Influence of oxygen, albumin and pH on copper dissolution in a simulated uterine fluid.

OBJECTIVES: The aim of this paper is to study the influence of albumin content, from 5 to 45 g/L, on copper dissolution and compounds composition in a simulated uterine solution. METHODS: Experiments were performed in atmospheric pressure conditions and with an additional oxygen pressure of 0.2 atmospheres, at 6.3 and 8.0 pH values, and at a temperature of 37 +/- 0.1 degrees C for 1, 3, 7, and 30 days experimentation time. RESULTS: The copper dissolution rate has been determined using absorbance measurements, finding the highest value for pH 8.0, 35 g/L albumin, and with an additional oxygen pressure of 0.2 atmospheres: 674 microg/day for 1 day, and 301 microg/day for 30 days. X-ray photoelectron spectroscopy (XPS) results show copper(II) as the main copper oxidation state at pH 8.0; and copper(I) and metallic copper at pH 6.3. CONCLUSIONS: The presence of albumin up to 35 g/L, accelerates copper dissolution. For high albumin content a stabilisation on the copper dissolution takes place. Corrosion product layer morphology is poorly protective, showing paths through which copper ions can release.

Potential regulation of nicotine and ethanol actions by alpha4-containing nicotinic receptors.

A restriction fragment length polymorphism (RFLP) associated with a major nicotinic receptor subunit (i.e., alpha4) has been identified in two mouse lines that were selectively bred for differences in sensitivity to ethanol. These mice, referred to as Long-Sleep (LS) and Short-Sleep (SS) mice, also differ in sensitivity to several effects of nicotine. The potential role of the alpha4 RFLP in regulating several responses to nicotine and ethanol was evaluated by using the LSxSS-derived recombinant inbred (RI) strains. Those RI strains that carried the LS-like alpha4 RFLP were more sensitive to the depressant effects of nicotine on Y-maze crossing and rearing activities and ethanol-induced increases in Y-maze crossing activity than were those RI strains that carry the SS-like alpha4 RFLP. The LS-like RI strains were also more sensitive to nicotine-induced hypothermia. The RFLP was not associated with strain differences in ethanol-induced body temperature or sleep time. The potential role of the RFLP in regulating ethanol and nicotine consumption was evaluated in heterogeneous stock (HS) mice. An association was found between the alpha4 RFLP and variation in ethanol consumption, but not in nicotine consumption, as measured in a four-bottle choice test. Recent studies of ethanol and tobacco abuse by human beings suggest that common genes may influence these two forms of substance abuse. The results of the studies reported here suggest that the alpha4 nicotinic receptor gene should be evaluated for its potential role in regulating ethanol and tobacco abuse in human beings.

In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a well-characterized cell surface antigen expressed by virtually all prostate cancers (PCas). PSMA has been successfully targeted in vivo with (111)In-labeled 7E11 monoclonal antibody (mAb; ProstaScint; Cytogen, Princeton, NJ), which binds to an intracellular epitope of PSMA. This work reports the in vitro characterization of three recently developed mAbs that bind the extracellular domain of PSMA (PSMAext). Murine mAbs J415, J533, J591, and 7E11 were radiolabeled with 131I and evaluated in competitive and saturation binding studies with substrates derived from LNCaP cells. J415 and J591 were conjugated to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid labeled with (111)In. The uptake and cellular processing of these antibodies were evaluated in viable LNCaP cells. All four mAbs could be labeled with 131I up to a specific activity of 350 MBq/mg with no or little apparent loss of immunoreactivity. Competition assays revealed that J415 and J591 compete for binding to PSMAext antigen. J533 bound to a region close to the J591 binding epitope, but J533 did not interfere with J415 binding to PSMA. mAb 7E11 did not inhibit the binding of J415, J533, or J591 (or vice versa), consistent with earlier work that these latter mAbs bind PSMAext whereas 7E11 binds the intracellular domain of PSMA. Saturation binding studies demonstrated that J415 and J591 bound with a similar affinity (Kds 1.76 and 1.83 nM), whereas J533 had a lower affinity (Kd, 18 nM). In parallel studies, all four mAbs bound to a similar number of PSMA sites expressed by permeabilized cells (1,000,000-1,300,000 sites/cell). In parallel studies performed with viable LNCaP cells, J415, J533, and J591 bound to a similar number of PSMA sites (i.e., 600,000-800,000 sites/cell), whereas 7E11 bound only to a subpopulation of the available PSMA sites (95,000 sites/cell). This apparent binding of 7E11 to viable cells can be accounted for by a 5-7% subpopulation of permeabilized cells produced when the cells were trypsinized and suspended. Up to five DOTA chelates could be bound to either J415 or J591 without compromising immunoreactivity. A comparison of the cellular uptake and metabolic processing of the 131I- and (111)In-labeled antibodies showed a rapid elimination of 131I from the cell and a high retention of (111)In. All four mAbs recognized and bound to similar numbers of PSMAs expressed by ruptured LNCaP cells (i.e., the exposed intracellular and extracellular domains of PSMA). By comparison to J415 and J591, J533 had a lower binding affinity. Both J415 and J591 recognized and bound to the same high number of PSMAs expressed by intact LNCaP. By contrast, 7E11 bound to fewer sites expressed by intact LNCaP cells (i.e., the exposed extracellular domain of PSMA). Both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides.

Preclinical evaluation of technetium-99m-labeled somatostatin receptor-binding peptides.

UNLABELLED: We report here the results of studies on the in vitro receptor binding affinity, in vivo tumor uptake and biodistribution of two 99m-Tc-labeled peptides. METHODS: Peptides P587 and P829 were synthesized by N-alpha-Fmoc peptide chemistry, purified by reversed-phase HPLC and characterized by fast-atom bombardment mass spectrometry. The peptides were labeled with 99mTc by ligand exchange from 99mTc-glucoheptonate. In vitro somatostatin receptors (SSTR)-binding affinities of P587, P829 and their oxorhenium complexes, [DTPA]octreotide and In-[DTPA]octreotide were determined in an inhibition assay using AR42J rat pancreatic tumor cell membranes and 125I-[Tyr3]somatostatin-14 as the probe. In vivo single- and dual-tracer studies of 99mTc peptides and 111In-[DTPA]octreotide were carried out using Lewis rats bearing CA20948 rat pancreatic tumor implants. RESULTS: Technetium-99m-P587 and 99mTc-P829 of high-specific activity (>60 Ci (2.2 TBq)/mmole) were prepared in >90% radiochemical yield. P587 and P829 had a Ki = 2.5 nM and 10 nM, respectively. [ReO]P587 and [ReO]P829, representing the 99mTc complexes, had Ki = 0.15 nM and 0.32 nM, respectively. In comparison, [DTPA]octreotide and In-[DTPA]octreotide had Ki = 1.6 and 1.2 nM, respectively. In vivo tumor uptake of 99mTc-P587 and 99mTc-P829 was high (4.1 and 4.9%ID/g at 90 min postinjection compared to 2.9% for 111In-[DTPA]octreotide). Tumor/blood and tumor/muscle ratios at 90 min postinjection were 6 and 33 for 99mTc-P587, 21 and 68 for 99mTcP829, and 22 and 64 for 111In-[DTPA]octreotide. CONCLUSION: The high SSTR-binding affinity and high receptor-specific and saturable in vivo tumor uptake indicate that 99mTc-P587 and 99mTc-P829 are promising radiotracers for the clinical detection of SSTR-expressing tumors and other tissues by 99mTc gamma scintigraphy.

Technetium-99m-white blood cell-specific imaging agent developed from platelet factor 4 to detect infection.

UNLABELLED: We have developed a leukocyte-avid, 99mTc-labeled peptide (P483H) as a potential imaging agent for infection. P483H contains the heparin-binding region of platelet factor-4 (PF-4) and a lysine-rich sequence for rapid renal clearance. Technetium-99m-P483H was evaluated for its ability to selectively label white blood cells (WBCs) in vitro and to detect focal E. coli infections in rabbits. METHODS: Technetium-99m-P483H was incubated with citrated whole human blood, layered onto WBC isolation media and subjected to density gradient centrifugation to measure WBC-associated radioactivity. Indium-111-WBCs and 99mTc-gluceptate were used as controls. In the in vivo model, E. coli infected rabbits were imaged and necropsied 4 hr after administration of 99mTc-P483H. Infected and contralateral control muscles were evaluated for %ID, %ID/g, Imax (muscle sample showing the highest uptake, i.e., %ID/g) and Imax-to-blood and Imax-to-control muscle ratios. Indium-111-WBCs, 111In-DTPA, 131I-albumin (HSA), 99mTc-nanocolloid, 67Ga and 99mTc-gluceptate were evaluated as in vivo controls. RESULTS: Technetium-99m-P483H associated predominantly with WBCs in vitro, and 99m-Tc-P483H provided high contrast images of infection in vivo. In vitro, 73% of 99mTc-P483H radioactivity was associated with WBCs. Technetium-99m-P483H outperformed 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate with an infection Imax average of 0.062 %ID/g (+/- 0.029; n = 48). Technetium-99m-P483H also outperformed all controls, including 111In-WBCs, 111In-DTPA, 131I-albumin, 99mTc-nanocolloid, 67Ga-citrate and 99mTc-gluceptate. The Imax-to-blood and Imax-to-control muscle ratios for 99mTc-P483H averaged 3.1 (+/- 2.4) and 26.8 (+/- 16.8), respectively, and again outperformed all controls. CONCLUSION: Technetium-99m-P483H associates predominantly with WBCs in vitro and identified focal infections in vivo within 4 hr versus conventional imaging agents. Additionally, the agent showed rapid blood clearance and exclusive renal excretion, which provides a clear abdominal field for imaging abdominal infections.