Potential role of growth factors and extracellular matrix in wound healing after laryngotracheal reconstruction.

Laryngotracheal reconstruction (LTR) has been used for more than 20 years to treat infants and children with subglottic stenosis. Results after pediatric LTR have been satisfactory; however, approximately 10% of children have recurrent airway narrowing after LTR. The purpose of our study was to determine whether a correlation existed between specific growth factors and extracellular matrix in patients with adequate wound healing capability as compared with patients with poor wound healing capability. Histologic sections from 27 patients who underwent LTR were cut, and immunohistochemical staining was performed for transforming growth factor-beta, platelet-derived growth factor, fibronectin, tenascin, transforming growth factor-alpha, and vascular endothelial growth factor. Results showed that patients with adequate wound healing capability had a positive correlation with vasculature fibronectin, vasculature tenascin, and stromal fibronectin. Patients with poor wound healing capability had a positive correlation with stromal vascular endothelial growth factor.

99mTc-monoclonal antibody radiolabeled via hydrazino nicotinamide derivative for imaging disialoganglioside G(D2)-positive tumors.

3F8 is a murine IgG3 monoclonal antibody (MAb) selective for the ganglioside G(D2). Previous studies using 131I-3F8 have shown great potential in the imaging of neuroectodermal tumors and the therapy of human neuroblastoma. 131I is commonly used in radioimmunodiagnosis, but its relatively long half-life (8 days) and its high energy gamma-emission (364 KeV) are suboptimal for imaging purposes when compared with 99mTc (6 h and 140 KeV, respectively). To label 3F8 with 99mTc, the antibody was first coupled with a heterobifunctional linker, succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH), obtaining a hydrazinonicotinamide-antibody conjugate. Using 99mTc-Tricine as the precursor complex, 3F8-SHNH was coupled efficiently to 99mTc, resulting in >90% radiometal incorporation, with a specific activity >10 mCi/mg and retaining full immunoreactivity. Immunoscintigraphy at 6, 22, and 46 h after intravenous injection of 1 mCi of 99mTc-3F8 showed selective neuroblastoma localization in xenografted nude mice, comparable to that obtained with the injection of 100 microCi of 131I-3F8. Biodistribution studies of 131I-3F8 and 99mTc-3F8 in mice demonstrated comparable %ID/g uptake in tumor (with a T/B ratio: approximately 2.5 at 24 h and approximately 3.5 at 48 h) and normal organs, including blood, except for spleen and liver which had about a three times higher uptake of the 99mTc conjugate. In conclusion, 99mTc can be coupled conveniently at high specific activity to 3F8 without compromising immunoreactivity. SHNH appears to be a useful linker for 99mTc in tumor diagnostic imaging and may have potential utility in coupling other radioisotopes (e.g., 94mTc) for positron imaging and therapy.

RP463: a stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging.

A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with (99m)Tc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with (99m)Tc produced a ternary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(2)] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [(99m)Tc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. The radiolabeling yield for RP463 was usually >/=90% using 10 microg of fMLFK-HYNIC and 100 mCi of [(99m)Tc]pertechnetate. Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h. [(99)Tc]RP463 was prepared and characterized by HPLC and electrospray mass spectrometry. In an in vitro assay, [(99)Tc]RP463 showed an IC(50) of 2 nM against binding of [(3)H]fMLF to receptors on PMNs. [(99)Tc]RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC(50) value of 0.2 +/- 0.2 nM. The localization of RP463 in the infection foci was assessed in a rabbit infection model. RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 +/- 0.2 at 15 min postinjection to 7.5 +/- 0.4 at 4 h postinjection. Visualization of the infected area could be as early as 2 h. A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit. This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes.

Imaging of apoptosis (programmed cell death) with 99mTc annexin V.

UNLABELLED: Apoptosis (programmed cell death) is a critical element in normal physiology and in many disease processes. Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of the plasma membrane. Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface. Annexin V, an endogenous human protein, has a high affinity for membrane-bound PS. This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro. We describe the use of radiolabeled annexin V to detect apoptosis in vivo. The results are compared to histologic and flow cytometric methods to identify cells and tissues undergoing apoptosis. METHODS: Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with 99mTc. Bioreactivity of 99mTc-HYNIC annexin V was compared with fluorescein isothiocyanate (FITC)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vivo thymic cell suspensions undergoing apoptosis in response to different stimuli. In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures. In vivo localization of annexin V was studied in Balb/c mice injected with 99mTc-HYNIC annexin V before and after induction of Fas-mediated hepatocyte apoptosis with intravenously administered antiFas antibody. RESULTS: Membrane-bound radiolabeled annexin V activity linearly correlated to total fluorescence as observed by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r2 = 0.987), antiFas antibody (N = 8, r2 = 0.836) and doxorubicin (N = 10, r2 = 0.804); and in ex vivo experiments on thymic cell suspensions with dexamethasone-induced apoptosis from Balb/c mice (N = 6, r2 = 0.989). Necrotic Jurkat T-cell cultures also demonstrated marked increases in radiopharmaceutical (4000-5000-fold) above control values. AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of annexin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting. The increase in hepatic uptake in antiFas antibody-treated mice correlated to histologic evidence of fulminant hepatic apoptosis. CONCLUSION: These data suggest that 99mTc-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.

Activity of different bicyclam derivatives against human immunodeficiency virus depends on their interaction with the CXCR4 chemokine receptor.

Bicyclams represent a novel class of selective anti-HIV inhibitors with potent activity against T-cell tropic strains of HIV. The prototype compound, the bicyclam AMD3100, has an EC50 of 1 to 10 ng/ml against different strains of HIV-1, including clinical isolates. AMD3100 was shown to interact with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-cell tropic strains of HIV. Here we describe the interaction of different bicyclam derivatives with CXCR4. A close correlation (r2 = 0.7) was found between the anti-HIV potency of the bicyclams and their ability to inhibit the binding of an anti-CXCR4 monoclonal antibody or the intracellular Ca++ signal induced by the stromal cell-derived factor-1alpha, the natural ligand of CXCR4. These results indicate that the mechanism of action of bicyclams is primarily mediated by their interaction with CXCR4. The most potent interaction with CXCR4 and thus anti-HIV activity was shown by bicyclam analogs with cyclam rings composed of fourteen members that are linked by an aromatic (phenyl) bridge. Elucidating the structural requirements for receptor interaction and the site(s) of interaction of bicyclams with CXCR4 will aid in the understanding of HIV-cell fusion.

In vivo detection and imaging of phosphatidylserine expression during programmed cell death.

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.

Perspectives on a state enacted hearing screening assessment program in the newborn population.

OBJECTIVE: A review of the early performance of Ohio's statewide infant hearing screening program was performed to provide insight as to the impact of the current medical and socioeconomic climate on its implementation. BACKGROUND: In March 1988, the State of Ohio enacted a law that required universal screening of newborn children for hearing loss through a program known as the Infant Hearing Screening and Assessment Program (IHSAP). The program design consisted of a universally applied high-risk questionnaire followed by a screening auditory assessment for those who fail. Although the value of such a program engendered little early public debate, the institution of such a program represented a significant challenge from a public health perspective. STUDY DESIGN: The program performance was analyzed using data from the index population of 160,000 live births per annum and hospital surveys. RESULTS: The questionnaires were found to be failing twice the number of newborns as originally projected, whereas completion rates and compliance were excellent. The assessment arm was plagued with poor compliance rates and limited resources. Lack of resources for effective data management has prevented an accurate evaluation of the program's sensitivity and specificity. CONCLUSION: IHSAP performance is being hampered by poor assessment follow-up and resource limitations, both in terms of screening equipment and habilitative follow-up services for infants identified as hearing impaired. The reasons for these problems are discussed in relation to existing legislative guidelines and medicoeconomic realities.

Tumor imaging with technetium-99m-labeled hydrazinonicotinamide-Fab' conjugates.

UNLABELLED: This study compares the in vivo properties of direct versus indirect 99mTc-labeling for two Fab' fragments from antibodies that recognize tumor-associated antigens. METHODS: Fab' fragments of two IgG2a monoclonal antibodies were either radiolabeled directly or via the linker bromoacetyl hydrazinonicotinamide hydrobromide (BAHNH) conjugated site specifically at protein thiols. A thiol assay was used to determine the number of thiols in the Fab' and to monitor their consumption during conjugation with BAHNH. Both preparations were labeled to > 95% incorporation of 99mTc, with the isotope tracking the single 50 kD absorbance peak seen on size-exclusion HPLC. The labeled preparations were tested in tumor-bearing and control mice, with dissections at 4 and 24 hr and gamma scintigraphy of the tumor-bearing mice. RESULTS: The major difference between the two labeled preparations for either antibody fragment was the greater accumulation of isotope in the tumor for the indirectly labeled preparations. This increase ranged from 1.5- and 2.7-fold at 4 hr to 2.6- and 3.2-fold at 24 hr for the two antibodies, respectively. Since blood clearance was similar for the two labeling methods, the higher tumor accumulation with the indirectly labeled fragments resulted in higher tumor to blood ratios. Tumors could be imaged with both antibodies with either type of labeling with greater clarity and sensitivity at the 24 hr time point. CONCLUSION: While both labeling methods resulted in tumor detection through imaging, the images obtained with the indirectly labeled antibody fragments were more easily visualized due to the combination of higher radioisotope accumulation in the tumor and similar blood clearances compared to the direct labeled fragment.

Biodistribution of model 105Rh-labeled tetradentate thiamacrocycles in rats.

105Rh(III)Cl2 complexes with a limited series of [14]ane- and [16]ane- thia macrocycles were prepared and their biodistributions in Sprague-Dawley rats studied. These studies demonstrate that modifications in the structure and composition of the 105Rh-thia macrocycle complexes produce significant differences in their uptake and retention in both the liver and kidneys. The results indicate that the cis-Rh(III)Cl2-[14]ane thiamacrocycles exhibit less kidney retention than the corresponding trans-Rh(III)Cl2-[16]ane thiamacrocycles. In addition, the presence of a side chain containing a carboxylate group will produce decreased retention of activity in the kidneys. HPLC analysis of urine from these animals indicates no observable in vivo metabolism or dissociation of these chelates in the blood stream.

Biodistribution and dosimetry of technetium-99m-hydrazino nicotinamide IgG: comparison with indium-111-DTPA-IgG.

UNLABELLED: The biological behavior of human polyclonal immunoglobulin G (IgG), radiolabeled with 99mTc via a nicotinyl hydrazine derivative (99mTc-HYNIC-IgG), was evaluated in normal human subjects. METHODS: Initial biodistribution and dosimetry studies were performed in six normal male volunteers. Additionally, 99mTc-IgG and 111In-DTPA-IgG were co-injected into six subjects and scintillation camera images were acquired at 6 and 18 hr later and serial blood and urine samples were collected. Biodistribution of both radiopharmaceuticals were measured by region of interest analysis. In the dual-injection group, images were crossover-corrected. RESULTS: All subjects tolerated injection of the radiolabeled IgG preparations without apparent ill effects. Biodistribution of the two antibody preparations were remarkably similar with an increase in liver and abdominal activity for the 111In preparation. Linear correlation of the tissue-to-blood ratios of 99mTc and 111In-labeled IgG was observed at both times (r2 > 0.98). The slopes of the regression line were 0.97 and 0.76 at 6 and 18 hr, respectively. The beta phase of the blood clearance of 99mTc-HYNIC-IgG was significantly delayed (p < 0.01) compared with 111In-IgG (t1/2: 51.9 +/- 6.5 versus 35.3 +/- 3.4 hr). In contrast, the volumes of redistribution and urinary excretions of the radiopharmaceuticals were not significantly different. CONCLUSION: These studies establish that the biodistribution of 99mTc-HYNIC-IgG in normal human subjects is nearly identical to 111In-DTPA-IgG.

A comparison of cleavable and noncleavable hydrazinopyridine linkers for the 99mTc labeling of Fab' monoclonal antibody fragments.

The design and synthesis of hydrazinopyridine bifunctional chelating agents (BCA's) featuring amide, ester, and disulfide groups are described. The BCA's site-specifically react with the free thiol groups of the tumor-specific monoclonal antibody fragment C46.3 using a one-pot in situ reduction and conjugation procedure from the F(ab')2 to give Fab'-linker conjugates. Molar substitution ratios (MSR's) of the hydrazinopyridine conjugates were comparable to the theoretical (maximum) number of thiols per fragment determined by free hydrazine and residual thiol assays. The series of C46.3 Fab'-linker conjugates were 99mTc-labeled in greater than 95% radiochemical purity by incubation with 99mTc-tricine for 1 h at room temperature. In order to evaluate the conjugates for radiopharmaceutical applications, the tumor localization and biodistribution properties of the radiolabeled Fab'-linker conjugates, compared to the direct labeled fragment, were tested in nude mice bearing LS174T xenografts. Depending upon the structure of the linker connecting the radiolabeled hydrazinopyridine group to the antibody fragment, we observed a variation in kidney uptake and whole-body clearance. Diester- and monoester-linked conjugates exhibited lower kidney uptake and faster whole-body clearance than the corresponding linker containing amide groups. This result may be interpreted as evidence for rapid metabolism of ester compared to amide groups in the kidney following uptake. At 24-h postinjection, the monoester-linked conjugate 99mTc-C46.3 Fab'-BA displayed the highest tumor: blood ratio (16.2) compared to the directly labeled conjugate (6.6) and is therefore a potential clinical candidate for imaging breast and ovarian cancer.

Synthesis and structure-activity relationships of phenylenebis(methylene)-linked bis-tetraazamacrocycles that inhibit human immunodeficiency virus replication. 2. Effect of heteroaromatic linkers on the activity of bicyclams.

A series of bicyclam analogs connected through a heteroaromatic linker have been synthesized and evaluated for their inhibitory effects on HIV-1 (IIIB) and HIV-2 (ROD) replication in MT-4 cells. The activity of pyridine- and pyrazine-linked bicyclams was found to be highly dependent upon the substitution of the heteroaromatic linker connecting the cyclam rings. For example, 2,6- and 3,5-pyridine-linked bicyclams were potent inhibitors of HIV-1 and HIV-2 replication, whereas the 2,5- and 2,4-substituted pyridine-linked compounds exhibited substantially reduced activity and, in addition, were found to be highly toxic to MT-4 cells. We have subsequently discovered that these effects are not unique; amino-substituted linkers also have the potential to deactivate phenylenebis(methylene)-linked bicyclams. A model is proposed to explain the deactivating effects of the pyridine group in certain substitution patterns based on the ability of the pyridine nitrogen to participate in pendant conformations (complexation) with the adjacent azamacrocyclic ring, which may involve hydrogen bonding or coordination to a transition metal. The introduction of a sterically hindering group such as phenyl at the 6-position of the 2,4-substituted pyridine-linked bicyclam appears to prevent pendant conformations, providing an analog with comparable anti-HIV-1 and anti-HIV-2 activities to the parent m-phenylenebis(methylene)-linked bicyclam. The results of this study have been used to develop a quantitative structure-activity relationship model with improved predictive capability in order to aid the design of antiviral bis-azamacrocyclic analogs.

[99mTc]tricine: a useful precursor complex for the radiolabeling of hydrazinonicotinate protein conjugates.

The stannous reduction of [99mTc]pertechnetate in the presence of tricine results in the formation of the new labeling precursor complex [[99m-Tc]tricine. This complex has improved efficacy for the 99mTc labeling of hydrazinonicotinate-modified IgG compared to [99mTc]glucoheptonate. FAB mass spectral analysis of the product formed by the reaction of [TcOCl4](-1) with tricine indicates the formation of [TcO(tricine x 2H)2](-1).

Synthesis and structure-activity relationships of phenylenebis(methylene)-linked bis-tetraazamacrocycles that inhibit HIV replication. Effects of macrocyclic ring size and substituents on the aromatic linker.

We have previously described the potent and selective inhibition of several strains of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) by JM2763, an n-propyl-linked dimer of the 1,4,8,11-tetraazamacrocyclic (cyclam) ring system. Upon further investigation, we have also found that incorporating an aromatic rather than aliphatic linker leads to analogs with higher antiviral potency. The prototype, JM3100 (19a, isolated as the octahydrochloride salt), which contains a p-phenylenebis(methylene) moiety linking the cyclam rings, inhibited the replication of HIV-1 (IIIB) and HIV-2 (ROD) at EC50's of 4.2 and 5.9 nM, respectively, while remaining nontoxic to MT-4 cells at concentrations exceeding 421 microM. In order to identify the structural features of bis-tetraazamacrocycles required for potent activity, we have prepared a novel series of phenylenebis(methylene)-linked analogs, in which the macrocyclic ring size was varied from 12 to 16 ring members. Depending upon the substitution of the phenylenebis(methylene) linker (para or meta), sub-micromolar anti-HIV activity was exhibited by analogs bearing macrocycles of 12-14 ring members but with varying cytotoxicity to MT-4 cells. Furthermore, while we found that identical macrocyclic rings are not required for activity, substituting an acyclic polyamine equivalent for one of the cyclam rings in 19a resulted in a substantial reduction in anti-HIV potency, clearly establishing the importance of the constrained macrocyclic structure. A short series of transition metal complexes of 19a were also prepared and evaluated. Complexes of low kinetic stability such as the bis-zinc complex retained activity comparable to that of the parent compound. Finally, the activity of bicyclam analogs appears to be insensitive to the electron-withdrawing or -donating properties of substituents introduced onto the linker, but sterically hindering groups such as phenyl markedly reduced activity. As a result, several analogs with anti-HIV potency comparable to that of 19a have been identified.

Imaging of focal sites of inflammation in rhesus monkeys with 99mTc-labeled human polyclonal IgG.

The biological behavior of human polyclonal immunoglobulin G (IgG) radiolabeled with 99mTc via a nicotinyl hydrazine derivative, was evaluated in Rhesus monkeys. 99mTc-IgG and 111In-MACROSCINT DTPA-IgG were co-administered to Rhesus monkeys with focal sites of sterile inflammation and scintillation camera images were acquired at 6 and 19 h after injection. The biodistribution of the two antibody preparations were similar, however, small differences were detected: 99mTc-IgG > 111In-IgG in spleen and lung; 99mTc-IgG in bone and skeletal muscle. A linear correlation of the tissue-to-blood ratios of 99mTc- and 111In-labeled IgG was observed at both times (r2 > 0.98). The slopes of the regression lines were not significantly different from unity: 6 h-0.982 +/- 0.018; 19 h 1.0334 +/- 0.0226. Also, at both 6 and 19 h after injection, the target-to-background ratios (T/B) for the sites of inflammation were remarkably similar for 111In and 99mTc. These studies establish that human polyclonal IgG labeled with 99mTc via a nicotinyl hydrazine modified intermediate is equivalent to 111In-MACROSCINT DTPA-IgG for imaging focal sites of inflammation in monkeys.

Technetium-99m-labeled hydrazino nicotinamide derivatized chemotactic peptide analogs for imaging focal sites of bacterial infection.

We synthesized and evaluated four hydrazino nicotinamide (HYNIC) derivatized chemotactic peptide analogs: For-NleLFK-HYNIC (HP1), For-MLFK-HYNIC (HP2), For-MLFNH(CH2)6NH-HYNIC (HP3), and For-MLF-(D)-K-HYNIC (HP4), for in vitro bioactivity and receptor binding. The peptides were radiolabeled with 99mTc via a glucoheptonate co-ligand and their biodistribution determined in rats (n = 6/time point) at 5, 30, 60 and 120 min after injection. Localization of the peptides at sites of deep thigh Escherichia coli infection was determined by radioactivity measurements on excised tissues in rats (n = 6/time point) and rabbits as well as scintillation camera imaging in rabbits (n = 6). All peptides maintained biological activity (EC50s for O2 production by human PMNs: 12-500 nM) and the ability to bind to the oligopeptide chemoattractant receptor on human PMNs (EC50s for binding: 0.12-40 nM). After incubation with 99mTc-glucoheptonate, radiolabeled peptides were isolated by HPLC at specific activities of > 10,000 mCi/microM. Technetium-99m-labeled peptides retained receptor binding with EC50s < 10 nM. Blood clearance of all four peptides was rapid. Biodistributions of the individual peptides were similar, with low levels of accumulation in most normal tissues. In rats, all of the peptides concentrated at the infection sites (T/B ratio: 2.5-3:1) within 1 hr of injection. In rabbits, outstanding images of the infection sites were obtained, with T/B ratios of > 20:1 at 15 hr after injection. This study demonstrates that 99mTc-labeled chemotactic peptide analogs are effective agents for the external imaging of focal sites of infection.

Metal compounds in therapy and diagnosis.

There is increasing interest in the use of metal-containing compounds in medicine. This review describes several therapeutic applications, such as the use of platinum complexes in cancer chemotherapy, gold compounds in the treatment of arthritis, gallium in hypercalcemia, bismuth in anti-ulcer medication, and sodium nitroprusside in hypertension. The use of metal radionuclides in diagnosis and radiotherapy and the role of paramagnetic metal complexes as contrast agents in magnetic resonance imaging are also discussed.