A lipophilic complex with 186Re188Re incorporated in liposomes suitable for radiotherapy

Liposomes with a 70 nm diameter were made by the detergent removal technique on a gel filtration column. The complex oxodichloroethoxy-bis-(triphenylphosphine)rhenium(V) = (Rephos) was irradiated by neutrons in the reactor at PSI. 45 ± 5% of the radioactive complex was incorporated into the bilayer of the liposomes during the liposome formation. The stability of these radioactive liposomes was tested by dialysis: a loss of 40% of the radioactivity identified as perrhenate was observed after 8 days. Addition of the antioxidant ascorbic acid diminished the loss to 20%. Such liposomes carrying the lipophilic radioactive Re-complex can potentially be used in β-radiotherapy. The γ-lines of the two rhenium isotopes are helpful for localizing them in therapy controls by a γ-camera, a big advantage compared to other nuclides proposed for therapy (e.g. ⁹⁰Yttrium).     

Antibodies labeled with 199Au: Potential of 199Au for radioimmunotherapy

We have investigated the direct labeling of antibodies with gold isotopes as an alternative to iodine and chelated metals for antibody guided therapy. Both ¹⁹⁹Au and ¹⁹⁵Au complex anions in citrate-buffered solutions (pH 3.8–7.2) are rapidly bound to sheep polyclonal anti-CEA with high efficiency. After purification by gel filtration, the antibody retained 80% of the bound gold for upto 6 days at 4 °C and was reactive with its target antigen in a solid phase assay. Incubation with the gold binding substance d-penicillamine reduced labeling efficiency and stability.     

Radiolabeled liposomes for scintigraphic imaging

Liposomes have been investigated extensively as carriers for drugs in attempts to achieve selective deposition and/or reduced toxicity. Liposomes radiolabeled with gamma emitters such as (67)Ga, (111)In and (99m)Tc, can be used for imaging purposes. Liposomes as formulated in the past, are rapidly taken up by cells of the mononuclear phagocyte system (MPS), primarily those located in liver and spleen. The recent development of long-circulating liposomes (LCLs), yielded liposomes that oppose recognition by the MPS. The development of these LCLs with enhanced circulatory half-lives has broadened the potential of liposomes to scintigraphically visualize pathologic processes in vivo. Liposomes have been proposed for tumor imaging, infection imaging and blood pool imaging. Strategies have been developed that allow rapid, easy and efficient labeling of preformed liposomes with (111)In and (99m)Tc. There is now a vast body of preclinical evidence showing that LCLs can be used to image a wide variety of tumors as well as inflammatory lesions. The first studies in patients show that radiolabeled liposomes can image tumor and inflammatory lesions with good sensitivity and good specificity. Here, the present status of liposome-based radiopharmaceuticals for scintigraphic application is reviewed.     

CRP: Cleavage of Radiolabeled Phosphoproteins

The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman sequencing of unseparated peptides. Traditionally, phosphorylation sites are determined by cleaving the phosphoprotein and separating the peptides for Edman 32P-phosphate release sequencing. CRP analysis of a phosphoprotein's sequence accelerates this process by omitting the separation step: given a protein sequence of interest, the CRP program performs an in silico proteolytic cleavage of the sequence and reports the predicted Edman cycles in which radioactivity would be observed if a given serine, threonine or tyrosine were phosphorylated. Experimentally observed cycles containing 32P can be compared with CRP predictions to confirm candidate sites and/or explore the ability of additional cleavage experiments to resolve remaining ambiguities. To reduce ambiguity, the phosphorylated residue (P-Tyr, P-Ser or P-Thr) can be determined experimentally, and CRP will ignore sites with alternative residues. CRP also provides simple predictions of likely phosphorylation sites using known kinase recognition motifs. The CRP interface is available at http://fasta.bioch.virginia.edu/crp.     

Radiolabeled hypoxic cell sensitizers: tracers for assessment of ischemia

Hypoxic, non-functional, but viable, tissue may exist in heart and brain following an arterial occlusion. Identification of such tissue in vivo is crucial to the development of effective treatment strategies. It has been suggested that certain compounds capable of sensitizing hypoxic tumor cells to killing by x-rays (i.e., misonidazole) might serve as in vivo markers of hypoxic tissue in ischemic myocardium or brain if properly radiolabeled. To this end we have radiolabeled two fluorinated analogs of nitroimidazole based hypoxic cell sensitizers with the 110 minute half-lived positron-emitting fluorine-18. The ability of these tracers to quantitate the presence of hypoxic tissue has been studied in a gerbil stroke model. The in vivo uptake of one of these tracers [F-18]-fluoronormethyoxymisonidazole is dependent on the extent of tissue hypoxia, and thus, appears to have potential as a diagnostic indicator of non-functional but viable tissue when the tracer is used in conjunction with positron emission tomography.     

Strategies for enhancement of radioimmunotherapy

Two new strategies for increasing tumor uptake have been investigated. First the effect of interleukin-2 (IL-2) on tumor uptake of ¹²⁵I-Lym-l antibody in nude mice was investigated. Secondly, the use of ⁶⁷Cu-labeled Lym-1 was evaluated in patients. In nude mice implanted with Raji human lymphoma, a greater than 2-fold enhancement of tumor uptake of ¹²⁵I-Lym-1 was observed after administration of PEG-interleukin-2 (PEG-IL-2). The macrocycle 1,4,8,11-tetraazacylcotetradecane-N,N′,N′,N'''-tetraacetic acid (TETA), synthesized specifically for copper chelation, has been conjugated to Lym-1 for ⁶⁷Cu labeling of the monoclonal antibody (MoAb). There was no evidence for bone or normal marrow uptake and the residence time in the tumor was prolonged. Surprisingly, a dose of 4.4 mCi that was intended for imaging induced substantial tumor regression in a patient.     

Novel cyanine dyes with vinylsulfone group for labeling biomolecules

Novel vinylsulfone cyanine dyes (em. 550-850 nm) were designed and synthesized for fluorescent labeling of biomolecules via 1,2-addition reaction in aqueous conditions. Due to the virtue of chemical structures of both fluorophore and reactive group, these dyes could be significantly stable and reactive in various aqueous/organic conditions. A wide variety of pH, temperature, buffer concentration, and protein were tested for the optimal labeling condition.     

Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology

Identifying the causes of past epidemics depends on the specific detection of pathogens in buried individuals; this field of research is known as paleomicrobiology, an emerging field that has benefited from technological advances in microbiology. For almost 15 years, the detection, identification, and characterization of microbes in ancient environmental and human specimens emerged on the basis of ancient DNA (aDNA) analyses. aDNA limitations due to potential contamination by modern DNA and altered aDNA led to the development of alternative methods for the detection and characterization of nonnucleotidic biomolecules, including mycolic acids (of ancient mycobacteria) and proteins. Accordingly, immunohistochemistry, immunochromatography, and enzyme-linked immunosorbent assay techniques have been developed for the specific detection of microbes from ancient human and environmental specimens. Protein analysis by mass spectrometry, a standard for ancient animal identification, has also recently emerged as a technique for ancient mycobacteria detection, while immuno-PCR is yet another promising technique. As with aDNA, strict protocols must be enforced to ensure authenticity of the data. Here we review the analysis of nonnucleotidic biomolecules from ancient microbes and the ability of these analyses to complement aDNA analyses, which opens new opportunities for identification of ancient microbes as well as new avenues to potentially resolve controversies regarding the cause of some historical pandemics and study the coevolution of microbes and hosts.     

Preparation and biological characterization of isomeric 188Re(V) oxocomplexes with tetradentate S4 ligands derived from meso-dimercaptosuccinic acid for labeling of biomolecules

A new type of tetradentate S4 ligand has been synthesized by bridging two molecules of meso-2,3-dimercaptosuccinic acid for stable binding and easy conjugation of rhenium-188 to tumor targeting structures. The stereoisomeric tetrathiolato S4 ligands form very robust anionic five-coordinated oxorhenium(V) and oxotechnetium(V) complexes. Two routes for the preparation of the (188)Re(V) oxocomplexes with (iBu)2N(O)C-C(SH)C(SH)C(O)NH(CH2)3NH(CH2)3NHC(O)C(SH)C(SH)C(O)N(iBu)2 (ligand 1) and its hydrophilic crown ether derivative (ligand 2) were tested and optimized. Several isomers were separated by HPLC from the preparation solutions and characterized in vitro and in vivo. The identity of the species obtained was determined by comparison with the HPLC profiles of reference (185/187)Re analogues and (99/99m)Tc complexes which were characterized by ESI-MS. All of them were absolutely stable in rat and human plasma solutions. Challenge experiments with cysteine corroborated the high inertness of the isomers toward ligand exchange reactions. Various in vivo samples, taken off at different times from blood, intestine, and urine of rats, confirmed the high in vivo stability of the (188)Re-S4 complexes. Biodistribution studies using male Wistar rats were performed and exhibited a high uptake and fast clearance from the liver of the more lipophilic cis and trans isomers of complex I (log P(o/w) between 1.5 and 1.7), whereas the isomers of the hydrophilic complex II (log P(o/w) about -1.75) were rapidly excreted via the renal and the hepatobiliary pathway. The low level of activity in the stomach confirms good in vivo stability. Thus, these new (188)Re-S4 complexes fulfill the requirements for a stable and high specific activity labeling of biomolecules with rhenium-188.     

Interaction of liposome-encapsulated cisplatin with biomolecules

We prepared liposomes by hydrating 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid with aqueous solutions of three "probe" molecules-cis-diamminedichloroplatinum(II) (cis-[Pt(II)(NH(3))(2)Cl(2)], cisplatin), guanosine 5'-monophosphate (5'-GMP), and 9-ethylguanine (9-EtG)-in phosphate-buffered saline as well as N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid buffer. The positively charged hydrolysis product of cisplatin, [Pt(II)(NH(3))(2)Cl(H(2)O)](+), is in the inner core of the liposomes and negatively charged 5'-GMP embeds in the lipid bilayer of liposomes. In the presence of cisplatin, the size of the liposomes remains unchanged, and for 5'-GMP-embedded liposomes the size increases significantly compared with that of empty or control liposomes. In contrast, the neutral biomolecule 9-EtG was found to be dispersed in the exterior bulk water and the size of the liposomes remained the same as that of empty or control liposomes. When cisplatin-containing liposomes mix with 5'-GMP-embedded liposomes or liposomes with 9-EtG, the N7 nitrogen atom of 5'-GMP or 9-EtG binds the cisplatin, thus replacing the "leaving groups" and forming a bisadduct. After 48?h of mixing, the size of the liposomes changes for the mixture of 5'-GMP-embedded liposomes and cisplatin-containing liposomes. We used (1)H and (31)P NMR spectroscopic techniques to monitor incorporation or association of cisplatin and biomolecules with liposomes and their subsequent reactions with each other. The dynamic light scattering technique provided the size distribution of the liposomes in the presence and absence of probe molecules.     

T-Pile--a package for thermodynamic calculations for biomolecules

Molecular dynamics and Monte Carlo, usually conducted in canonical ensemble, deliver a plethora of biomolecular conformations. Proper analysis of the simulation data is a crucial part of biophysical and bioinformatics studies. Sequence alignment problem can be also formulated in terms of Boltzmann distribution. Therefore tools for efficient analysis of canonical ensemble data become extremely valuable. T-Pile package, presented here provides a user-friendly implementation of most important algorithms such as multihistogram analysis and reweighting technique. The package can be used in studies of virtually any system governed by Boltzmann distribution. AVAILABILITY: T-Pile can be downloaded from: http://biocomp.chem.uw.edu.pl/services/tpile. These pages provide a comprehensive tutorial and documentation with illustrative examples of applications. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.