Synthesis of phosphine and antibody-azide probes for in vivo Staudinger ligation in a pretargeted imaging and therapy approach.

The application of intact monoclonal antibodies (mAbs) as targeting agents in nuclear imaging and radioimmunotherapy is hampered by the slow pharmacokinetics of these molecules. Pretargeting with mAbs could be beneficial to reduce the radiation burden to the patient, while using the excellent targeting capacity of the mAbs. In this study, we evaluated the applicability of the Staudinger ligation as pretargeting strategy using an antibody-azide conjugate as tumor-targeting molecule in combination with a small phosphine-containing imaging/therapeutic probe. Up to 8 triazide molecules were attached to the antibody without seriously affecting its immunoreactivity, pharmacokinetics, and tumor uptake in tumor bearing nude mice. In addition, two (89)Zr- and (67/68)Ga-labeled desferrioxamine (DFO)-phosphines, a (177)Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-phosphine and a (123)I-cubyl phosphine probe were synthesized and characterized for their pharmacokinetic behavior in nude mice. With respect to the phosphine probes, blood levels at 30 min after injection were <5% injected dose per gram tissue, indicating rapid blood clearance. In vitro Staudinger ligation of 3.33 µM antibody-azide conjugate with 1 equiv of radiolabeled phosphine, relative to the azide, in aqueous solution resulted in 20-25% efficiency after 2 h. The presence of 37% human serum resulted in a reduced ligation efficiency (reduction max. 30% at 2 h), while the phosphines were still >80% intact. No in vivo Staudinger ligation was observed in a mouse model after injection of 500 µg antibody-azide, followed by 68 µg DFO-phosphine at t = 2 h, and evaluation in blood at t = 7 h. To explain negative results in mice, Staudinger ligation was performed in vitro in mouse serum. Under these conditions, a side product with the phosphine was formed and ligation efficiency was severely reduced. It is concluded that in vivo application of the Staudinger ligation in a pretargeting approach in mice is not feasible, since this ligation reaction is not bioorthogonal and efficient enough. Slow reaction kinetics will also severely restrict the applicability of Staudinger ligation in humans.

The relationship between the daily dose, the plasma concentration of blonanserin, and its plasma anti-dopamine D2 and anti-serotonin 5-HT2A activity.

OBJECTIVE: Blonanserin (BNS) possesses anti-serotonin 5-HT(2A) activity in addition to anti-dopamine D(2) activity, which is characteristic of second-generation antipsychotics, little information is available on its pharmacologic profile in vivo. We investigated the BNS daily dose, plasma concentration, plasma anti-D(2) activity, and plasma anti-5-HT(2A) activity in schizophrenia in a total of 14 subjects. METHODS: Blood samples were taken 14 days after the BNS dose was fixed, and the plasma concentration was measured by means of high-performance liquid chromatographic (HPLC) method. In addition, the plasma anti-D(2) activity and anti-5-HT(2A) activity were measured by means of radioreceptor assays in which [(3)H]-spiperone and [(3)H]-ketanserin were used. RESULTS: The results revealed a statistically significant correlation between the daily dose and the plasma concentration (p = 0.04). Statistically significant correlations were also observed between the plasma concentration and the anti-D(2) activity and between the plasma concentration and the anti-5-HT(2A) activity (p = 0.003 and 0.04). CONCLUSIONS: It is therefore believed that both the anti-D(2) activity in plasma and the anti-5-HT(2A) activity in plasma are regulated almost solely by the unchanged principal. Moreover, the mean plasma serotonin/dopamine (S/D) ratio was 0.9 and BNS exhibited both anti-D(2) activity and also anti-5-HT(2A) activity in vivo, as well, so it was clear that the in vitro pharmacological profile was retained in vivo.

Changes in in vivo [(3)H]-Ro15-4513 binding induced by forced swimming in mice.

Mice were forced to swim for 5 min in water at a temperature of 12 degrees C (cold water swim stress) or 32 degrees C (warm water swim stress), and stress-induced analgesia (SIA) was measured using the tail-flick test. The cold water swim stress induced non-opioid SIA as well as hypothermia, whereas the warm water swim stress caused opioid SIA. The in vivo binding of [(3)H]-Ro15-4513 was measured in the stressed mice and compared with that in control mice. The specific binding of [(3)H]-Ro15-4513 in the cerebral cortex, hippocampus, and cerebellum was significantly altered by forced swimming in cold water. Apparent association and dissociation rate of [(3)H]-Ro15-4513 binding were decreased, and the change in the dissociation rate was most pronounced in the hippocampus. In contrast, no significant alterations were observed in in vitro binding. The hypothermia induced by the cold water swim stress seems to be the main reason for alterations in the specific binding of [(3)H]-Ro15-4513. The kinetics of a saturable amount of [(3)H]-Ro15-4513 in the blood and brain were also measured. The relative ratio of the radioactivity concentration in the brain to that in the blood was significantly decreased by forced swimming in cold water, indicating that the cold water swim stress induced changes in the nonspecific binding of [(3)H]-Ro15-4513 in the brain. These results together with previous reports suggested that non-opioid SIA induced by the cold water swim stress might be related to alterations in the rates of general ligand-receptor interactions including GABA(A)/benzodiazepine system. Changes in the nonspecific binding might be also involved in non-opioid SIA.

Metabolite analysis of [11C]Ro15-4513 in mice, rats, monkeys and humans.

We performed in vitro and in vivo assays of the metabolism of [(11)C]Ro15-4513 over time in the plasma of mice, rats, monkeys and humans, using a radio-HPLC equipped with a sensitive positron detector, in order to compare the metabolic rates of the radiopharmaceutical agent among the different animal species and to establish a highly sensitive analytical method for the radiotracer agent. We also examined the metabolism of [(11)C]Ro15-4513 in the brain tissue of mice and rats. The analytical method used in this study permitted detection of even extremely low levels of radioactivity (approximately 5,000 dpm). In vitro experiments revealed that [(11)C]Ro15-4513 in the blood was metabolized to hydrolysate [(11)C]A. The species were classified in descending order of the metabolic rate of the radiotracer in vitro as follows; mice, rats, and monkeys/humans. In the in vitro experiment, the percentage of the unchanged drug in the plasma at 60 minutes postdose was 9% in mice, 70% in rats, 97% in monkeys, and 98% in humans. In vivo metabolite analysis in the blood showed the presence of two radioactive metabolites, consisting of one hydrolysate [(11)C]A and another unidentified substance. The species were classified in descending order of the metabolic rate of the radiotracer in vivo as follows; mice, rats/humans, and monkeys. The percentage of the unchanged drug in the plasma was 6% in mice, 21% in rats, 26% in humans, and 40% in monkeys. Furthermore, the in vitro and in vivo experiments conducted to analyze the metabolism of [(11)C]Ro15-4513 in the brain tissue of mice and rats revealed that the radiotracer was metabolized to some extent in the brain tissue of these animals. In the in vivo experiment, the percentage of the unchanged drug at 60 min postdose was 86% in the brain tissue of mice and 88% in the brain tissue of rats, while in the in vitro experiment, the corresponding percentage was 93% in mice, and 91% in rats.

Application of a new analytical method using gas chromatography and gas chromatography-mass spectrometry for the azide ion to human blood and urine samples of an actual case.

We have established a practical and reliable method to identify and quantify the azide ion in human whole blood and human urine by transforming the ion into pentafluorobenzyl azide (PFBN3). PFBN3 was simply derived from a reaction of the ion with an excess amount of pentafluorobenzyl bromide (PFBBr). The excess amount of PFBBr was removed from the products by its reaction with sodium thiosulfate. PFBN3 in the sample was detected in high sensitivity by gas chromatography with nitrogen-phosphorus detector (GC-NPD) and gas chromatography-mass spectrometry (GC-MS). The lower detection limits of the ion by GC-NPD were 5 ng/ml for human whole blood sample and 0.5 ng/ml for human urine sample at S/N=3. On the other hand, they were 100 ng/ml for human whole blood sample and 10 ng/ml for human urine sample by the full-scan mode of GC-MS. The analytical method was applied to identification and quantification of the ion in the actual whole blood and urine samples of the victims in an actual criminal case.

Effects of sodium azide on motor activity, motor coordination, and learning.

Because of the proposed importance of cytochrome oxidase in some neurological disorders, an inhibitor of this enzyme was evaluated in a battery of tests measuring exploration, motor coordination, and learning. Mice injected with sodium azide (6 or 12 mg/kg) were slower to initiate a response in a T maze and had less rears in a small chamber than mice injected with placebo. Drugged mice did not alternate spontaneously even at a minimal retention interval (0 min), but were not impaired in water maze spatial and visual discrimination learning tasks. No group differences emerged in terms of horizontal motor activity and its habituation, number of grooming episodes, and motor coordination. These results indicate that azide-induced slowing of motor activity is situation-specific and is accompanied by abnormalities in choice behavior in a T maze.

Analytical findings in a suicide involving sodium azide.

A 47-year-old laboratory assistant ingested approximately 9 g of sodium azide powder and died 4 h later at a hospital. A high-performance liquid chromatographic method using diode-array detection has been developed for the determination of an azide benzoyl derivative in blood (after a simple deproteinization) and in several tissues (after homogenization in a neutral buffer and deproteinization of the supernatant). The blood concentration in this case was lower than those previously published. The highest azide concentration was found in lung tissue. A complete toxicological screening revealed the presence of cyanide in blood, which has been previously reported twice, but for the first time, it was confirmed by mass spectrometry. Whether the production of cyanide in the presence of azide took place in vivo or postmortem remains unknown; the nature of the metabolic pathway involved also remains unknown.

Detection of oxidants in uremic plasma by electron spin resonance spectroscopy.

Depletion of antioxidants and the presence of products of free radical damage in plasma suggest that oxidative stress is increased in uremia. We have developed an application of electron spin resonance spectroscopy, and used this method to show that a stable oxidizing component or components of plasma accumulate in uremia. No oxidizing activity was detectable in plasma from subjects with normal renal function. The oxidant was detected by its capacity to oxidize the spin trap 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS). The oxidant was dialyzable from plasma, had an upper molecular weight limit of about 3,000 Daltons and was stable over many months. Physiological plasma concentrations of vitamin C, a water soluble congener of vitamin E and reduced glutathione were unable to inhibit the oxidizing capacity of uremic plasma. Thus, uremia is associated with accumulation of an endogenous oxidizing activity at much higher concentrations than in subjects with normal renal function.

Acute neurotoxicity of sodium azide and nitric oxide.

Sodium azide is a chemical of rapidly growing commercial importance with a high acute toxicity and an unknown mechanism of action. Although it has some chemical properties and biological effects in common with cyanide, its lethality does not appear to be due to inhibition of cytochrome oxidase. Unlike cyanide it is a potent vasodilator and inhibitor of platelet aggregation presumably by virtue of its conversion to nitric oxide in vivo and in isolated preparations of blood vessels and thrombocytes. It is not clear whether the high toxicity of azide is due to nitric oxide or to the parent anion. Of a number of possible azide antagonists tested in intact mice only phenobarbital in both anesthetic and subanesthetic doses afforded statistically significant protection against death. Diazepam, phenytoin, and an anesthetic dose of a ketamine/xylazine combination had no effect. Major motor seizures are sometimes seen in human azide poisoning, and these are a regular feature of azide poisoning in laboratory rodents. Solutions of nitric oxide given systemically to mice produced no signs of toxicity, but doses 1,000-fold lower placed in the cerebroventricular system of rats produced brief but violent tonic convulsive episodes. A dose of 0.61 mmol/kg azide as given systemically regularly produced convulsions whereas a dose of 6 mumol/kg given icv produced seizures in rats. The icv convulsive dose of azide was 50-fold larger than the icv dose of nitric oxide. These results suggest that azide lethality is due to enhanced excitatory transmission in the central nervous system perhaps after its conversion to nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Embryotoxic effects of sodium azide infusions in the Syrian hamster.

Pairs of osmotic minipumps containing 400 mg/ml (6.15 M) sodium azide in distilled water were subcutaneously implanted in timed pregnancy Syrian golden hamsters. The total delivered dose was calculated as 6 X 10(-2) mmol kg-1 hr-1 at the maximal pumping rate. Most dams exhibited obvious signs of toxicity during the period of pump implantation which was Days 7 through 9 of gestation. After removal of the pumps the dams were euthanized on Day 13 of gestation, and the uteri were removed for counting of the number of living, malformed, and resorbed fetuses. This dose rate resulted in a significantly increased incidence of resorptions of embryos over that in a control group implanted with pumps delivering only distilled water. The incidence of gross malformations exclusively in the form of encephaloceles was not different between control and azide-infused groups. The extent of nitrosylation of circulating hemoglobin was followed with time and found to involve only about 0.1% of the total blood pigment. Thus, this commercially important and widely distributed chemical with high acute toxicity is not considered to be teratogenic in hamsters, and it produces embryotoxicity only at dose rates that result in toxic signs in the dams.

Photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor.

The synthesis, binding and photoincorporation of a thromboxane A2/prostaglandin H2 (TXA2/PGH2) analog (9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-azidophenyl )-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2) [( 125I]PTA-Azido) to washed human platelets was characterized. Kinetic analysis of the binding of [125I]PTA-Azido at 30 degrees C yielded a k1 of 1.83.10(7) M-1.min-1 and k -1 of 0.195 min-1, Kd = k -1/k1 = 11 nM. Incubation of washed human platelets with [125I]PTA-Azido followed by photolysis resulted in the radiolabelling of a number of platelet proteins as assessed by SDS-PAGE autoradiography. The radiolabelling of three of these protein bands could be either uniformly blocked or reduced with a series of structurally dissimilar TXA2/PGH2 receptor antagonists or agonists and corresponded to proteins with a molecular mass of 43, 39 and 27 kDa. In addition, the incorporation of [125I]PTA-Azido into the three proteins was stereoselectively blocked by a pair of optically active stereoisomers that are TXA2/PGH2 receptor antagonists. Two-dimensional gel electrophoresis indicated that the 43 kDa protein possessed a pI value of 5.6 and that the 27 kDa protein exists in at least three isoforms with pI values of 4.9, 5.1 and 5.3. The labelling pattern was not altered by a mixture of proteinase inhibitors. The data suggest that one or more of these specifically radiolabelled proteins may represent the human platelet TXA2/PGH2 receptor.

Lysosomotropic behavior of adrenergic antagonists in interactions with human neutrophils.

Autonomic neurohormones affect the secretory activity of neutrophils by modulating release of lysosomal enzymes in response to immunologic stimuli. Autonomic agents are also weak bases which might modify cell function by accumulating in the acidic interior of the lysosome, in addition to their receptor-mediated activity. We examined the association of the beta-adrenergic antagonist [3H]dihydroalprenolol with human neutrophils and lysosome and membrane fractions derived from neutrophils, and the subcellular distribution of the photoaffinity-labeled beta-adrenergic ligand [3H]azidobenzylcarazolol after incubation with intact cells. Isolated neutrophil lysosomes accumulated significantly more [3H]dihydroalprenolol than isolated membrane preparations. Decreasing the transmembrane pH gradient markedly reduced [3H]dihydroalprenolol accumulation by intact cells or lysosomes but not by membranes. Since [3H]dihydroalprenolol accumulated by intact cells remained rapidly exchangeable, the photoaffinity ligand [3H]azidobenzylcarazolol was used to assess ligand distribution after association with whole cells. After cell disruption, 18.5 +/- 1.3% of this ligand appeared in the lysosome fraction as compared to 2.2 +/- 0.6% in the membrane fraction. The secretagogue phorbol myristate acetate caused release of the ligand as well as lysosomal enzymes from cells. These findings suggest that there is significant pH-dependent lysosomal accumulation of beta-antagonists. This lysosomotropic interaction may be important both as it affects the sequestration and redistribution of the drugs, and as it might in some circumstances affect host defense functions of the neutrophil.

Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin.

Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.

Comparison of the equilibrium exchange of nucleosides and 3-O-methylglucose in human erythrocytes and of the effects of cytochalasin B, phloretin and dipyridamole on their transport.

Because of similarities in the physical and molecular properties of the nucleoside and sugar transporters of human erythrocytes and the photoaffinity labeling of the sugar transporter by 8-azidoadenosine (Jarvis et al. (1986) J. Biol. Chem. 261, 11077-11085), we have directly compared the equilibrium exchange of uridine and 3-O-methylglucose in these cells as measured by rapid kinetic techniques under identical experimental conditions. Both the Michaelis-Menten constant and maximum velocity were about 100-fold higher for 3-O-methylglucose exchange than for uridine exchange so that the first order rate constants for both transporters were about the same. When calculated on the basis of the number of nucleoside and sugar carriers per red cell estimated by equilibrium binding of nitrobenzylthioinosine and cytochalasin B, respectively, the turnover numbers for the sugar and nucleoside carriers with 3-O-methylglucose and uridine, respectively, as substrates were quite similar. Various sugars up to concentrations of 108 mM had no effect on the exchange of 500 microM uridine or adenosine, and uridine up to a concentration of 50 mM had no effect on the exchange of 10 mM 3-O-methylglucose. Adenosine, on the other hand, inhibited 3-O-methylglucose exchange in a concentration dependent manner, though not very effectively (IC50 approximately equal to 3 mM). Both uridine and 3-O-methylglucose exchange were inhibited in a concentration dependent manner by cytochalasin B, phloretin and dipyridamole, but cytochalasin B and phloretin were 100-times more effective in inhibiting 3-O-methylglucose than uridine exchange, whereas the opposite was the case for the inhibition by dipyridamole.

Anion binding to the four-copper form of bovine erythrocyte superoxide dismutase: Mechanistic implications.

The binding of thiocyanate, azide, and other anions to superoxide dismutase (SOD) containing copper in both the copper and zinc sites of the native enzyme (Cu2Cu2SOD) has been studied. Electron spin resonance spectroscopy was used to show that binding of SCN- to Cu2Cu2SOD breaks the imidazolate bridge between the two copper centers. Thiocyanate displaces the bridging histidine from the Cu2+ ion in the copper site and also replaces the aspartic acid ligand from the Cu2+ ion in zinc site. This conclusion is supported by studies with Ag2Cu2SOD, where silver is in the native copper site and copper is in the native zinc site. At low concentrations, N3- ion displaces axially coordinated water from Cu2Cu2SOD. At higher concentrations, N3- also breaks the imidazolate bridge. Parallel behavior was observed for SCN- and N3- binding to a model compound for Cu2Cu2SOD, showing that the difference between the two anions is a consequence of their copper binding properties and is not due to secondary interactions with the protein active site. The SCN- complex of Cu2Cu2SOD is catalytically active, indicating that the intact imidazolate bridge is not essential to the mechanism of superoxide dismutase action. Phosphate ion breaks the histidine bridge in Cu2Cu2SOD, whereas cyanate, formate, and fluoride ions do not. At about pH 11, hydroxide ion promotes the irreversible formation of a copper complex of deprotonated peptide nitrogen atoms for Cu2Cu2SOD but not for Cu2Zn2SOD in the same pH range.

Pulse radiolytic investigation of single heme group reduction in hum an methemoglobin.

Reduction of one of the four heme groups of human aquomethemoglobin A has been investigated by the pulse radiolysis method. The reactivity of e-a-q, the hydrated electron, with methemoglobin was determined by observing this species directly. The separate reactions of the hydroxy yl radical and hydrogen atom, as well as of e-a-q, were studied by observing absorbance changes in the protein spectrum over the wavelength range 290 to 600nm, with appropriate scavengers in solution...