Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

Technetium-99m-labeled platelets: Comparison of labeling with a new lipid-soluble Sn(II)-mercaptopyridine-N-oxide and 99mTc-HMPAO

Platelets pretinned with a neutral Sn(II)-2-mercaptopyridme-N-oxide (SN-MPO) were labeled with ^99mTc and compared to those labeled with ^99mTc-HMPAO. The conditions of labeling platelets, e.g. concentrations of platelets and Sn(II)-MPO, ^99mTc in ACD-saline or ACD-plasma media, pH and incubation time, were optimized using canine platelets. Moderate labeling efficiency was obtained with 20 μg of tin(II) chloride and 30 min incubation with Sn-MPO and pertechnetate. The viability of labeled platelets was determined by platelet recovery and platelet survival times in Beagle dogs. The labeling efficiency with platelets from 43 mL of blood was 62.8 ± 7.6%. The platelet recovery was 35.7 ± 5.0% and exponential survival time was 34.6 ± 3.1 h compared to 43.3 ± 12.0% and 29.5 ± 3.3 h for ^99mTc-HMPAO-labeled platelets. These values were significantly (P < 0.01) lower than ¹¹¹In-labeled platelets. Biodistribution in dogs indicates lower retention in blood, spleen and liver after some initial ^99mTc excretion in urine. The platelet deposition with ^99mTc platelets (Sn-MPO method) on polyurethane angio-catheters was similar to ^99mTc-HMPAO-labeled platelets. This study indicates that the platelets could be successfully labeled with pertechnetate in a cost-effective manner for the evaluation of thromboembolic complications.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

A monoclonal antibody reacting with platelets for monitoring thrombolysis

Thrombus immunoscintigraphy with radiolabeled monoclonal antibodies are presently undergoing intense clinical evaluations. Reports on clinical trials of radiolabeled antifibrins are very encouraging and results of antiplatelet antibody evaluations are forthcoming. Animal studies with antiplatelet antibodies indicate that a diagnosis can be made within the critical “lytic window” of 4–6 h, and thus the imaging procedure may be used as an adjunct to thrombolytic therapy, i.e. screening of patients.We now report on a potentially new application of monoclonal antibodies, immunoimaging for monitoring thrombolysis. In vitro studies were performed with “standardized clots” incubated with ^99mTc 50H.19 and re-incubated with streptokinase (SK), urokinase (UK) or recombinant tissue plasminogen activator (rt-PA). The decrease in clot-bound ^99mTc 50H.19 activity after SK, UK or rt-PA incubation was proportional to the decrease in clot weight (r = 0.90–0.98). The direct effects of these thrombolytic agents on the labeled antibody and the possible interference of aspirin, warfarin and heparin in thrombus immunoimaging were also investigated. Aspirin, heparin and warfarin did not interfere with clot-binding of ^99mTc 50H.19. Thrombolytic agents did not affect the stability of the radiolabel or immunoreactivity of 50H.19. These results indicate that ^99mTc 50H.19 is a promising agent that may enable monitoring thrombolysis in addition to thrombus immunoimaging.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, ^99mTc, and an aqueous marker, ¹²⁵I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped ¹²⁵I-labelled poly(vinyl pyrrolidone) were labelled with ^99mTc by the SnCl₂ method [2]. ^99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, ^99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than ¹²⁵I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60–70% of ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of ^99mTc-radioactivity was co-eluted with liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all ^99mTc-radioactivity does not represent the 60–70% of intact liposomes present in lymph nodes. Using the aqueous marker ¹²⁵I-labelled poly(vinyl pyrrolidone), the lymphnode localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that ¹²⁵I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier [2], where the authors used ^99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became ‘leaky’ to low-molecular-weight drugs, for example, methotrexate (M_r 454) to a much greater extent than with a large-molecular-weight substance, ¹²⁵I-labelled poly(vinyl pyrrolidone) (M_r 30 000–40 000), when incubated with rat lymph at 37°C. Using the two markers ^99mTc and ¹²⁵I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining λ-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with ^99mTc rather than using the SnCl₂ technique [2], or using other radionuclides as markers for γ-scan imaging.     

Direct 99mTc labeling of monoclonal antibodies: Radiolabeling and in vitro stability

Direct labeling involves ^99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind ^99mTc. The direct ^99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability.Stable direct antibody labeling with ^99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for ^99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the ^99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution.Very good ^99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab′ fragment, which makes these substances possible candidates for immunoscintigraphy use.     

Transchelation of 99mTc from low affinity sites to high affinity sites of antibody

An exclusive labeling of high affinity sites of IgG and its F(ab′)₂ fragments with ^99mTc was accomplished. Antibody was first labeled in 0.1 M acetate buffer at pH 4.5, using stannous chloride as a reducing agent. Thus, high capacity, low affinity sites and low capacity, high affinity sites were both labeled. These ^99mTc complexes were stable at pH 4.5 and 7.0; however, they became destabilized at pH 8.2 and 9.0. Transchelation of ^99mTc to DTPA took place at the higher pH values and leveled off at 54% ^99mTc-F(ab′)₂ and 73% ^99mTc-IgG. These results indicate that the majority of ^99mTc bound to the low affinity sites was transchelated to the high affinity sites rather than to DTPA since low affinity sites account for 84% of total F(ab′)₂ sites and 76% of IgG sites. Biodistribution data in mice at 2.5 h postinjection were consistent with this hypothesis in that tissue concentrations of ¹¹¹In-DTPA-F(ab′)₂ were similar to the reequilibrated ^99mTc-F(ab′)₂ but were much higher than that of the unequilibrated ^99mTc-F(ab′)₂.     

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody

A rat monoclonal IgG_2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca²⁺ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca²⁺-dependent and Ca²⁺-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled ¹²⁵I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a ¹²⁵I-labeled band of M_r 88 000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca²⁺ and 1 mM Mg²⁺, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Isolation of human platelet glycoproteins

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of M_r ≈ 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of M_r ≈ 165 000.Treatment of whole platelets by periodate oxidation and sodium[³H]borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of M_r ≈ 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with M_r ≈ 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others.Phosphorylation of intact platelets with ³²P_i also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the     

Direct labeling of proteins with 99mTc

The direct labeling of antibodies and antibody fragments to form a highly stable bond between technetium and the sulfide groups of proteins is now well established. To optimize this reaction, the antibody protein must have sufficient reactive sulfides available to accept that technetium metal ions that are formed by the reduction of pertechnetate in the presence of a weak complexing agent. The reactive sulfide groups are provided by first reducing a small fraction of the disulfide bridges in the antibody protein or by starting with Fab′ fragments, which already have reactive sulfide groups. When the antibody protein has been appropriately reduced, and the reactive sulfide groups protected by a metal ion with a lower binding affinity than technetium, such as tin or zinc, very high labeling yields of high-affinity-bonded ^99mTc can be achieved. This can be accomplished without loss of immunoreactivity, measured as either affinity or immunoreactive fraction.Side reactions can produce radiochemical impurities such as low-affinity, bound ^99mTc; ^99mTc colloids; ^99mTc peptides or antibody aggregates; or ^99mTc-complexes. Also, pertechnetate ions may be an impurity if the sodium pertechnetate solution added to the reduced antibodies is not completely reduced. The specifics of minimizing these side reactions have not been extensively discussed in the prior literature; however, it is clear that appropriate reduction of the protein prior to labeling and complete removal of the reducing agent, particularly if it contains reactive sulfide groups or is toxic, are critical.One- or two-step ^99mTc-labeling kits for preparing ^99mTc-labeled antibody or antibody fragments are rapidly being introduced for use in clinical nuclear medicine studies. These direct labeling methods employ a common sequence of chemical reactions, although the reducing agents for both the antibody and the [^99mTc]pertechnetate may vary. Different ^99mTc transfer agents may be used, but all transfer agents have the common feature of quickly forming weak to moderately strong complexes with reduced technetium. Most use Sn(II) to reduce the pertechnetate, although other reducing agents can be used.     

Technetium-99m-labeled antibodies

It is essential in any method for radiolabeling antibody with^99mTc that the labeling procedure is rapid and reliable, producing a highly stable^99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (^99mTc and¹²⁵I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (^99mTc or¹²⁵I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of^99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr<IgG) that were not detected by the same analysis of¹²⁵I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the^99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of^99mTc-labeled material with a Mr<IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the^99mTc equivalent corresponds to free iodide.     

Epinephrine and shear stress synergistically induce platelet aggregation via a mechanism that partially bypasses vWF-Gp Ib interactions

Elevated shear stress levels in pathologically stenosed vessels induce platelet activation and aggregation, and may play a role in the pathogenesis of arterial disease. Increased plasma catecholamine concentrations have also been implicated in the onset of acute coronary ischemic syndromes. This study was designed to examine the synergistic interaction of shear stress and epinephrine in the activation of platelets. Platelets (in PRP) sheared at 60 dyn/cm² showed little or no aggregation unless pretreated with epinephrine. Pretreatment with 250 nM epinephrine followed by shear at 60 dyn/cm² induced >60% platelet aggregation. The specific α₂-adrenergic receptor antagonist yohimbine inhibited the synergistic aggregation, as did the ADP scavenging system phosphocreatine/creatine phosphokinase, indicating a three-way synergism with ADP. Chemical or monoclonal antibody blockade of von Willebrand factor (vWF) interactions with either platelet glycoprotein (Gp) Ib or Gp IIb/IIIa completely inhibited platelet aggregation induced by activating levels of shear stress alone. However, the combination of epinephrine and shear stress induced platelet aggregation that was blocked by 10E5, a monoclonal antibody that inhibits vWF binding to Gp IIb/IIIa, but not by aurin tricarboxylic acid or the monoclonal antibody 6D1, both of which inhibit vWF binding to Gp Ib. Synergistic platelet aggregation in response to epinephrine and shear stress was observed in washed platelets, platelet-rich plasma and whole blood in vitro, and also ex vivo following exercise to elevate endogenous levels of catecholamines. These results indicate that epinephrine synergizes with shear stress to induce platelet aggregation. This synergistic response requires functional Gp IIb/IIIa complexes, but is at least partially independent of vWF-Gp Ib interactions.     

99mTc-tricarbonyl labeled agents for cell labeling: Development,biodistribution in normal mice and preliminary in vitro evaluation

We have developed four ^99mTc(CO)₃-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. ^99mTc(CO)₃-hexadecylamino-N,N′-diacetic acid (negatively charged), ^99mTc(CO)₃-hexadecylamino-N-α-picolyl-N′-acetic acid (uncharged), ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine (positively charged), ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with ^99mTc-d,l-HMPAO and [¹⁸F]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine after 60 min incubation (compared to 55% and 23% for ^99mTc-d,l-HMPAO and [¹⁸F]FDG, respectively) while in plasma-rich medium, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine was best bound (54%, similar to the binding of ^99mTc-d,l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent (^99mTc(CO)₃-N,N′-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent ^99mTc-d,l-HMPAO, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.     

Synthesis and biological evaluation of a novel 99mTc(CO)3 complex of ciprofloxacin dithiocarbamate as a potential agent to target infection

The ciprofloxacin dithiocarbamate (CPFXDTC) was radiolabeled with [^99mTc(CO)₃(H₂O)₃]⁺ intermediate to form the ^99mTc(CO)₃–CPFXDTC complex in high yield. The ^99mTc(CO)₃–CPFXDTC complex was characterized by HPLC and its stability in serum was studied. Its partition coefficient indicated that it was a lipophilic complex. The bacterial binding efficiency of ^99mTc(CO)₃–CPFXDTC was almost the same as that of ^99mTcN–CPFXDTC, and was higher than that of ^99mTc–ciprofloxacin. Biodistribution results in induced infection mice showed ^99mTc(CO)₃–CPFXDTC had higher uptake at the sites of infection and better abscess/blood and abscess/muscle ratios than those of ^99mTc–ciprofloxacin and ^99mTcN–CPFXDTC. Single photon emission computed tomography (SPECT) static imaging study in infected rabbits demonstrated the uptake in the left thigh infection lesion was observable, while no accumulation in the right thigh muscle was found. These results suggested ^99mTc(CO)₃–CPFXDTC would be a promising candidate for further evaluation as infection imaging agent.     

Synthesis and evaluation of technetium-99m monocationic mixed ligand complexes of phenyl substituted/condensed tetradentate schiff's bases and trimethylphosphine

Tc-99m monocationic mixed ligand complexes of phenyl substituted/condensed Schiff's bases, N,N′-ethylene-bis-(benzoylacetone imine) (L_b) or N,N′-ethylene-bis-(salicylaldehyde imine) (L_c) or N,N′-ethylene-bis-(2-hydroxyacetophenone imine) (L_d) and trimethylphosphine were synthesized to determine the influence of the presence of a phenyl group in these tracers on their heart uptake in rats. A new formulation procedure using aq. β-hydroxypropylcyclodextrin (HPB) solution was developed for intravenous administration of nonpolar ^99mTc complexes. Comparison of biodistribution data for the reference ^99mTc complex from N,N′-ethylene-bis-(acetylacetone imine) and trimethylphosphine using HPB formulation and alternate formulation (0.9% saline) showed the same results. Biodistribution of the title ^99mTc complexes, [^99mTc L_b (PMe₃)₂]⁺, [^99mTc L_c (PMe₃)₂]⁺ and [^99mTc L_d (PMe₃)₂]⁺ showed heart-to-blood activity ratios of 1.7, 2.1 and 1.7, respectively, at 15 min post-injection in rats.     

Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Analysis of elimination mechanisms of some 99mTc-complexes

The biological behaviour of complexes of ^99mTc with aminopolycarboxylic and aminocarbohydroxamic ligands EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), EDTAH (ethylenediaminetetraacetohydroxamic acid) and HIDAmH (N-2-hydroxyethyl-N-carboxymethyl-aminoacetohydroxamic acid) was studied in rabbits. The pharmacokinetic parameters determined in intact rabbits were compared with the results obtained in the study of renal and hepatic clearance of the complexes under study. Hepatobiliary excretion, which in [^99mTc]EDTA forms 20–30% of the total excreted amount, is of negligible magnitude in the other ^99mTc-complexes studied (<2%). Their renal clearance is not influenced by the inhibition of tubular secretion with probenecid. Binding to plasma proteins increases in the order [^99mTc]DTPA < [^99mTc]EDTA <[^99mTc]HIDAmH <[^99mTc]EDTAH and the elimination half-life increases in the same order. The value of renal clearance of the complexes studied related to inulin clearance correlates well with the fraction of the free drug in the plasma. In rabbits the complexes under study are excreted mainly by the mechanism of glomerular filtration in the kidney.     

Vasopressin inhibits the adenylate cyclase activity of human platelet particulate fraction through V1-receptors

Arg⁸-vasopressin inhibited the adenylate cyclase activity of human platelet particulate fraction up to a maximum of 27% (IC₅₀ = 1.2 nM). This inhibition required the presence of 10 μM GTP and was optimal with 100 mM NaCl. Orn⁸-vasopressin had similar effects. 1-Deamino-Val⁴, D-Arg⁸-vasopressin did not by itself affect adenylate cyclase activity but competitively inhibited the action of Arg⁸-vasopressin (pA₂ = 7.74). Arg⁸-vasopressin did not inhibit adenylate cyclase in intact platelets but instead caused platelet aggregation, an effect that was also competitively inhibited by 1-deamino-Val⁴, D-Arg⁸-vasopressin (pA₂ = 7.82). Thus, platelets possess vasopressin receptors of the V₁ type that, under appropriate conditions, can mediate either inhibition of platelet adenylate cyclase or platelet aggregation.     

Common identity of substrate binding subunit of vacuolar h-translocating inorganic pyrophosphatase of higher plant cells

There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H⁺-translocating inorganic pyrophosphatase (tonoplast H⁺-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (M_r) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinity-labeling, sequencing, and immunological procedures, that the major polypeptide associated with the H⁺-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H⁺-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under identical conditions (M_r(Beta) = 64,500; M_r(Vigna) = 66,000) and exhibit identical kinetics of irreversible inhibition and ligand-modified labeling by [¹⁴C]-N-ethylmaleimide. Similarly, the M_r 64,500 and 67,000 polypeptides isolated from Beta by independent methods (cf. C.J. Britten, J.C. Turner, P.A. Rea [1989] FEBS Lett 256: 200-206 versus V. Sarafian and R.J. Poole [1989] Plant Physiol 91: 34-38) are indistinguishable: the two polypeptides comigrate when electrophoresed under the same conditions and yield tryptic fragments with identical overlapping sequences. Because both the N-terminal sequence of the M_r 66,000 subunit of the H⁺-PPase isolated from Vigna and the direct sequence data from Beta align precisely with the deduced amino acid sequence of cDNAs encoding the H⁺-PPase of Arabidopsis, all three enzymes are inferred to be highly conserved structurally. Accordingly, immunoblots of membranes prepared from Arabidopsis, Beta, Vigna, and Zea, probed with antibody affinity purified against the magnesium inorganic pyrophosphate-binding, M_r 66,000 polypeptide of Vigna, reveal a single immunoreactive band at M_r 64,500 to 67,000 in all four preparations. The M_r 66,000 polypeptide of Zea membranes is, however, prone to proteolysis during membrane fractionation and selective aggregation during sample denaturation for SDS-PAGE. The anomalous M_r 37,000 to 45,000 subunit pattern previously ascribed to the H⁺-PPase from Zea (A. Chanson and P.E. Pilet [1989] Plant Physiol 90: 934-938) is attributed to loss of the M_r 66,000 subunit and the appearance of polypeptide fragments of M_r 44,700 and 39,000 through the combined effects of sample aggregation before SDS-PAGE and proteolysis, respectively. It is, therefore, concluded that the substrate-binding subunit of the tonoplast H⁺-PPase has a common identity in all four organisms.