Pradeep K. Garg

Recent Trends in Antibody-based Oncologic Imaging

Antibodies, with their unmatched ability for selective binding to any target, are considered as potentially the most specific probes for imaging. Their clinical utility, however, has been limited chiefly due to their slow clearance from the circulation, longer retention in non-targeted tissues and the extensive optimization required for each antibody-tracer. The development of newer contrast agents, combined with improved conjugation strategies and novel engineered forms of antibodies (diabodies, minibodies, single chain variable fragments, and nanobodies), have triggered a new wave of antibody-based imaging approaches. Apart from their conventional use with nuclear imaging probes, antibodies and their modified forms are increasingly being employed with non-radioisotopic contrast agents (MRI and ultrasound) as well as newer imaging modalities, such as quantum dots, near infra red (NIR) probes, nanoshells and surface enhanced Raman spectroscopy (SERS). The review article discusses new developments in the usage of antibodies and their modified forms in conjunction with probes of various imaging modalities such as nuclear imaging, optical imaging, ultrasound, MRI, SERS and nanoshells in preclinical and clinical studies on the diagnosis, prognosis and therapeutic responses of cancer.

Kinetics of brain nicotine accumulation in dependent and nondependent smokers assessed with PET and cigarettes containing 11C-nicotine

Tobacco smoking is a chronic, relapsing disorder that constitutes one of the primary preventable causes of death in developed countries. Two of the popular hypotheses to explain the development and maintenance of strong nicotine dependence in cigarette smokers posit (i) a rapid brain nicotine accumulation during cigarette smoking and/or (ii) puff-associated spikes in brain nicotine concentration. To address these hypotheses, we investigated the dynamics of nicotine accumulation in the smokers brain during actual cigarette smoking using PET with 3-s temporal resolution and 11C-nicotine loaded into cigarettes. The results of the study, performed in 13 dependent smokers (DS) and 10 nondependent smokers (NDS), suggest that puff-associated spikes in the brain nicotine concentration do not occur during habitual cigarette smoking. Despite the presence of a puff-associated oscillation in the rate of nicotine accumulation, brain nicotine concentration gradually increases during cigarette smoking. The results further suggest that DS have a slower process of brain nicotine accumulation than NDS because they have slower nicotine washout from the lungs and that DS have a tendency to compensate for their slower rate of brain nicotine accumulation compared with NDS by inhaling a larger volume of smoke. For these reasons, smokers’ dependence on cigarette smoking, or the resistance of NDS to becoming dependent, cannot be explained solely by a faster brain nicotine accumulation.

Effect of Menstrual Cycle Phase on Dopamine D2 Receptor Availability in Female Cynomolgus Monkeys

Sex differences have been reported in a variety of affective and neurodegenerative disorders that involve dysfunctional dopamine (DA) neurotransmission. In addition, there is evidence for differences in sensitivity to the abuse-related effects of psychostimulants across the menstrual cycle which may result from effects of ovarian hormones on DA function. The goal of the present study was to extend previous work examining menstrual cycle-related changes in DA D2 receptor availability in humans to drug-naive female cynomolgus monkeys (n=7) using the selective D2-like receptor ligand [18F]fluoroclebopride (FCP) and a high-resolution microPET P4 scanner. Menstrual cycle phase was characterized by daily vaginal swabs and measurements of serum progesterone levels. PET studies were conducted once during the luteal phase and once during the follicular phase. Regions of interest in the caudate nucleus, putamen, and cerebellum were defined on coregistered MRIs. Distribution volumes were calculated for FCP in each structure and the distribution volume ratio (DVR) for both brain regions relative to the cerebellum was used as a measure of D2 receptor availability. FCP DVRs were significantly higher in the luteal phase compared to the follicular phase in both the caudate nucleus (11.7% difference, p=0.02) and putamen (11.6% difference, p=0.03). These findings extend earlier work in humans and suggest that changes in DA receptor availability may be involved in the variation in symptoms of various neuropsychiatric disorders across the menstrual cycle, including differences in sensitivity to the abuse-related effects of stimulants.

Enhanced binding and inertness to dehalogenation of alpha-melanotropic peptides labeled using N-succinimidyl 3-iodobenzoate.

Two peptides of potential utility for targeting melanoma cells, alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analogue [Nle4,D-Phe7]-alpha-MSH, were radioiodinated in 45-65% yield using N-succinimidyl 3-[125I]iodobenzoate (SIB). To determine whether this labeling method resulted in improved in vitro and in vivo characteristics, these peptides also were labeled with 131I by direct iodination with the iodogen method. For alpha-MSH, the rapid tissue clearance of both radionuclides in mice was consistent with rapid degradation of the peptide; however, significantly lower levels of 125I were observed in thyroid and stomach, reflecting a greater inertness to deiodination. More extensive comparisons were performed with [Nle4,D-Phe7]-alpha-MSH. The in vitro binding of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH (prepared using SIB) to the murine B-16 melanoma cell line, 34.1 +/- 4.7%, was more than twice as high as that for [Tyr2(131I),Nle4,D-Phe7]-alpha-MSH (15.0 +/- 0.1%), and its KD was more than 10-fold lower than that for conventionally labeled peptide (10 +/- 5 versus 140 +/- 14 pM). The normal tissue clearance of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH in mice was faster than that of [Tyr2(131I),-Nle4,D-Phe7]-alpha-MSH. The 19-40-fold lower activity concentrations of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH in tissues accumulating free iodide (thyroid and stomach) suggest a greater inertness of this peptide to deiodination. The primary urinary catabolite of [Nle4,D-Phe7, Lys11-(125I)IBA]-alpha-MSH was the lysine conjugate of iodobenzoic acid, whereas radioiodide was the chief catabolite generated from [Tyr2(131I),Nle4,D-Phe7]-alpha-MSH. We conclude that further evaluation of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH for targeting alpha-MSH receptors is warranted and that SIB may be a useful method for the radioiodination of peptides.

Effects of Cocaine and MDMA Self-Administration on Serotonin Transporter Availability in Monkeys

Although serotonin (5-HT) can interact with dopamine (DA) systems to modulate the subjective and reinforcing effects of psychostimulants such as cocaine and 3,4-methyldioxymethamphetamine (MDMA, ecstasy), the long-term effects of exposure to psychostimulants on brain 5-HT systems are not well characterized. The present study assessed 5-HT transporter (SERT) availability using positron emission tomography (PET) in rhesus monkeys with the SERT-specific radioligand [11C]3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile (DASB). SERT availability was assessed in regions of interest including the caudate nucleus, putamen, anterior cingulate cortex, and cerebellum. [11C]DASB distribution volume ratios (DVRs) were calculated using the cerebellum as the reference region. DVRs were calculated in control monkeys and in cocaine or MDMA self-administering monkeys approximately 24 h after the last self-administration (SA) session. SERT availability did not differ between monkeys with a history of MDMA SA and control monkeys in any region examined. In contrast, monkeys with a history of cocaine SA showed significantly higher levels of SERT availability in the caudate nucleus and putamen compared to control subjects. These results suggest that chronic SA of cocaine, but not MDMA, leads to alterations in serotonergic function in brain areas relevant to drug abuse. The higher level of SERT availability in cocaine-experienced monkeys may lead to a reduced inhibitory tone of 5-HT on the DA system, which may explain, in part, differences in the abuse liability between cocaine and MDMA.

Social Dominance in Female Monkeys: Dopamine Receptor Function and Cocaine Reinforcement

BACKGROUND Brain imaging and behavioral studies suggest an inverse relationship between dopamine (DA) D2/D3 receptors and vulnerability to cocaine abuse, although most research has used males. For example, male monkeys that become dominant in a social group have significant elevations in D2/D3 receptor availability and are less vulnerable to cocaine reinforcement. METHODS DA D2/D3 receptor availability was assessed in female cynomolgus monkeys (n = 16) with positron emission tomography (PET) while they were individually housed, 3 months after stable social hierarchies had formed, and again when individually housed. In addition, PET was used to examine changes in dopamine transporter (DAT) availability after social hierarchy formation. After imaging studies were complete, monkeys received implantation with indwelling intravenous catheters and self-administered cocaine (.001-.1 mg/kg/injection) under a fixed-ratio 30 schedule of reinforcement. Acquisition of cocaine reinforcement occurred when response rates were significantly higher than when saline was self-administered. RESULTS Neither DAT nor D2/D3 receptor availability in the caudate nucleus and putamen was predictive of social rank, but both significantly changed after formation of social hierarchies. DA D2/D3 receptor availability significantly increased in females that became dominant, whereas DAT availability decreased in subordinate females. Dominant female monkeys acquired cocaine reinforcement at significantly lower doses than subordinate monkeys. CONCLUSIONS The relationship between D2/D3 receptor availability and vulnerability to cocaine reinforcement seems, on the basis of these findings, opposite in females and males. These data indicate that the social environment profoundly affects the DA system but does so in ways that have different functional consequences for females than for males.

Behavioral and neurobiological characteristics influencing social hierarchy formation in female cynomolgus monkeys.

Socially housed monkeys have been used as a model to study human diseases. The present study examined behavioral, physiological and neurochemical measures as predictors of social rank in 16 experimentally naïve, individually housed female cynomolgus monkeys (Macaca fascicularis). The two behavioral measures examined were novel object reactivity (NOR), as determined by latency to touch an opaque acrylic box placed in the home cage, and locomotor activity assessed in a novel open-field apparatus. Serum cortisol concentrations were evaluated three times per week for four consecutive weeks, and stress reactivity was assessed on one occasion by evaluating the cortisol response to adrenocorticotropic hormone (ACTH) following dexamethasone suppression. Measures of serotonin (5-HT) function included whole blood 5-HT (WBS) concentrations, cerebrospinal fluid (CSF) concentrations of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) and brain 5-HT transporter (SERT) availability obtained using positron emission tomography (PET). After baseline measures were obtained, monkeys were assigned to four social groups of four monkeys per group. The two measures that correlated with eventual social rank were CSF 5-HIAA concentrations, which were significantly higher in the animals who eventually became subordinate, and latency to touch the novel object, which was significantly lower in eventual subordinate monkeys. Measures of 5-HT function did not change as a consequence of social rank. These data suggest that levels of central 5-HIAA and measures of novel object reactivity may be trait markers that influence eventual social rank in female macaques.

Design, Synthesis, and Preliminary in Vitro and in Vivo Evaluation of N-(2-diethylaminoethyl)-4-[18F]fluorobenzamide ([18F]-DAFBA): A Novel Potential PET Probe to Image Melanoma Tumors

In order to develop a PET radiopharmaceutical to image malignant melanoma, we synthesized N-(2-diethylaminoethyl)-4-[(18)F]fluorobenzamide ([(18)F]-DAFBA). In vitro studies show a high uptake of [(18)F]-DAFBA by the B16F1 melanoma cells. No significant binding was seen for DAFBA to the sigma-1 and sigma-2 receptors in vitro. The in vivo biodistribution studies performed in normal ICR mice showed a low uptake in the normal tissues followed by further elimination of radioactivity from these tissues with time. The biodistribution studies performed in C57 mice bearing the melanoma tumor xenograft showed a rapid uptake of radioactivity in the tumor that reached a plateau within 30 min postinjection. The F-18 uptake in the tumor was 7.00 +/- 2.76, 6.57 +/- 1.66, and 5.80 +/- 0.98%ID/g at 60, 120, and 180 min, respectively. A steady uptake of radioactivity in the tumor and low uptake in normal tissues resulted in high tumor to normal tissue ratios. For example, at 180 min postinjection, the tumor to tissue ratios were 14.90 +/- 6.47, 21.90 +/- 4.68, 32.91 +/- 6.11, 39.73 +/- 11.78, and 6.33 +/- 1.9, for the spleen, lungs, muscle, blood, and liver, respectively. The radioactivity rapidly cleared from the blood pool, and it decreased from 0.68 +/- 0.21%ID/g at 60 min to 0.13 +/- 0.03%ID/g at 180 min. The F-18 uptake in the bones at 60, 120, and 180 min was 0.91 +/- 0.27, 0.57 +/- 0.32, and 0.17 +/- 0.05%ID/g, respectively. This low uptake in the bones reflects its in vivo resistance toward defluorination. A low residual activity in normal tissues and a high tumor uptake signifies the superior imaging potential of this compound. Because of these positive traits, [(18)F]-DAFBA could help delineate the tumor and its metastases when used for imaging applications. Further in vivo studies are underway to assess the potential of [(18)F]-DAFBA as a promising PET imaging probe.

Chimeric anti-tenascin antibody 81C6: Increased tumor localization compared with its murine parent

When labeled using the Iodogen method, a chimeric antibody composed of the human IgG2 constant region and the variable regions of murine anti-tenascin 81C6 exhibited superior uptake in human glioma xenografts compared with its murine parent. In the current study, three paired-label experiments were performed in athymic mice with subcutaneous D-54 MG human glioma xenografts to evaluate further the properties of radioiodinated chimeric 81C6. These studies demonstrated that (a) the enhanced tumor uptake of chimeric 81C6 is specific; (b) when labeling was performed using N-succinimidyl 3-iodobenzoate, chimeric 81C6 again showed preferential accumulation in tumor compared with murine 81C6; and (c) the tumor uptake advantage observed previously with murine 81C6 for N-succinimidyl 3-iodobenzoate compared with Iodogen labeling did not occur with chimeric 81C6.

Measuring astatine-211 distributions with SPECT

We have investigated standard SPECT techniques (rotating gamma cameras, multi-hole collimators, and filtered backprojection reconstruction) for imaging astatine-211 distributions. Since 211At emits alpha particles, this nuclide has potential for use in radiotherapy. The capability of imaging this nuclide would allow in vivo evaluation of the distribution and stability of potential 211At-labelled radiotherapeutic agents. 211At decay yields x-rays in the 77-92 keV range in addition to 500-900 keV gamma rays. This study evaluates the feasibility of SPECT imaging using the x-ray emissions of 211At. We have evaluated several collimators, with the determination that the medium-energy collimators we used are suitable, with 7% penetration (uncollimated counts versus collimated counts). Several phantoms were imaged and attenuation coefficients were measured (narrow-beam mu = 0.182 cm-1 for 77-80 keV x-rays in water). Reconstructed images demonstrate qualitative capabilities and a simple quantitative study demonstrates good correction for attenuation and scatter (approximately 10% error), at low count densities, at least for the phantom geometries used in this study.