissue/organ

99m

Tc-labeled radioligands in normal mice.

Time of sacrifice after administration of 5 min

Heart Spleen Lungs Liver Kidney Stomach Intestines Muscle Ovum / Sperm Brain

15 min

2.34 ± 0.02 16.9 ± 0.32 13.3 ± 4.21 9.81 ± 3.41 2.81 ± 0.05 0.55 ± 0.02 0.99 ± 0.03 0.27 ± 0.08 0.18 ± 0.09 1.23 ± 0.11

99m

Tc-NCAM

30 min

0.81 ± 0.03 9.99 ± 1.03 8.63 ± 2.21 13.2 ± 1.75 1.05 ± 0.06 0.31 ± 0.03 0.50 ± 0.02 0.15 ± 0.02 0.38 ± 0.12 0.87 ± 0.02

0.51 ± 0.03 13.6 ± 1.22 15.0 ± 4.32 23.9 ± 5.33 1.17 ± 0.24 0.38 ± 0.02 1.73 ± 0.13 0.12 ± 0.02 0.28 ± 0.07 0.52 ± 0.12

Time of sacrifice after administration of

Heart Spleen Lungs Liver Kidney Stomach Intestines Muscle Ovum / Sperm Brain

1039

99m

60 min

120 min

180 min

240 min

360 min

0.39 ± 0.03 15.9 ± 2.08 10.1 ± 2.53 20.0 ± 4.94 1.27 ± 0.48 0.57 ± 0.05 1.11 ± 0.15 0.10 ± 0.03 0.22 ± 0.14 0.26 ± 0.04

0.24 ± 0.03 10.4 ± 2.25 8.48 ± 1.54 21.6 ± 5.07 0.84 ± 0.23 0.52 ± 0.04 0.70 ± 0.07 0.06 ± 0.04 0.19 ± 0.08 0.15 ± 0.03

0.18 ± 0.12 9.97 ± 3.24 7.55 ± 2.38 18.6 ± 6.25 0.72 ± 0.27 0.45 ± 0.18 0.58 ± 0.23 0.05 ± 0.03 0.16 ± 0.05 0.11 ± 0.05

0.11 ± 0.03 8.81 ± 3.42 6.43 ± 1.04 15.5 ± 3.61 0.68 ± 0.23 0.32 ± 0.03 0.35 ± 0.11 0.03 ± 0.02 0.13 ± 0.16 0.09 ± 0.03

0.12 ± 0.05 5.34 ± 2.36 4.58 ± 1.24 11.2 ± 4.86 0.36 ± 0.12 0.25 ± 0.08 0.19 ± 0.13 0.02 ± 0.03 0.08 ± 0.02 0.06 ± 0.01

Tc-NHAM

5 min

15 min

30 min

60 min

120 min

180 min

240 min

360 min

3.82 ± 0.78 3.35 ± 1.01 11.41 ± 0.8 16.69 ± 0.6 12.02 ± 0.8 1.21 ± 0.28 8.66 ± 1.87 1.76 ± 0.18 0.64 ± 0.24 1.47 ± 0.01

1.91 ± 0.11 3.72 ± 0.41 11.89 ± 0.83 14.31 ± 1.51 10.65 ± 1.06 1.41 ± 0.11 7.98 ± 1.41 1.16 ± 0.23 0.45 ± 0.14 0.99 ± 0.03

1.38 ± 0.10 4.39 ± 0.58 7.71 ± 0.58 12.12 ± 2.13 7.96 ± 1.12 1.61 ± 0.34 5.44 ± 0.43 0.75 ± 0.08 0.98 ± 0.17 0.76 ± 0.01

1.33 ± 0.11 3.17 ± 0.59 4.52 ± 0.68 8.98 ± 2.78 6.32 ± 0.63 1.65 ± 0.25 5.96 ± 1.18 0.84 ± 0.28 1.50 ± 0.97 0.29 ± 0.02

0.50 ± 0.05 1.77 ± 0.36 1.67 ± 0.33 6.06 ± 0.98 2.72 ± 0.46 0.56 ± 0.22 2.92 ± 1.11 0.40 ± 0.07 0.48 ± 0.04 0.11 ± 0.01

0.41 ± 0.05 1.54 ± 0.32 1.22 ± 0.14 6.19 ± 1.06 3.09 ± 0.31 0.81 ± 0.20 2.36 ± 0.50 0.24 ± 0.04 0.74 ± 0.55 0.07 ± 0.01

0.54 ± 0.11 1.15 ± 0.32 2.54 ± 0.89 6.17 ± 0.99 3.52 ± 0.30 0.80 ± 0.09 1.39 ± 0.40 0.32 ± 0.04 0.76 ± 0.32 0.06 ± 0.01

0.43 ± 0.04 0.87 ± 0.09 0.77 ± 0.11 5.25 ± 1.47 2.60 ± 0.40 0.40 ± 0.09 0.61 ± 0.37 0.20 ± 0.06 0.42 ± 0.10 0.04 ± 0.00

Values are mean ± SD (n = 5, %ID/g) after tail vein injection of

99m

Tc-NCAM or

99m

Tc-NHAM (10 MBq, 0.2 ml).

3.2. Rate of uptake into SH-SY5Y cells To investigate the NMDA receptor binding properties of 99mTclabeled memantine derivatives, in vitro experiments were performed with SH-SY5Y cells, a human neuroblastoma cell line. Uptake of both 99m Tc-NCAM and 99mTc-NHAM into SH-HY5Y nerve cell was significantly higher compared to 99mTc (Fig. 3), with peaks of 2.7% and 3.8% at 60 min, respectively. Uptake of 99mTc in the control SH-HY5Y cells was 1% and this did not increase with extension of incubation time. The uptake of both complexes decreased slowly with prolonged incubation, which is related to metabolism endocytosis and exocytosis of the cell. High concentrations of glutamate can injure normal nerve cells and even cause cell death. The protective effect of the precursors on SH-HY5Y cells treated with glutamate (Glu, 5 mmol/l) is shown in Fig. 3B. Based on OD values, the survival of cells treated with NHAM, NCAM, and memantine was 159%, 143%, and 151%, respectively, compared to the cells treated with Glu alone (Fig. 3B).

NHAM into the brain (1.23 and 1.47%ID/g, respectively) at 5 min, indicating that the two ligands successfully crossed the blood–brain barrier. High initial uptake was seen in the spleen and lungs for 99mTcNCAM and in the liver and kidney for 99mTc-NHAM, both of which then decreased quickly over time. The brain regional distribution of 99m Tc-NCAM and 99mTc-NHAM was studied by isolating the region of interest including the frontal (FT), occipital (OT), parietal (PT), and temporal (TR) lobes, and the striatum (ST), hippocampus (HP), and cerebellum (CB). The cortex (especially PT) and HP were used as the target regions since they have the highest density of NMDA receptors, and the cerebellum (CB) was used as a nontarget region since it has the lowest density of NMDA. The ratios of the target to nontarget counts for the complexes are showed in Fig. 5. 99mTc-NCAM had a good target to nontarget ratio (FT/CB) which peaked at about 2.98 at 60 min after injection (Fig. 5). The ratio HP/CB reached 2.52 at 60 min. In contrast, 99mTc-NHAM was randomly distributed in the

3.3. In vitro binding affinity Competitive binding curves are shown in Fig. 4. Inhibition of 99mTc-NCAM and 99mTc-NHAM binding by three antagonists was studied: ketamine and dizocilpine, two NMDA receptor-specific antagonists; and aripiprazole, a dopamine D2 and 5HT1A receptor partial agonist that does not bind to the NMDA receptor. Specific binding of both 99mTc-NCAM and 99mTc-NHAM to the NMDA receptor could be inhibited by ketamine and dizocilpine, but not by aripiprazole. The IC50 (50% inhibitory concentration) was calculated according to the equation, Ki = IC50/(1 + LT/Kd), where Ki is the inhibition constant of ketamine or dizocilpine, and LT is ligand concentration (Table 2). The Ki values also indicated that 99mTc-NCAM and 99mTc-NHAM have high affinity for the NMDA receptor. 3.4. In vivo biodistribution In vivo distribution of 99mTc-NCAM and 99mTc-NHAM in mice is shown in Table 3. There was good uptake of 99mTc-NCAM and 99mTc-

Fig. 5. Ratio of ligand in the frontal lobe and hippocampus to cerebellum are represented. 99mTc-NCAM achieves a higher target to nontarget ratio than 99m Tc-NHAM.

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X. Zhou et al. / Nuclear Medicine and Biology 39 (2012) 1034–1041 Table 4 Pharmacokinetic parameters of

99m

Tc-NCAM in mice.

Parameters

99m

t1/2a/min t1/2b/min K12/min−1 K21/min−1 AUC/cpm·min·l−1 CL/cpm·min−1

2.204 157.38 0.2275 0.0192 79.948 1.2508

Values are means obtained after injecting 0.2 ml of

Fig. 6. Regional brain distribution of

99m

Tc-NCAM in mice.

brain without clear regional targeting. The highest total regional brain uptake of 99mTc-NCAM was in the cortex and HP, and the lowest was in the CB (Fig. 6). Intermediate levels of 99mTc-NCAM uptake were seen in the ST (Fig. 6). Significant abnormalities have been reported in the superior frontal gyrus in schizophrenia [26]. The prefrontal cortex was demonstrated to play a crucial role in modulating cognitive functions [27]. In rodents, systemic administration of NMDA receptor antagonists is associated with significant cognitive impairment and obvious signs of neuronal damage in the prefrontal cortex [28]. Uptake of 99m Tc-NCAM was higher than 99mTc-NHAM specifically in the frontal cortex, where the NMDA receptor is very abundant. 3.5. Blood kinetics The kinetics of 99mTc-NCAM fit a two-compartment model by regression analysis (Fig. 7). The pharmacokinetic equation was C = 5.492e −0.3141t + 0.275e −0.0042t. The absorption half life (t1/2) of 99m Tc-NCAM was 2.20 min. The pharmacokinetic parameters of 99mTc-NCAM are summarized in Table 4. 4. Discussion Studies of central nervous system receptor imaging agents generally progress from in vitro to in vivo, and include lipid solubility, affinity, and regional uptake in the brain. [ 11C]GMOM was a potential

99m

Tc-NCAM

Tc-NCAM (10 MBq, n = 6).

NMDA/PCP receptor PET tracer (Ki = 5.2 ± 0.3 nM; logP = 2.34), but it was uniformly distributed in the brain and lacked specific binding [29]. [ 11C]-3MPICA, another PET ligand for the NMDA/glycine site with a Ki = 4.8 nM in vitro, had low brain uptake in vivo, and did not appear to be a promising PET tracer for NMDA receptor research [11]. [ 11C] methyl-BIII277CL, a new benzomorphan derivative, with a Kd of 6 nmol/l and a Bmax of 670 fmol/mg protein also lacked specific binding and had unfavorable pharmacokinetics on PET studies in rats [30]. [ 11C]-AcL703 was an NMDA receptor antagonist targeted to the glycine site that achieved the highest radioactivity concentration in the cortex and lowest in the white matter, but was limited by its low brain uptake [31,32]. Another agent, [ 125I] MK-801 had good affinity and liposolubility in vitro; but in vivo, it was not suitable for NMDA receptor imaging due to lack of affinity for the NMDA receptor [7]. [ 18F]-memantine was also a good antagonist of the NMDA receptor in vitro, but the distribution in the human brain did not reflect the regional distribution of NMDA receptor concentration, making it unsuitable for the PET imaging of the NMDA receptor in vivo [33]. Even though these tracers were not successful, binding specificity can be improved in the future by modifying the structure of NMDA antagonists. Thus, developing high affinity NMDA radiotracers is in progress and desirable. In this paper, 99mTc-NCAM a potentially new compound for SPECT imaging of NMDA receptors in the brain was evaluated. 99m Tc-NCAM had relatively fast clearance from the blood into other organs (t1/2 = 2.20 min) and rapid uptake in mice brain (1.23%ID/ organ at 5 min). Regional brain distribution showed higher concentration in the frontal cortex and specific intake in the hippocampus. 99mTc-NCAM reached a higher target to nontarget ratio than 99mTc-NHAM (Figs. 5 and 6). 99m Tc-NCAM bound to a single site on the NMDA receptor with a Bmax of 62.47 nmol/mg and a Kd of 701.21 nmol/l, which was close to memantine (Kd 700 nM) [34]. The results indicated that introduction of the S2N2 ring did not significantly affect the affinity of 99mTc-NCAM for the NMDA receptor. In competitive binding experiments, the Ki of ketamine and dizocilpine was 8.43 × 10 −6 mol/l and 3.15 × 10 −8 mol/l, respectively, which are the same order of magnitude as previously reported values (3100 nM and 79 nM, respectively) [35,36]. Aripiprazole had no significant inhibitory effect on the complex. The uptake of 99mTc-NCAM by SH-HY5Y nerve cells was significantly higher than that of control compounds. These results suggest that 99m Tc-NCAM has some specific targeting to the NMDA receptor. 5. Conclusion

Fig. 7. Blood kinetic curve of

99m

Tc-NCAM in mice.

A new technetium-labeled ligand memantine dinitrogen disulfide derivative 99mTc-NCAM was prepared successfully with the NMDA receptor antagonist memantine as a lead compound for structural modification. These preclinical data indicate good affinity for the NMDA receptor, and uptake in the frontal lobe and hippocampus. Furthermore, NCAM was able to increase the survival of SH-HY5Y cells after glutamate-induced injury by targeting NMDA receptors on the nerve cells. 99mTc-NCAM crosses the blood–brain barrier well, which makes it a potential radiotracer for monitoring changes in central

X. Zhou et al. / Nuclear Medicine and Biology 39 (2012) 1034–1041

nervous system NMDA receptors in various neurodegenerative diseases, such as schizophrenia and Alzheimer's disease. Investigations aimed at determining the maximum specific activity that could be obtained after 99mTc labeling are under way as this is an important parameter in receptor imaging.

Acknowledgments This work was supported by the National Natural Science Foundation of China (30770602) and the Natural Science Foundation of Jiangsu Province, China (BK2010157, BK2008111, and BK2011167).

Reference [1] Ganapathy PS, White RE, Ha Y, Bozard BR, McNeil PL, Caldwell RW, et al. The role of N-methyl-D-aspartate receptor activation in homocysteine-induced death of retinal ganglion cells. Invest Ophthalmol Vis Sci 2011;52(8):5515–24. [2] Ohnuma T, Arai H. Significance of NMDA receptor-related glutamatergic amino acid levels in peripheral blood of patients with schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 2011;35(1):29–39. [3] Tsai GE, Lin PY. Strategies to enhance N-methyl-D-aspartate receptor-mediated neurotransmission in schizophrenia, a critical review and meta-analysis. Curr Pharm Des 2010;16(5):522–37. [4] Yang JH, Wada A, Yoshida K, Miyoshi Y, Sayano T, Esaki K, et al. Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain. J Biol Chem 2010;285(53):41380–90. [5] Frasca A, Aalbers M, Frigerio F, Fiordaliso F, Salio M, Gobbi M, et al. Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity. Neurobiol Dis 2011;43(2):507–15. [6] Sobrio F, Gilbert G, Perrio C, Barre L, Debruyne D. PET and SPECT imaging of the NMDA receptor system: an overview of radiotracer development. Mini Rev Med Chem 2010;10(9):870–86. [7] Owens J, Wyper DJ, Patterson J, Brown DR, Elliott AT, Teasdale GM, et al. First SPET images of glutamate (NMDA) receptor activation in vivo in cerebral ischaemia. Nucl Med Commun 1997;18(2):149–58. [8] Shiue CY, Vallabhahosula S, Wolf AP, Dewey SL, Fowler JS, Schlyer DJ, et al. Carbon11 labelled ketamine-synthesis, distribution in mice and PET studies in baboons. Nucl Med Biol 1997;24(2):145–50. [9] Brown DR, Wyper DJ, Owens J, Patterson J, Kelly RC, Hunter R, et al. 123Iodo-MK801: a SPECT agent for imaging the pattern and extent of glutamate (NMDA) receptor activation in Alzheimer's disease. J Psychiatr Res 1997;31(6):605–19. [10] Haradahira T, Maeda J, Okauchi T, Zhang MR, Hojo J, Kida T, et al. Synthesis, in vitro and in vivo pharmacology of a C-11 labeled analog of CP-101,606, (+/−)threo1-(4-hydroxyphenyl)-2-[4-hydroxy-4-(p-[11C]methoxyphenyl)piperi dino]-1propanol, as a PET tracer for NR2B subunit-containing NMDA receptors. Nucl Med Biol 2002;29(5):517–25. [11] Waterhouse RN, Sultana A, Laruelle M. In vivo evaluation of [11C]-3-[2-[(3methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxylic acid ([11C]3MPICA) as a PET radiotracer for the glycine site of the NMDA ion channel. Nucl Med Biol 2002;29(8):791–4. [12] Kegeles LS, Abi-Dargham A, Zea-Ponce Y, Rodenhiser-Hill J, Mann JJ, Van Heertum RL, et al. Modulation of amphetamine-induced striatal dopamine release by ketamine in humans: implications for schizophrenia. Biol Psychiatry 2000;48(7): 627–40. [13] Bressan RA, Erlandsson K, Stone JM, Mulligan RS, Krystal JH, Ell PJ, et al. Impact of schizophrenia and chronic antipsychotic treatment on 123ICNS-1261 binding to Nmethyl-D-aspartate receptors in vivo. Biol Psychiatry 2005;58(1):41–6. [14] Stone JM. Imaging the glutamate system in humans: relevance to drug discovery for schizophrenia. Curr Pharm Des 2009;15(22):2594–602.

1041

[15] Erlandsson K, Bressan RA, Mulligan RS, Gunn RN, Cunningham VJ, Owens J, et al. Kinetic modelling of 123ICNS 1261—a potential SPET tracer for the NMDA receptor. Nucl Med Biol 2003;30(4):441–54. [16] Stone JM, Erlandsson K, Arstad E, Bressan RA, Squassante L, Teneggi V, et al. Ketamine displaces the novel NMDA receptor SPET probe [(123)I]CNS-1261 in humans in vivo. Nucl Med Biol 2006;33(2):239–43. [17] Owens J, Tebbutt AA, McGregor AL, Kodama K, Magar SS, Perlman ME, et al. Synthesis and binding characteristics of N-(1-naphthyl)-N′-(3-[(125)I]-iodophenyl)-N′-methylguanidine ([(125)I]-CNS 1261): a potential SPECT agent for imaging NMDA receptor activation. Nucl Med Biol 2000;27(6):557–64. [18] Ohe Y, Nakazato Y, Ohkuma A, Tamura N, Takahashi Y, Araki N. A case of occipital epilepsy with anti-GluRepsilon2 antibody in cerebrospinal fluid, presenting as repeated visual disturbance and headache. Rinsho Shinkeigaku 2011;51(7): 505–9. [19] Lipton SA. Paradigm shift in NMDA receptor antagonist drug development: molecular mechanism of uncompetitive inhibition by memantine in the treatment of Alzheimer's disease and other neurologic disorders. J Alzheimers Dis 2004;6(6 Suppl):S61–74. [20] Zhou XQ, Zhang Jiankang. A preliminary study on a new memantine derivative [99Tcm]-N-3,5-dimethyltricyclo[3,3,1]decanyl-2-((2-mercaptoethyl) (2-(2-mercaptoethylamino)-2-oxoethyl)amino) Acetamide. J Nucl Med 2008;49(Suppl 1): 310. [21] Meegallas K, PlÖssl K, Kung MP, et al. Synthesis and characterization of technetium-99m-labeled tropanes as dopamine transporter-imaging agents. J Med Chem 1997;40(1):9–17. [22] Takahashi K, Sakano H, Rytting HJ, et al. Influence of pH on the permeability of p-toluidine and aminopyrine through shed snake skin as a model membrane. Drug Dev Ind Pharm 2001;27:159–64. [23] Han FZ, Peng T, Wang L, et al. Protection of rhodioside derivative SO1 against damage of SH-SY5Y cells induced by glutamate and oxygen-glucose deprivation. Chin J Pharmacol Toxicol 2008;22(3):180–5. [24] Arbab AS, Koizumi K, Toyama K, et al. Uptake of technetium-99m-tetrofosmin, technetium-99m-MIBl and thallium-201 in tumor cell line. J Nud Med 1996;37: 1551–6. [25] Reynolds IJ, Sharma TA. Methods. Mol Biol 1999;128:93–102. [26] Katsel P, Davis KL, Gorman JM, Haroutunian V. Variations in differential gene expression patterns across multiple brain regions in schizophrenia. Schizophrenia Research 2005;77:241–52. [27] Benes FM. Emerging principles of altered neural circuitry in schizophrenia. Brain Res Brain Res Rev 2000;31:251–69. [28] Olney JW. Excitatory amino acids and neuropsychiatric disorders. Biol Psychiatry 1989;26:505–25. [29] Waterhouse RN, Slifstein M, Dumont F, Zhao J, Chang RC, Sudo Y, et al. In vivo evaluation of [11C]N-(2-chloro-5-thiomethylphenyl)-N′-(3-methoxy-phenyl)-N ′-methylguanidine ([11C]GMOM) as a potential PET radiotracer for the PCP/NMDA receptor. Nucl Med Biol 2004;31(7):939–48. [30] Kokic M, Honer M, Kessler LJ, Grauert M, Schubiger PA, Ametamey SM. Synthesis and in vitro and in vivo evaluation of [11C]methyl-BIII277CL for imaging the PCPbinding site of the NMDA receptor by pet. J Recept Signal Transduct Res 2002;22(1-4):123–39. [31] Matsumoto R, Haradahira T, Ito H, et al. Measurement of glycine binding site of Nmethyl-D–asparate receptors in living human brain using 4-acetoxy derivative of L-703,717,4-acetoxy-7-chloro-3-[3-(4-[11C] methoxybenzyl) phenyl]-2(1H)quinolone (AcL703) with positron emission tomography. Synapse 2007;61(10): 795–800. [32] Haradahira T, Zhang MR, Maeda J, et al. A strategy for increasing the brain uptake of a radioligand in animals: use of a drug that inhibits plasma protein binding. Nucl Med Biol 2000;27(4):357–60. [33] Ametamey SM, Bruehlmeier M, Kneifel S, et al. PET studies of [18F]-memantine in healthy volunteers. Nucl Med Biol 2002;29:227–31. [34] Xie Q, Hao J, Qiu Z. Advanced research on novel analgesic targets of ion channels. Chin J Pharm 2004;35(5):304–9. [35] Seeman P, Ko F, Tallerico T. Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics. Mol Psychiatry 2005;10(9):877–83. [36] Waterhouse RN. Imaging the PCP site of the NMDA ion channel. Nucl Med Biol 2003;30(8):869–78.