Uday Mukhopadhyay

Molecular–Genetic PET Imaging Using an HSV1-tk Mutant Reporter Gene with Enhanced Specificity to Acycloguanosine Nucleoside Analogs

Imaging 2 different molecular–genetic events in a single subject by PET is essential in a variety of in vivo applications. Using herpes simplex virus-1 thymidine kinase (HSV1-tk) mutants with narrower substrate specificities in combination with wild-type HSV1-tk (wtHSV1-tk) would enable differential imaging with corresponding radiotracers, namely 2′-deoxy-2′-18F-fluoro-5-ethyl-1-β-d-arabinofuranosyl-uracil (18F-FEAU) and the acycloguanosine derivative 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG). In this study, we evaluated wtHSV1-tk and the A168H mutant, which has been reported to exhibit enhanced acycloguanosine substrate catalytic activity and diminished pyrimidine phosphorylating activity, as PET reporter genes. Methods: Computational analysis was performed to assess the binding mode of FHBG and FEAU to wtHSV1-tk and the A168H variant. U87 cells were stably transduced with wtHSV1-tk or HSV1-tk(A168H) fused with green fluorescent protein and sorted to obtain equivalent transgene expression. In vitro uptake studies were performed to determine rates of substrate accumulation and retention. Nude mice bearing tumors expressing HSV1-tk variants were subsequently imaged using 18F-FHBG and 18F-FEAU. Results: Docking results indicate that binding of FHBG to the A168H variant is unaffected whereas the binding of FEAU is hindered because of a steric clash with the bulkier mutant residues. U87 cells expressing HSV1-tk(A168H) accumulated 18F-FHBG in in vitro uptake studies at a 3-fold higher rate than did cells expressing wtHSV1-tk without any detectable accumulation of 3H-FEAU. Furthermore, HSV1-tk(A168H) demonstrated no thymidine phosphorylation activity. In contrast, U87 cells expressing wtHSV1-tk preferentially accumulated 3H-FEAU at an 18-fold higher rate than they did 18F-FHBG. Tumors expressing wtHSV1-tk or HSV1-tk(A168H) were distinctly imaged with 18F-FEAU or 18F-FHBG, respectively. Hence, tumors expressing HSV1-tk(A168H) accumulated 8.4-fold more 18F-FHBG than did tumors expressing wtHSV1-tk. In addition, wtHSV1-tk tumors, compared with HSV1-tk(A168H)–expressing tumors (which retained baseline levels of the radiotracer), preferentially accumulated 18F-FEAU. Conclusion: The FEAU and FHBG substrate discrimination capacity of the wtHSV1-tk and HSV1-tk(A168H) reporter enzymes was validated in vivo by PET of mice with tumor xenografts established from U87 cells expressing these different reporters. Thus, HSV1-tk(A168H) may potentially be used as a second reporter gene in combination with wtHSV1-tk to achieve differential PET.

Detection of pancreatic carcinomas by imaging lactose-binding protein expression in peritumoral pancreas using [18F] fluoroethyl-deoxylactose PET/CT

Background Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size (∼2–3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with β-O-D-galactopyranosyl-(1,4′)-2′-deoxy-2′-[18F]fluoroethyl-D-glucopyranose ([18F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [18F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells. Methodology/Principal Findings Dynamic PET/CT imaging demonstrated rapid accumulation of [18F]FEDL in peritumoral pancreatic tissue (4.04±2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65±0.50 min and 14.14±3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [18F]FEDL in peritumoral pancreatic tissue was estimated as 3.57±0.60 and 0.94±0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [18F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2–4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm. Conclusion/Significance We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [18F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [18F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.

Preparation, characterization, and antitumor activity of new cisplatin analogues with 1-methyl-4-(methylamino)piperidine: crystal structure of [PtII(1-methyl-4-(methylamino) piperidine)(oxalate)].

A series of new platinum(II) complexes of the type [Pt(II)(mmap)X] (where mmap, 1-methyl-4-(methylamino)piperidine and X, 1,1-cyclobutanedicarboxylato (CBDCA), oxalato, malonato, methylmalonato, dimethylmalonato, ethylmalonato, diethylmalonato or 2,3-naphthalene dicarboxylato (NDCA)) have been synthesized and characterized by elemental analysis, infrared (IR), and 13C and 195Pt nuclear magnetic resonance (NMR) spectroscopy. The crystal structure of the analogue [Pt(II)(mmap)(oxalate)] was determined using the single crystal X-ray diffraction method. Based upon a total of 4964 collected reflections, we determined that the compound crystallizes in the monoclinic space group P2(1)/c (with a=11.890(2) A, b=9.6695(19) A, c=9.875(2) A, beta=102.03(3) degrees, Z=4, and R=0.0428). In this complex, platinum has a slightly distorted square planar geometry with the two adjacent corners being occupied by two nitrogen atoms of the mmap ligand, whereas the remaining cis positions are occupied by two oxygen atoms of the oxalate molecule. The mmap ligand is in a boat conformation and forms six-membered chelating rings as well as the oxalate molecule forms five-membered chelating rings with platinum. The complexes were evaluated for their cytotoxic potential against the sensitive A2780 tumor model and cisplatin-resistant clone derived in vitro from potential cells.

Whole-Body Biodistribution Kinetics, Metabolism, and Radiation Dosimetry Estimates of 18F-PEG6-IPQA in Nonhuman Primates

We recently developed the radiotracer 4-[(3-iodophenyl)amino]-7-(2-[2-{2-(2-[2-{2-(18F-fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide) (18F-PEG6-IPQA) for noninvasive detection of active mutant epidermal growth factor receptor kinase-expressing non–small cell lung cancer xenografts in rodents. In this study, we determined the pharmacokinetics, biodistribution, metabolism, and radiation dosimetry of 18F-PEG6-IPQA in nonhuman primates. Methods: Six rhesus macaques were injected intravenously with 141 ± 59.2 MBq of 18F-PEG6-IPQA, and dynamic PET/CT images covering the thoracoabdominal area were acquired for 30 min, followed by whole-body static images at 60, 90, 120, and 180 min. Blood samples were obtained from each animal at several time points after radiotracer administration. Radiolabeled metabolites in blood and urine were analyzed using high-performance liquid chromatography. The 18F-PEG6-IPQA pharmacokinetic and radiation dosimetry estimates were determined using volume-of-interest analysis of PET/CT image datasets and blood and urine time–activity data. Results: 18F-PEG6-IPQA exhibited rapid redistribution and was excreted via the hepatobiliary and urinary systems. 18F-PEG6 was the major radioactive metabolite. The critical organ was the gallbladder, with an average radiation-absorbed dose of 0.394 mSv/MBq. The other key organs with high radiation doses were the kidneys (0.0830 mSv/MBq), upper large intestine wall (0.0267 mSv/MBq), small intestine (0.0816 mSv/MBq), and liver (0.0429 mSv/MBq). Lung tissue exhibited low uptake of 18F-PEG6-IPQA due to the low affinity of this radiotracer to wild-type epidermal growth factor receptor kinase. The effective dose was 0.0165 mSv/MBq. No evidence of acute cardiotoxicity or of acute or delayed systemic toxicity was observed. On the basis of our estimates, diagnostic dosages of 18F-PEG6-IPQA up to 128 MBq (3.47 mCi) per injection should be safe for administration in the initial cohort of human patients in phase I clinical PET studies. Conclusion: The whole-body and individual organ radiation dosimetry characteristics and pharmacologic safety of diagnostic dosages of 18F-PEG6-IPQA in nonhuman primates indicate that this radiotracer should be acceptable for PET/CT studies in human patients.

PET imaging of T cells derived from umbilical cord blood

Progress in understanding tumor-specific immune responses, genetic engineering and ex vivo manufacturing, have led to improvements in the safety and feasibility of adoptive transfer of genetically modified T cells. However, rational design, application and evaluation of T-cell therapy requires monitoring methods that can detect, locate and serially quantify these cell-mediated immune responses. Currently, such monitoring methods are chiefly limited to invasive techniques to investigate recovered cell populations for in vitro measurements including histology, flow cytometry, Q-PCR or the detection of cytokines. These assays provide episodic glimpses of the bio distribution of T cells and are limited by the number and sites of sampling. In contrast, imaging provides a methodology for quantitative, non-invasive, longitudinal and spatial in vivo information about the dynamic processes of infused T cells.

The structures of cyclic dihydronium cations

Recent experimental discoveries have revealed the existence of hitherto unexpected cyclic hydronium di-cations trapped within crystal structures. The molecular formulas are (H14O6)2+, present as two isomers, four- and six-member cyclic structures, and (H18O8)2+, an eight-member cyclic structure. As these unprecedented hydronium species are stabilized by the crystal structures in which they are captured, the question arises whether they could be stable as independent species as, for example, in solution or gas phase. Quantum mechanical density functional theory calculations are used to investigate this question. We find these doubly charged cyclic hydronium structures to be energetically stable and, as between the four- and six-member structures, the former has more binding energy than the latter. We also determine the theoretically optimized structures for all three ions and give their calculated atomic charges for both their crystal and optimized geometries.

Semi-automated production of 89Zr-oxalate/89Zr-chloride and the potential of 89Zr-chloride in radiopharmaceutical compounding

Interest in using (89)Zr is rapidly increasing for immuno-PET applications due to its unique characteristics and increased availability. The focus of this study was to develop an optimized semi-automated methodology for producing (89)Zr-oxalate/(89)Zr-chloride, and evaluate the potential application of (89)Zr-chloride for radiopharmaceutical compounding. The data presented herein will be useful for the production of (89)Zr-labeled radiopharmaceuticals and their compliance with regulatory issues for both preclinical and clinical use.

Synthesis and characterization of cis-bis-heptamethyleneimine platinum(II) dicarboxylate complexes: crystal structure of cis-[Pt(heptamethyleneimine)2(malonate)]·H2O

Abstract A series of new platinum complexes of the type cis-[Pt(L)2X] (where L=heptamethyleneimine and X=1,1-cyclobutanedicarboxylate (CBDCA), oxalate, malonate, methylmalonate, ethylmalonate, dimethylmalonate, or diethylmalonate ligand) were synthesized and characterized by elemental analysis, infrared, and 195Pt nuclear magnetic resonance spectroscopy. The crystal structure of cis-[Pt(L)2(malonate)]·H2O was determined by X-ray crystallography. In all of the molecules, the platinum atom adopts a distorted square-planar geometry. Two of the coordination sites of the metal center are occupied by heptamethyleneimine ligands, which are arranged in a cis orientation. The coordination sphere of the metal is completed through interaction of the platinum with two of the oxygen atoms of the malonate ligand, resulting in the formation of a six-membered chelate ring. In the solid state, an intricate network of hydrogen bonds is found to exist between carbonyl oxygen atoms, amine hydrogen atoms and included solvent water.

Improved detection and measurement of low levels of [ 18F]fluoride metabolized from [ 18F]-labeled pyrimidine nucleoside analogues in biological samples

INTRODUCTION It is important to identify all circulating metabolites, including free fluoride, for accurate pharmacokinetic modeling of [(18)F]-labeled radiotracers. We sought to determine the most efficient method to detect and quantify low levels of free [(18)F]fluoride in biological samples. METHODS Low levels of [(18)F]fluoride were analyzed using two methods: (A) an ion-exchange cartridge and gamma counting, and (B) radio-HPLC, to compare the detection limits of these two analytical methods. Twenty microliters of [(18)F]fluoride solution was loaded onto an ion-exchange cartridge, then eluted with 20% MeCN/water (5 ml) and radioactivity trapped in the cartridge counted on a gamma counter. [(18)F]Fluoride was also determined in plasma and urine from mice injected with [(18)F]-labeled thymidine analogues using Method A. RESULTS The detection sensitivity of Method A was 9.4-fold higher than that of Method B (0.075±0.004 vs. 0.71±0.02 nCi). With Method A, [(18)F]fluoride was determined in plasma for [(18)F]FLT, [(18)F]FMAU, [(18)F]FEAU and N(3)-[(18)F]FPrT as 1.4±0.31% (n=4), 0.17±0.49% (n=3), 4.88±1.62% (n=3) and 12.94±0.48% (n=4), respectively. The amount of [(18)F]fluoride determined in the urine was 11.49±1.60% (n=4) from [(18)F]FLT, 5.36±2.34% (n=3) from [(18)F]FMAU, 13.57±1.96% (n=3) from [(18)F]FEAU and 11.19±1.98% (n=4) from N(3)-[(18)F]FPrT. CONCLUSION Low levels of [(18)F]fluoride in biological samples can be detected and quantified using an ion-exchange cartridge and gamma counting. This methodology is simple, accurate and superior to the standard use of radio-HPLC on a C(18) column for metabolite analysis, and it should be useful in pharmacokinetic modeling for animal imaging studies using an [(18)F]-labeled radiotracer and PET.

Homopiperazine platinum(II) complexes containing substituted disulfide groups: crystal structure of [PtII(homopiperazine)(diphenylsulfide)Cl]NO3

Abstract A series of new cationic platinum(II) complexes of the type [Pt(L)(R′R″S)Cl]NO3 (where L=homopiperazine or 1-methylhomopiperazine and R′R″S=dimethylsulfide, diethylsulfide, dipropylsulfide, diisopropylsulfide, dibutylsulfide, diphenylsulfide, dibenzylsulfide, methylphenylsulfide, or methyl p-tolylsulfide) were synthesized and characterized by elemental analysis and infrared, 1H and 195Pt nuclear magnetic resonance spectroscopy. Among the complexes synthesized, [PtII(homopiperazine)(diphenylsulfide)Cl]NO3 was examined by single-crystal X-ray diffraction. The slightly distorted square plane of the platinum complex included the amino groups of the homopiperazine molecule in a cis orientation, the sulfur atom of diphenyl sulfide, and a chloride ion. The homopiperazine molecule adopts a boat conformation and forms five- and six-membered chelating rings with platinum. Hydrogen bonding plays an important part in holding the crystal together.