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Biomedical subjects

H F Kung

Publications and source records attributed to H F Kung.

At least 55 records · Page 3Linked to original sources
PubMed2000

Quantitative autoradiographic mapping of rat brain dopamine D3 binding with [(125)I]7-OH-PIPAT: evidence for the presence of D3 receptors on dopaminergic and nondopaminergic cell bodies and terminals.

The regional distribution and cellular localization of dopamine D3 receptors in the rat brain was examined using quantitative autoradiography. [(125)I]7-OH-PIPAT bound in a saturable and reversible manner and exhibited subnanomolar affinity for a single population of GTP-insensitive sites. The pharmacological profile was characteristic of cloned D3 receptors and nonspecific binding was uniformly low. The highest levels of D3 receptors were measured in the islands of Calleja, nucleus accumbens, ventral pallidum, substantia nigra, and lobules 9 and 10 of the cerebellum. The high specific activity of this ligand also allowed detection of D3 receptors in other regions, including the serotonergic dorsal and median raphe nuclei, indicating that the distribution of this receptor is more widespread than previously appreciated. The cellular localization of D3 receptors in regions containing dopaminergic cells and terminals was examined by discrete injection of neurotoxins. Lesion of dopaminergic neurons with 6-hydroxydopamine produced 50% decreases in [(125)I]7-OH-PIPAT binding in the nucleus accumbens and substantia nigra. Quinolinic acid lesion of neurons originating in the nucleus accumbens also produced approximately 50% decreases in D3 receptors in the nucleus accumbens, substantia nigra, and ventral pallidum. 5, 7-Dihydroxytryptamine lesion of serotonergic cells and processes produced no changes in [(125)I]7-OH-PIPAT binding. These results demonstrate the presence of D3 receptors in several brain regions not previously identified and suggest that D3 receptors are expressed at somatodendritic and terminal levels of both dopaminergic and nondo-paminergic cells within the mesolimbic dopamine system.

5,7-Dihydroxytryptamine↗
PubMed1999

Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active.

We analyzed the structural and functional organization of anti-HIV and anti-tumor proteins MAP30 and GAP31 by limited proteolysis with endopeptidases Lys-C and Glu-C (V8). MAP30 and GAP31 are resistant to proteolytic digestion under conditions of as much as 5% (w/w) proteases. In the presence of 10% (w/w) protease, the central regions of the proteins are still resistant to proteolysis, whereas the N- and C-termini are accessible. Peptide fragments were purified by FPLC on Superdex 75 columns, characterized by gel electrophoresis, identified by amino acid sequencing, and analyzed for anti-HIV, anti-tumor, and other biochemical activities. We report here that limited proteolysis yields biologically active fragments of both MAP30 and GAP31. These fragments are active against HIV-1 and tumor cells with EC(50)s in the sub-nanomolar ranges, 0.2-0.4 nM. At the dose levels used in the assays, little cytotoxicity to normal cells was observed. In addition, these fragments remain fully active in HIV-integrase inhibition and HIV-LTR topological inactivation, but not ribosome inactivation. These results demonstrate that the antiviral and anti-tumor activities of MAP30 and GAP31 are independent of ribosome inactivation activity. In addition, we demonstrate that portions of the N- and C-termini are not essential for antiviral and anti-tumor activities, but do appear to be required for ribosome inactivation. These results may provide novel strategies for rational design and targeted development of mimetic antiviral and anti-tumor therapeutics.

Amino Acid Sequence↗
PubMed1999

Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) preparations contain activity against HIV type 1 (HIV-1). However, there has been controversy about whether some biological activities of hCG beta-subunit (hCGbeta) preparations are caused by the beta-subunit itself or other proteins present in the preparations. We report here the purification, characterization, and identification of three enzymes with anti-HIV activity present in the beta-core fraction of hCGbeta prepared from the urine of pregnant women. The N-terminal amino acid sequence of one protein is identical to human urinary lysozyme C, and those of the other two are identical to human RNase A and urinary RNase U. We thus refer to these proteins as AVL (antiviral lysozyme) and AVR (antiviral RNases). In addition to HIV-1 inhibition, AVL is capable of lysing Micrococcus lysodeikticus. AVR digests a variety of RNA substrates, including RNA from HIV-1-infected cells. We also find that lysozyme from chicken egg white, human milk, and human neutrophils and RNase A from bovine pancreas possess activity against HIV-1. These findings may offer additional strategies for the treatment of HIV-1 infection.

Amino Acid Sequence↗
PubMed1999

The presence and absence of histocompatibility antigens in HIV type 1 produced by autologous blood-derived macrophages and peripheral blood lymphoblasts.

Acquisition of cellular proteins by HIV-1 virions is known to alter the physiology of the virus in vitro. Reported studies of this aspect have been largely limited to transformed T cell lines. In this study, we investigated the incorporation of major histocompatibility antigens (HLAs) on a primary macrophage-tropic isolate, HIV-1ADA, grown from autologous monocyte-derived macrophages (MDMs) and peripheral blood mononuclear cells (PBMCs). A virus precipitation assay (VPA) demonstrated that HIV-1ADA grown from PBMCs incorporated substantial amounts of HLA class I (alpha chain and beta2m) and DR antigens, comparable with a laboratory strain, HIV-1MN, grown from the same host cells. HIV-1ADA, however, grown from MDMs incorporated significantly lower amounts of HLAI and -II antigens despite the fact that the infected MDMs were found to express significant amounts of HLA antigens. The lack of incorporation of these important immunomodulatory cell surface proteins may be yet another unique characteristic of macrophage-tropic isolates and suggests a possible role in their biology and or immunology.

Chemical Precipitation↗
PubMed1999

In vivo imaging of serotonin transporters with [99mTc]TRODAT-1 in nonhuman primates.

[99mTc]TRODAT-1 was the first 99mTc-labeled imaging agent to show specific binding to dopamine transporters (DAT) in the striatum (STR) of human brain. Additionally, in vitro binding and autoradiographic experiments demonstrated that this tracer also binds to serotonin transporters (SERT) in the midbrain/hypothalamus (MB) area. In this study, [99mTc]TRODAT-1 was investigated as a potentially useful ligand to image SERT in the MB of living brain. A total of eight single-photon emission tomography (SPET) scans were performed in two baboons (Papio anubis) after intravenous (i.v.) injection of 740 MBq (20 mCi) of [99mTc]TRODAT-1 using a triple-head gamma camera equipped with ultra-high-resolution fan-beam collimators (scan time: 0-210 min). In four blocking studies, baboons were pretreated with (+)McN5652 (1 mg/kg, i.v.) or methylphenidate (1 mg/kg, i.v.) to specifically block SERT or DAT, respectively. After co-registration with magnetic resonance images of the same baboon, a region of interest analysis was performed using predefined templates to calculate specific uptake in the midbrain area and the striatum, with the cerebellum as the background region [(MB-CB)/CB, (STR-CB)/CB]. Additionally, two PET scans of the same baboons were performed after i.v. injections of 74-111 MBq (2-3 mCi) of [11C](+)McN5652 to identify the SERT sites. In [99mTc]TRODAT-1/SPET scans, the SERT sites in the MB region were clearly visualized. Semiquantitative analysis revealed a specific uptake in MB ([MB-CB]/CB) of 0.30+/-0.02, which was decreased to 0. 040+/-0.005 after pretreatment with nonradioactive (+)McN5652, a selective SERT ligand. Pretreatment with methylphenidate reduced the specific binding of [99mTc]TRODAT-1 to DAT sites [(STR-CB)/CB] from 2.45+/-0.13 to 0.32+/-0.04 without any effect on its binding to SERT sites [(MB-CB)/CB], which was confirmed by the co-registration of the [11C](+)McN5652/PET scans. This preliminary study suggests that specific binding of [99mTc]TRODAT-1 to SERT sites can be detected by in vivo SPET imaging despite the low target to background ratio. These findings provide impetus for further development of similar compounds with improved binding affinity and selectivity to SERT sites.

Adrenergic Uptake Inhibitors↗
PubMed1999

Simplified reference region model for the kinetic analysis of [99mTc]TRODAT-1 binding to dopamine transporters in nonhuman primates using single-photon emission tomography.

Accurate quantification of neuroreceptors requires full kinetic modeling of the dynamic single-photon emission tomography (SPET) or positron emission tomography (PET) images, with highly invasive arterial blood sampling. This study investigated the application of a reference region kinetic model to the dynamics of [99mTc]TRODAT-1 in nonhuman primates, obviating the need for blood sampling. A series of dynamic SPET scans were performed on two baboons following the injection of approximately 700 MBq of [99mTc]TRODAT-1. Rapid arterial blood samples were taken automatically during scanning. Reconstructed SPET images were coregistered with magnetic resonance imaging (MRI) scans of the baboons, and regions of interest (ROIs) placed on the striatum, cerebellum and cerebral hemispheres. The ROI data were combined with metabolite-corrected blood data, and fitted to a three-compartment kinetic model using nonlinear least squares techniques. The same data were also used in a simplified reference region model, in which the input function was derived from the nondisplaceable tissue compartment. In addition, semiquantitative blinded analysis was performed by three raters to determine the point of transient equilibrium in the specific binding curves. All methods generated values for the ratio of the kinetic rate constants k3/k4, which gives an estimate of the binding potential, BP. These were compared with the full kinetic model. The mean values of k3/k4 for the three different analysis techniques for each baboon were: 1.17 +/- 0.21 and 1.12 +/- 0.13 (full kinetic model), 0.93 +/- 0.13 and 0.90 +/- 0.07 (reference region model), and 0.97 +/- 0.18 and 0.92 +/- 0.08 (equilibrium method). The reference region method gave significantly lower results than the full kinetic model (P = 0.01), but it also produced a much smaller spread and better quality fits to the kinetic data. The reference region model results for k3/k4 correlated very strongly with the full kinetic analysis (r2 = 0.992, P < 0.001), and with the equilibrium model (r2 = 0.88, P = 0.002). The subjectivity inherent in the equilibrium method produces inferior results compared with both kinetic analyses. It is suggested that the self-consistency of the reference region model, which requires no arterial blood sampling, provides a more precise and reliable estimate of the binding of [99mTc]TRODAT-1 to dopamine transporters than full kinetic modeling. The reference region method is also better suited to a routine clinical environment, and would be able to distinguish smaller differences in dopaminergic function between patient groups.

Animals↗
PubMed1999

Automatic segmentation of dynamic neuroreceptor single-photon emission tomography images using fuzzy clustering.

The segmentation of medical images is one of the most important steps in the analysis and quantification of imaging data. However, partial volume artefacts make accurate tissue boundary definition difficult, particularly for images with lower resolution commonly used in nuclear medicine. In single-photon emission tomography (SPET) neuroreceptor studies, areas of specific binding are usually delineated by manually drawing regions of interest (ROIs), a time-consuming and subjective process. This paper applies the technique of fuzzy c-means clustering (FCM) to automatically segment dynamic neuroreceptor SPET images. Fuzzy clustering was tested using a realistic, computer-generated, dynamic SPET phantom derived from segmenting an MR image of an anthropomorphic brain phantom. Also, the utility of applying FCM to real clinical data was assessed by comparison against conventional ROI analysis of iodine-123 iodobenzamide (IBZM) binding to dopamine D2/D3 receptors in the brains of humans. In addition, a further test of the methodology was assessed by applying FCM segmentation to [123I]IDAM images (5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio] benzyl alcohol) of serotonin transporters in non-human primates. In the simulated dynamic SPET phantom, over a wide range of counts and ratios of specific binding to background, FCM correlated very strongly with the true counts (correlation coefficient r2>0.99, P<0.0001). Similarly, FCM gave segmentation of the [123I]IBZM data comparable with manual ROI analysis, with the binding ratios derived from both methods significantly correlated (r2=0.83, P<0.0001). Fuzzy clustering is a powerful tool for the automatic, unsupervised segmentation of dynamic neuroreceptor SPET images. Where other automated techniques fail completely, and manual ROI definition would be highly subjective, FCM is capable of segmenting noisy images in a robust and repeatable manner.

Animals↗
PubMed1999

Characterization of [(123)I]IDAM as a novel single-photon emission tomography tracer for serotonin transporters.

Development of selective serotonin transporter (SERT) tracers for single-photon emission tomography (SPET) is important for studying the underlying pharmacology and interaction of specific serotonin reuptake site inhibitors, commonly used antidepressants, at the SERT sites in the human brain. In search of a new tracer for imaging SERT, IDAM (5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol) was developed. In vitro characterization of IDAM was carried out with binding studies in cell lines and rat tissue homogenates. In vivo binding of [(125)I]IDAM was evaluated in rats by comparing the uptakes in different brain regions through tissue dissections and ex vivo autoradiography. In vitro binding study showed that IDAM displayed an excellent affinity to SERT sites (K(i)=0.097 nM, using membrane preparations of LLC-PK(1) cells expressing the specific transporter) and showed more than 1000-fold of selectivity for SERT over norepinehrine and dopamine (expressed in the same LLC-PK(1) cells). Scatchard analysis of [(125)I]IDAM binding to frontal cortical membrane homogenates prepared from control or p-chloroamphetamine (PCA)-treated rats was evaluated. As expected, the control membranes showed a K(d) value of 0.25 nM+/-0.05 nM and a B(max) value of 272+/-30 fmol/ mg protein, while the PCA-lesioned membranes displayed a similar K(d), but with a reduced B(max) (20+/-7 fmol/ mg protein). Biodistribution of [(125)I]IDAM (partition coefficient =473; 1-octanol/buffer) in the rat brain showed a high initial uptake (2.44%dose at 2 min after i.v. injection) with the specific binding peaked at 60 min postinjection (hypothalamus-cerebellum/cerebellum =1.75). Ex vivo autoradiographs of rat brain sections (60 min after i.v. injection of [(125)I]IDAM) showed intense labeling in several regions (olfactory tubercle, lateral septal nucleus, hypothalamic and thalamic nuclei, globus pallidus, central gray, superior colliculus, substantia nigra, interpeduncular nucleus, dorsal and median raphes and locus coeruleus), which parallel known SERT density. This novel tracer has excellent characteristics for in vivo SPET imaging of SERT in the brain.

Animals↗
PubMed1999

Single-photon emission tomography imaging of serotonin transporters in the non-human primate brain with the selective radioligand [(123)I]IDAM.

A new radioligand, 5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol ([(123)I]IDAM), has been developed for selective single-photon emission tomography (SPET) imaging of SERT. In vitro binding studies suggest a high selectivity of IDAM for SERT (K(i)=0.097 nM), with considerably lower affinities for norepinephrine and dopamine transporters (NET K(i)= 234 nM and DAT K(i)>10 microM, respectively). In this study the biodistribution of SERT in the baboon brain was investigated in vivo using [(123)I]IDAM and SPET imaging. Dynamic sequences of SPET scans were performed on three female baboons (Papio anubis) after injection of 555 MBq of [(123)I]IDAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 90-120 min after injection of [(123)I]IDAM. Similar studies were performed using a NET inhibitor, nisoxetine, and a DAT blocker, methylphenidate. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT, with the highest uptake in the midbrain area (hypothalamus, raphe nucleus, substantia nigra), and the lowest uptake in the cerebellum (an area presumed free of SERT). Peak specific binding in the midbrain occurred at 120 min, with a ratio to the cerebellum of 1.80+/-0.13. At 30 min, 85% of the radioactivity in the blood was metabolite. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited rapid washout from areas with high concentrations of SERT (dissociation rate constant in the midbrain, averaged over three baboons, k(off)=0. 025+/-0.002 min(-1)), while the cerebellar activity distribution was undisturbed (washout rate 0.0059+/- 0.0003 min(-1)). Calculation of tracer washout rate pixel-by-pixel enabled the generation of parametric images of the dissociation rate constant. Similar studies using nisoxetine and methylphenidate had no effect on the distribution of [(123)I]IDAM in the brain. These results suggest that [(123)I]IDAM is suitable for selective SPET imaging of SERT in the primate brain, with high contrast, favorable kinetics, and negligible binding to either NET or DAT.

Animals↗
PubMed1999

Logistic discriminant parametric mapping: a novel method for the pixel-based differential diagnosis of Parkinson's disease.

Positron emission tomography (PET) and single-photon emission tomography (SPET) imaging of the dopaminergic system is a powerful tool for distinguishing groups of patients with neurodegenerative disorders, such as Parkinson's disease (PD). However, the differential diagnosis of individual subjects presenting early in the progress of the disease is much more difficult, particularly using region-of-interest analysis where small localized differences between subjects are diluted. In this paper we present a novel pixel-based technique using logistic discriminant analysis to distinguish between a group of PD patients and age-matched healthy controls. Simulated images of an anthropomorphic head phantom were used to test the sensitivity of the technique to striatal lesions of known size. The methodology was applied to real clinical SPET images of binding of technetium-99m labelled TRODAT-1 to dopamine transporters in PD patients (n=42) and age-matched controls (n=23). The discriminant model was trained on a subset (n=17) of patients for whom the diagnosis was unequivocal. Logistic discriminant parametric maps were obtained for all subjects, showing the probability distribution of pixels classified as being consistent with PD. The probability maps were corrected for correlated multiple comparisons assuming an isotropic Gaussian point spread function. Simulated lesion sizes measured by logistic discriminant parametric mapping (LDPM) gave strong correlations with the known data (r(2)=0. 985, P<0.001). LDPM correctly classified all PD patients (sensitivity 100%) and only misclassified one control (specificity 95%). All patients who had equivocal clinical symptoms associated with early onset PD (n=4) were correctly assigned to the patient group. Statistical parametric mapping (SPM) had a sensitivity of only 24% on the same patient group. LDPM is a powerful pixel-based tool for the differential diagnosis of patients with PD and healthy controls. The diagnosis of disease even before clinical symptoms become apparent may be possible, and ultimately this technique could be most useful in differentiating between several neurodegenerative disorders, incorporating images of multiple neuroreceptor systems.

Aged↗
PubMed1999

Single-photon emission tomography imaging of serotonin transporters in the nonhuman primate brain with [(123)I]ODAM.

We have described previously a selective serotonin transporter (SERT) radioligand, [(123)I]IDAM. We now report a similarly potent, but more stable IDAM derivative, 5-iodo-2-[2-[(dimethylamino)methyl]phenoxy]benzyl alcohol ([(123)I]ODAM). The imaging characteristics of this radioligand were studied and compared against [(123)I]IDAM. Dynamic sequences of single-photon emission tomography (SPET) scans were obtained on three female baboons after injection of 375 MBq of [(123)I]ODAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 120 min after injection of [(123)I]ODAM. Total integrated brain uptake of [(123)I]ODAM was about 30% higher than [(123)I]IDAM. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT. Peak specific binding in the midbrain occurred 120 min after injection, with an equilibrium midbrain to cerebellar ratio of 1. 50+/-0.08, which was slightly lower than the value for [(123)I]IDAM (1.80+/- 0.13). Both the binding kinetics and the metabolism of [(123)I]ODAM were slower than those of [(123)I]IDAM. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited washout from areas with high concentrations of SERT, with a dissociation kinetic rate constant k(off)=0.0085+/-0.0028 min(-1) in the midbrain. Similar studies using nisoxetine and methylphenidate showed no displacement, consistent with its low binding affinity to norepinephrine and dopamine transporters, respectively. These results suggest that [(123)I]ODAM is suitable for selective SPET imaging of SERT in the primate brain, with higher uptake and slower kinetics and metabolism than [(123)I]IDAM, but also a slightly lower selectivity for SERT.

Animals↗
PubMed1999

Neutral and stereospecific Tc-99m complexes: [99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT).

Technetium-99m-labeled radiopharmaceuticals are currently the most commonly used agents in nuclear medicine. To prepare binding site-specific small molecules containing a Tc-99m complexing core, it is important to consider a ligand system, which selectively forms only one stereoisomer. A novel series of bisaminoethanethiol (BAT) derivatives as a model system were prepared. Stereoisomers of N-benzyl-3,4-di(N-2-mercaptoethyl)-amino pyrrolidines (P-BAT): (3R,4R)-P-BAT (R,R-4) and (3,4)meso-P-BAT (8), the trans and meso isomer, respectively, as a chelating group were prepared successfully. The desired Tc-99m P-BAT complexes were obtained by using Sn(II)/glucoheptonate as the reducing agent for [99mTc]pertechnetate. As predicted, after complexation with [99mTc]Tc'O, the trans isomer, (3R,4R)-P-BAT (R,R-4), showed only one isomer; whereas the corresponding meso isomer, (3,4)meso-P-BAT (8), produced two distinctive complexes isolated readily by high performance liquid chromatography (HPLC). The [99mTc](R,S) meso-P-BAT (8) isomers showed a different lipophilicity (partition coefficient [P.C.] = 54.3 and 55.4 for peak A and peak B, respectively), as compared with that of the corresponding [99mTc](3R,4R)-P-BAT (R,R-4), trans isomer (P.C. = 163). Results of the biodistribution study in rats of these isomers show different heart and brain uptake, suggesting that the intrinsic differences in biodistribution are due to structural and stereospecific factors. Examples in this report confirm that it is possible to design stereospecific Tc-99m complexes based on the bisaminoethanethiol (N2S2, BAT) ligand system. Consideration on stereoselectivity of site-specific agents labeled with Tc-99m is likely an essential requirement on developing binding-site specific radiopharmaceuticals.

Animals↗
PubMed1999

An improved kit formulation of a dopamine transporter imaging agent: [Tc-99m]TRODAT-1.

Recently, [Tc-99m]TRODAT-1, the first Tc-99m-labeled tracer for imaging CNS dopamine transporters in humans, was reported. This tracer displayed excellent specific binding to dopamine transporters in the basal ganglia region of the brain, thus it is potentially useful for the diagnosis of deficit of dopamine transporters in neurodegenerative diseases, such as Parkinson's disease. Preparation of [Tc-99m]TRODAT-1 was previously achieved by a multistep kit formulation. It is highly desirable to further improve the preparation by developing a simplified one-vial formulation with a reduced amount of TRODAT-1 ligand for routine clinical use. To achieve this goal, a series of studies to optimize labeling efficiency by varying a combination of factors (amount of free ligand, reaction reagents, and reaction pH) was carried out. [Tc-99m]TRODAT-1 prepared by this new kit formulation was evaluated by assessing the brain uptake and target (striatum) versus nontarget (cerebellum) ratios in rats. Appropriate amounts of various ingredients for a one-vial kit formulation providing > or =90% radiolabeling yields were identified. The most consistent and reliable formulation contained 10 microg of TRODAT-1 (a reduction of free ligand from 200 microg to 10 microg), 32 microg of SnCl2, 10 mg of sodium glucoheptonate, and 840 microg of disodium EDTA in one vial as a lyophilized kit. It is feasible to reconstitute the vial with [Tc-99m]pertechnetate (0.5-2 mL, < or =1110 MBq, 30 mCi), resulting in a final solution with a pH value of 4.5-5.0. [Tc-99m]TRODAT-1, prepared by this new kit, was stable at room temperature for 6 h. Biodistribution studies of this agent in rats with the new formulation showed similar regional brain distribution as compared with those obtained with the previous preparation (high striatum-to-cerebellum ratio). In conclusion, using this lyophilized one-vial kit formulation, [Tc-99m]TRODAT-1 can be prepared with greater than 90% radiochemical purity. This simplified kit will significantly improve the reliability of preparation of this agent for routine clinical use.

Animals↗
PubMed1999

Synthesis of radioiodinated 1-deoxy-nojirimycin derivatives: novel glucose analogs.

Novel lipophilic and neutral glucose analogs, which are potentially useful for tumor imaging, have been developed. They are designed to circumvent Glut-facilitated transport mechanism, and direct the localization by either hexokinase binding or enzyme reactions (phosphorylation) as potential metabolic markers of tumor cells. Syntheses of tetraacetylated N-(3'-iodo-2'-propenyl)-1-deoxy-nojirimycin (11) and N-(3'-iodo-benzyl)-1-deoxy-nojirimycin (14) were achieved by reacting 1-deoxy-nojirimycin with appropriate alkylating agents. The corresponding tri-butyltin derivatives were also prepared as the starting materials for preparation of I-125-labeled compounds for biodistribution study in rats. Biodistribution in rats showed that [123I]14 exhibited a modest initial brain uptake and retention at a later time (0.59, 0.38, 0.30, and 0.30% dose/organ at 2, 30, 60, and 120 min after an intravenous [i.v.] injection, respectively), whereas [125I]11 displayed a lower brain uptake (0.35, 0.27, 0.20, and 0.18% dose/organ at 2, 30, 60, and 120 min). In addition, compounds with free hydroxyl groups (12 and 13) were also obtained. As expected, after an i.v. injection, these free hydroxyl compounds showed a dramatic decrease in brain uptake in rats. It appears that both of the acetylated agents (11 and 14), which display higher lipophilicity (partition coefficient [P.C.] = 57.9 and 1,462, respectively), can penetrate the blood-brain barrier (BBB) by a simple diffusion mechanism whereas the free hydroxy compounds (12 and 13), with lower lipophilicity (P.C. = 0.43 and 6.8), showed no brain uptake. A similar pair of glucose derivatives, fluorodeoxyglucose (FDG) and tetraacetylated FDG (AFDG), displayed a dramatic difference in brain uptake in rats. While the lipophilic AFDG (P.C. = 3.79) may penetrate the intact BBB, due to its relatively low P.C. value, the first pass extraction due to simple diffusion mechanism may be low (brain uptake at 2 min was 0.68% dose/organ). The FDG itself has a very low lipophilicity (P.C. = 0.22) but it can be taken up into the brain by a glucose transporter mediated mechanism to cross the BBB (brain uptake at 2 min was 2.53% dose/organ). Preliminary data of these glucose derivatives suggest that further studies are needed to elucidate the uptake and retention mechanisms and their potential application as tumor imaging agents.

1-Deoxynojirimycin↗
PubMed1999

A simplified method to determine [99mTc]TRODAT-1 in human plasma.

[99mTc]TRODAT-1 is a useful imaging agent in evaluating changes in presynaptic dopamine transporters (DAT) for Parkinson's disease and other central nervous system (CNS) neurodegenerative diseases, for which a reduction of dopamine neurons is indicated. As part of an effort to establish a quantitative single-photon emission tomography (SPECT) procedure for imaging CNS DAT, measurement of nonmetabolized [99mTc]TRODAT-1 in human plasma was investigated. After an intravenous injection of [99mTc]TRODAT-1, there are three possible radioactive components in human plasma: hydrophilic compounds (pertechnetate, etc.), lipophilic metabolite(s), and unchanged [99mTc]TRODAT-1. Based on the differences in lipophilicity of [99mTc]TRODAT-1 and its lipophilic metabolite [99mTc]BAT, a new quantitative method for measuring [99mTc]TRODAT-1 with a simple solvent extraction method was developed. Various organic solvents or mixtures of solvents were tested, among which cyclohexane gave the best extraction yield for [99mTc]TRODAT-1 (76.06 +/- 3.32%) with a low extraction for [99mTc]BAT (2.43 +/- 0.82%). Extractions of [99mTc]TRODAT-1 and [99mTc]BAT mixtures in different predetermined ratios to simulate the actual human plasma samples with cyclohexane from phosphate buffer (5 mM, pH 8.0) were evaluated. The experimentally obtained ratios were in good agreement with the theoretical ratios. To investigate further the possibility of replacing the previously established high performance liquid chromatography (HPLC) method with the new solvent extraction method for the clinical application, both HPLC and extraction methods were used side by side to determine the unchanged [99mTc]TRODAT-1 in human plasma samples during [99mTc]TRODAT-1/SPECT imaging studies. The results from four human subjects showed that both methods consistently produced similar values for unchanged [99mTc]TRODAT-1 in the plasma samples. This improved solvent extraction method provides an easy and reliable technique to quantify unchanged [99mTc]TRODAT-1 in human plasma, thus making the clinical application of this agent readily available for quantitation of the DAT binding sites in the brain by SPECT imaging.

Brain↗
PubMed1999

Immunodetection of Xenopus bone morphogenetic protein-1 in adult and embryonic cells.

In order to analyze biochemical properties of Xenopus bone morphogenetic protein-1 (XBMP-1), rabbit antiserum (alpha-B1) was raised against a synthetic peptide (P1) corresponding to a hydrophilic N-terminal region. XBMP-1B (Xtld) synthesized in the reticulocyte lysate was successfully immunoprecipitated by this antiserum. This precipitation was completely blocked when P1 was added to the reaction, indicating that alpha-B1 recognized XBMP-1B specifically. In Western blot analysis, two distinct sizes of protein (107 and 34 kD) were detected in hind limbs in metamorphosing animals. Both proteins were detected in various adult tissues such as lung, liver, kidney, heart, muscle, intestine, brain, and testis. The mixing of the liver and muscle extracts, and the following detection of immunoreactive proteins suggested that the 34 kD band was a proteolytic product of the 107 kD protein. In the embryonic extracts from the unfertilized egg (stage 0) to swimming tadpoles (stage 40), a 63 kD protein was detected in addition to the 107 kD protein. We also showed that the 107 kD protein was much more expressed in the animal half of the unfertilized eggs than in the vegetal half, but that it was ubiquitously expressed in the gastrula embryos. We suggest that the 63 and 107 kD proteins correspond to full-length proteins encoded by XBMP-1A and XBMP-1B genes, and these proteins are expressed in embryo and in various adult tissues.

Animals↗
PubMed1999

Simultaneous SPECT studies of pre- and postsynaptic dopamine binding sites in baboons.

UNLABELLED: The central nervous system dopamine transporters (DATs) and dopamine D2/D3 receptors are implicated in a variety of neurological disorders. Both sites are also targets for drug treatment. With the successful development of [99mTc]TRODAT-1, single-isotope imaging studies using this ligand for DAT imaging can be complemented by additional use of 123I-labeled D2/D3 receptor ligand co-injected to assess both pre- and postsynaptic sites of the dopaminergic system simultaneously. METHODS: Twelve SPECT scans of the brain were obtained in two baboons after intravenous administration of 740 MBq (20 mCi) [99mTc]-TRODAT-1 (technetium, [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3,2,1]oct-2-yl]methyl ](2-mercaptoethyl) amino]ethyl]-amino]ethanethiolato (3-)]- oxo-[1R-(exo-exo)]) and 185 MBq (5 mCi) [123I]iodobenzamide or [123I]iodobenzofuran. SPECT data were acquired by a triple-head gamma camera equipped with ultra-high-resolution fanbeam collimators (scan duration = 210 min). Two sets of SPECT data were obtained using energy windows of 15% centered on 140 keV for 99mTc and 10% asymmetric with a lower bound at 159 keV for 123I. After coregistration with MRI, region-of-interest analysis was performed using predefined templates from coregistered MRI. In blocking studies, baboons were pretreated with N-methyl-2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT, 14 mg) or raclopride (14 mg) to block DAT or D2/D3 binding site, respectively. RESULTS: Image quality of dual-isotope studies was similar to that obtained from single-isotope studies. When one site was blocked with CFT or raclopride, the binding of the respective ligand to the other site was not affected. CONCLUSION: This is the first example that clearly demonstrates the feasibility of simultaneous imaging of both pre- and postsynaptic sites of the dopaminergic system in baboons with dual-isotope SPECT studies. With or without corrections for cross-contamination of 123I into the 99mTc window, striatum-to-cerebellum ratios (target-to-nontarget) of dual-isotope experiments did not differ significantly from single-isotope experiments. This method may be a valuable and cost-effective tool for gaining comprehensive information about the dopaminergic system in one SPECT imaging session.

Animals↗