A "dose on demand" Biomarker Generator for automated production of [(18)F]F(-) and [(18)F]FDG.

The University of Oklahoma-College of Pharmacy has installed the first Biomarker Generator (BG75) comprising a self-shielded 7.5-MeV proton beam positive ion cyclotron and an aseptic automated chemistry production and quality control module for production of [(18)F]F(-) and clinical [(18)F]FDG. Performance, reliability, and safety of the system for the production of "dose on demand" were tested over several months. No-carrier-added [(18)F]F(-) was obtained through the (18)O(p,n)(18)F nuclear reaction by irradiation (20-40 min) of a >95% enriched [(18)O]H2O target (280 µl) with a 7.5-MeV proton beam (3.5-5.0 µA). Automated quality control tests were performed on each dose. The HPLC-based analytical methods were validated against USP methods of quality control. [(18)F]FDG produced by BG75 was tested in a mouse tumor model implanted with H441 human lung adenocarcinoma cells. After initial installment and optimization, the [(18)F]F(-) production has been consistent since March 2011 with a maximum production of 400 to 450 mCi in a day. The average yield is 0.61 mCi/min and 0.92 mCi/min at 3.8 µA and 5 µA, respectively. The current target window has held up for over 25 weeks against >400 bombardment cycles. [(18)F]FDG production has been consistent since June 2012 with an average of six doses/day in an automated synthesis mode (RCY≈50%). The release criteria included USP-specified limits for pH, residual solvents (acetonitrile/ethanol), kryptofix, radiochemical purity/identity, and filter integrity test. The entire automated operation generated minimal radiation exposure hazard to the operator and environment. As expected, [(18)F]FDG produced by BG75 was found to delineate tumor volume in a mouse model of xenograft tumor. In summary, production and quality control of "[(18)F]FDG dose on demand" have been accomplished in an automated and safe manner by the first Biomarker Generator. The implementation of a cGMP quality system is under way towards the ANDA submission and first clinical use of [(18)F]FDG produced by BG75.

Liposome-encapsulated EF24-HPßCD inclusion complex: a preformulation study and biodistribution in a rat model.

3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24) is an anti-proliferative diphenyldifluoroketone analog of curcumin with more potent activity. The authors describe a liposome preparation of EF24 using a "drug-in-CD-in liposome" approach. An aqueous solution of EF24 and hydroxypropyl-ß-cyclodextrin (HPßCD) inclusion complex (IC) was used to prepare EF24 liposomes. The liposome size was reduced by a combination of multiple freeze-thaw cycles. Co-encapsulation of glutathione inside the liposomes conferred them with the capability of labeling with imageable radionuclide Tc-99m. Phase solubility analysis of EF24-HPßCD mixture provided k(1:1) value of 9.9 M(-1). The enhanced aqueous solubility of EF24 (from 1.64 to 13.8 mg/mL) due to the presence of HPßCD helped in the liposome preparation. About 19% of the EF24 IC was encapsulated inside the liposomes (320.5 ± 2.6 nm) by dehydration-rehydration technique. With extrusion technique, the size of 177 ± 6.5 nm was obtained without any effect on encapsulation efficiency. The EF24-liposomes were evaluated for anti-proliferative activity in lung adenocarcinoma H441 and prostate cancer PC-3 cells. The EF24-liposomes demonstrated anti-proliferative activity superior to that of plain EF24 at 10 µM dose. When injected in rats, the Tc-99m-labeled EF24-liposomes cleared from blood with an α-t(1/2) of 21.4 min and ß-t(1/2) of 397 min. Tissue radioactivity counting upon necropsy showed that the majority of clearance was due to the uptake in liver and spleen. The results suggest that using "drug-in-CD-in liposome" approach is a feasible strategy to formulate an effective parenteral preparation of EF24. In vitro studies show that the liposomal EF24 remains anti-proliferative, while presenting an opportunity to image its biodistribution.

Kinetics of liposome-encapsulated hemoglobin after 25% hypovolemic exchange transfusion.

Liposome-encapsulated hemoglobin (LEH) is being developed as an oxygen therapeutic. In this work, we evaluated a neutral formulation of PEGylated LEH for its circulation and distribution properties in rodent models of 25% hypovolemic exchange transfusion. About 25% of blood in rats and rabbits was exchanged with LEH that had been previously labeled with 99mTc radionuclide. The distribution of 99mTc-LEH was followed by gamma camera imaging and intermittent blood sampling during 48 h, and counting the tissue-associated radioactivity after necropsy at 48 h. On the basis of circulation kinetics, the half-life of 99mTc-LEH in blood was 30 and 39.8 h in rats and rabbits, respectively. Apart from blood, major organs of accumulation of LEH after 48 h included liver (rats, 10.3% and rabbits, 5.4% of injected dose) and spleen (rats, 2.4% and rabbits, 0.8% of injected dose). The results demonstrate that LEH circulates for a prolonged time after administration and that the animals tolerate at least 25% of blood exchange without any distress. Subsequent to the enhanced uptake in the RES, the rats clear LEH from the circulation faster than the rabbits.

Neutral and anionic liposome-encapsulated hemoglobin: effect of postinserted poly(ethylene glycol)-distearoylphosphatidylethanolamine on distribution and circulation kinetics.

To prepare long-circulating liposomes, poly(ethylene glycol) (PEG)-lipid is usually mixed with other lipid components before vesicle formation. PEG-lipids can also be postinserted in the outer layer of liposomes after the preparation. In this study, PEG-distearoylphosphatidylethanolamine was incorporated by postinsertion technique into liposome-encapsulated hemoglobin (LEH) carrying neutral or negative charge. Postinsertion technique improved the encapsulation efficiency of hemoglobin from about 0.0017 to 0.017 (hemoglobin/phospholipid, molar ratio) for a similar lipid composition. Thus, neutral, anionic, PEG-neutral, and PEG-anionic LEHs were made and labeled with technetium-99m to follow their biodistribution. A small dose of LEH (approximately 15 mg of phospholipid) was injected intravenously in rabbits, and its distribution was monitored by blood sampling, gamma camera imaging, and tissue radioactivity counting on necropsy. The 24-h blood levels of neutral, PEG-neutral, anionic, and PEG-anionic LEHs were 14, 40.3, 13.1, and 35.7% of injected dose, respectively; calculated T(1/2) values of circulation were 8.9, 19.3, 9.6, and 16.5 h, respectively. PEGylation also influenced accumulation of LEH in the reticuloendothelial system. Liver uptake of neutral LEH dropped from 52.1 to 19.1%, whereas that of anionic LEH came down from 35.3 to 11.5% on PEGylation. In contrast, PEGylation increased the spleen uptake by 8.5- and 2.5-fold for neutral and anionic LEH, respectively. The results demonstrate that PEGylation by postinsertion not only improves the circulation t1/2 of LEH but also enhances hemoglobin content inside the vesicles for better oxygen-carrying capacity.

Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits.

To determine the largest size of liposomes that can retain stealth behavior conferred by poly(ethylene glycol)-DSPE, neutral liposomes were studied in rabbits for their circulation and distribution. Five sizes (136.2, 165.5, 209.2, 275 and 318 nm) of liposomes (DSPC, Cholesterol, PEG-DSPE and alpha-tocopherol, 90:80:4.5:3.9 molar ratio) were made by extrusion technique and radiolabeled with technetium-99m (Tc-99m) to follow their distribution through 24 h. Although all liposomes showed prolonged circulation in blood, the amount still in circulation at 24 h was dependent on their size. Radioactivity accumulation in spleen progressively increased with increase in size of the liposomes. In the size range of approximately 160-220 nm, liver uptake was minimum, spleen uptake was moderate while the amount of circulating liposomes was maximum. Gamma camera scintigraphy corroborated the distribution pattern of liposomes on necropsy. Images within 1h showed high blood pool activities for liposomes of all sizes. However, at 24h, the blood pool activity was diminished for 275 nm and negligible for 308 nm liposomes; the smaller sized liposomes (136.2-209.2 nm) continued to show high blood pool activity. The amounts of radioactivity still circulating at 24h were 46.4, 50.4, 46.8, 36.2 and 14.5% for 136.2, 165.5, 209.2, 275 and 318 nm liposomes, respectively. Corresponding circulation T(1/2)s were 21.7, 26.5, 24.9, 18.7 and 8.9h, respectively. Thus, the optimum size of PEG-liposomes for prolonged circulation in rabbits is 160-220 nm. Beyond this range, the stealth property of PEG-liposomes is significantly compromised and the distribution is characterized by high RES accumulation.

Accumulation of PEG-liposomes in the inflamed colon of rats: potential for therapeutic and diagnostic targeting of inflammatory bowel diseases.

Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with severe toxicity related to the nonspecific and ubiquitous interaction of drugs with the organs and tissues. In order to prevent side effects from aggressive and prolonged treatment with glucocorticoids and immunosuppressive agents, preferential accumulation of these potent drugs in diseased tissue is desired. In this work, we report that liposomes show a remarkable tendency to accumulate in inflamed colon of rats with experimental colitis. The disposition of liposomes was monitored by labeling them with Tc-99m followed by gamma camera imaging, and determining biodistribution of radioactivity in various organs. The images showed distinct accumulation of radioactivity in the colon of rats with colitis, while the abdomen of normal rats was conspicuously free of any visible radioactivity. Although images acquired 4 h after Tc-99m-liposome injection were clear enough for diagnostic indication, the real potential of liposomes for drug delivery was evident in 24 h images where the major organs of liposome accumulation were dwarfed by intense colon activity in animals with colitis. On necropsy, 13.5% +/- 5.48 of the activity accumulated in the inflamed colon as compared to only 0.1% in the normal colon, giving a target-to-nontarget ratio of 135. The blood borne radioactivity was 9% +/- 2.12 (colitis) and 25.7% +/- 4.27 (normal), indicating that the decrease in circulating liposomes is associated with an increase in liposome accumulation in the inflammatory site. The other two major organs that accumulated liposomes were spleen (10.7% normal vs. 11% colitis) and liver (8% normal vs. 10.1% colitis). In conclusion, this study demonstrates the innate propensity of liposomes to accumulate in the sites of inflammation and potential of liposomes loaded with therapeutic drugs or diagnostic agents for targeting colitis.

CTP:phosphocholine cytidylyltransferase inhibition by ceramide via PKC-alpha, p38 MAPK, cPLA2, and 5-lipoxygenase.

In a companion paper (Vivekananda J, Smith D, and King RJ. Am J Physiol Lung Cell Mol Physiol 281: L98-L107, 2001), we demonstrated that tumor necrosis factor (TNF)-alpha inhibited the activity of CTP:phosphocholine cytidylyltransferase (CT), the rate-limiting enzyme in the de novo synthesis of phosphatidylcholine (PC), and that its actions were likely exerted through a metabolite of sphingomyelin. In this paper, we explore the signaling pathway employed by TNF-alpha using C2 ceramide as a cell-penetrating sphingolipid representative of the metabolites induced by TNF-alpha. We found that in H441 cells, as reported in other cell types, cytosolic phospholipase A2 (cPLA2) is activated by TNF-alpha. We also observed that the inhibiting action of C2 ceramide on CT requires protein kinase C-alpha, p38 mitogen-activated protein kinase, and cPLA2. The actions of C2 ceramide on CT activity can be duplicated by adding 2 microM lysoPC to these cells. Furthermore, we found that the effects of C2 ceramide are dependent on 5-lipoxygenase but that cyclooxygenase II is unimportant. We hypothesize that CT activity is inhibited by the lysoPC generated as a consequence of the activation of cPLA2 by protein kinase C-alpha and p38 mitogen-activated protein kinase. The other product of the activation of cPLA2, arachidonic acid, is a substrate for the synthesis of leukotrienes, which raise intracellular Ca2+ levels and complete the activation of cPLA2.

PKC, p42/p44 MAPK, and p38 MAPK are required for HGF-induced proliferation of H441 cells.

In this paper, we studied the signaling pathway used by hepatocyte growth factor/scatter factor (HGF) to stimulate mitosis. We show, using H441 cells, that 1) HGF activates membrane-associated protein kinase C (PKC); the activity is transient and peaks within 30 min; 2) HGF activates p42/p44 and p38 mitogen-activated protein kinases (MAPKs); maximum activity in both is within 10 min; and 3) the activation of neither p38 nor p42/p44 MAPK is dependent on PKC, indicating that HGF uses separate and nonintersecting pathways to activate these two classes of kinase. However, phorbol 12-myristate 13-acetate also activates both MAPKs as well as PKC, but this activation is abolished in cells pretreated with the PKC inhibitor GF-109203X. HGF was found to significantly increase [(3)H]thymidine incorporation within 5 h; peak thymidine incorporation was observed at 16 h. However, when cells were pretreated with inhibitors of p42/p44 (PD-98059), p38 (SB-203580), or PKC (GF-109203X, Gö-6983, or myristoylated inhibitor peptide(19-27)), HGF-induced thymidine uptake was diminished in a dose-dependent manner. Taken together, these results demonstrate that HGF activates PKC and both MAPKs simultaneously through parallel pathways and that the activation of the MAPKs does not depend on PKC. However, p38 and p42/p44 MAPKs and PKC may all be essential for HGF-induced proliferation of H441 cells.

Hepatocyte growth factor is elevated in chronic lung injury and inhibits surfactant metabolism.

Adult respiratory distress syndrome may incorporate in its pathogenesis the hyperplastic proliferation of alveolar epithelial type II cells and derangement in synthesis of pulmonary surfactant. Previous studies have demonstrated that hepatocyte growth factor (HGF) in the presence of serum is a potential mitogen for adult type II cells (R. J. Panos, J. S. Rubin, S. A. Aaronson, and R. J. Mason. J. Clin. Invest. 92: 969-977, 1993) and that it is produced by fetal mesenchymal lung cells (J. S. Rubin, A. M.-L. Chan, D. P. Botarro, W. H. Burgess, W. G. Taylor, A. C. Cech, D. W. Hirschfield, J. Wong, T. Miki, P. W. Finch, and S. A. Aaronson. Proc. Natl. Acad. Sci. USA 88: 415-419, 1991). In these studies, we expand on this possible involvement of HGF in chronic lung injury by showing the following. First, normal adult lung fibroblasts transcribe only small amounts of HGF mRNA, but the steady-state levels of this message rise substantially in lung fibroblasts obtained from animals exposed to oxidative stress. Second, inflammatory cytokines produced early in the injury stimulate the transcription of HGF in isolated fibroblasts, providing a plausible mechanism for the increased amounts of HGF seen in vivo. Third, HGF is capable of significantly inhibiting the synthesis and secretion of the phosphatidylcholines of pulmonary surfactant. Fourth, HGF inhibits the rate-limiting enzyme in de novo phosphatidylcholine synthesis, CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15). Our data indicate that fibroblast-derived HGF could be partially responsible for the changes in surfactant dysfunction seen in adult respiratory distress syndrome, including the decreases seen in surfactant phosphatidylcholines.

Polyethylene glycol-modified liposome-encapsulated hemoglobin: a long circulating red cell substitute.

A major obstacle in the development of red cell substitutes has been overcoming their short circulation persistence. In this study, distearoyl phosphoethanolamine polyethylene glycol 5000 (PEG-PE) (10 mol%) was added to the formulation of liposome-encapsulated hemoglobin (LEH) to decrease reticuloendothelial system uptake and prolong LEH circulation persistence. PEG-LEH was radiolabeled with technetium-99m, infused into rabbits (25% of blood pool at 1 ml/min) (n = 5), and monitored by scintigraphic imaging at various times out to 48 h. At 48 h, animals were sacrificed, and tissue samples were collected for counting in a scintillation well counter. Tissue distribution data at 48 h revealed that 51.3 +/- 3.4% of the technetium-99m-PEG-LEH remained in circulation, a greater than 3-fold increase in the circulation half-life compared with circulation half-lives previously reported for non-PEG-containing LEH formulations. The liver had the greatest accumulation at 48 h (12.7 +/- 0.7%), followed by bone marrow (6.2 +/- 0.1%), whereas the spleen had only 1.4 +/- 0.2%. The addition of PEG-PE to the LEH formulation greatly prolongs the circulation persistence of LEH and represents a significant step in the development of red cell substitutes with prolonged oxygen delivery.

Imaging experimental osteomyelitis using radiolabeled liposomes.

UNLABELLED: We evaluated radiolabeled liposomes (liposomes labeled both with 99mTc and 111In) for the early detection of osteomyelitis in an experimental model. METHODS: Liposomes, containing 5% polyethylene glycol-distearoyl phosphatidylethanolamine with encapsulated glutathione and deferoxamine, were prepared and labeled with 99mTc and 111In by a previously described method. Acute osteomyelitis was induced in male New Zealand rabbits by intramedullary injection of sodium-morrhuate and Staphylococcus aureus in the tibial bone marrow. Serial imaging studies, consisting of radiolabeled liposome imaging (2-4 mCi 99mTc and 75-125 microCi 111In), 99mTc-methylene diphosphonate (MDP) (3-5 mCi) and 67Ga-citrate (500 microCi), were performed starting at the third day after injection. Each radionuclide study was separated by at least 2 days. The animals also underwent radiography of the lower extremities. The animals were then killed and the infected tibia was excised for histopathology. RESULTS: For interpreting relative efficacy of individual radiopharmaceuticals, only animals showing positive histopathological findings (n = 9) were considered. Radiographs (Days 12, 13) were conclusive for osteomyelitis in only 3 rabbits. Radiolabeled liposome imaging (Days 4-6) showed positivity in 8 cases and was equivocal in 1. Though the lesion could be delineated as early as 8 hr postinjection in the 99MTc window, the best target-to-nontarget ratio (T/NT) of 1.86 +/- 0.19 was obtained at 48 hr in the 111In window. Three-phase 99mTc-MDP scan (Day 7) was positive in only 5 rabbits with 3 hr T/NT of 1.6 +/- 0.23. Galium-67-citrate images (Days 9-11) were positive in 8 cases and equivocal in 1, the mean 48 hr T/NT being 1.74 +/- 0.24. These results show liposomes are better than 99mTc-MDP for imaging bone infection. Given the early localization and better quality of the images, radiolabeled liposomes also exhibited advantages over 67Ga-citrate for detection of acute osteomyelitis.

Dual radiolabeled liposomes: biodistribution studies and localization of focal sites of infection in rats.

Liposomes encapsulating both glutathione and deferoxamine were labeled with 99mTc-HMPAO and 111In-oxine at the same time. These dual radiolabeled liposomes were intravenously injected in rats with S. aureus infection in thigh. The target-to-background ratio (T/BG) increased from 2.9 at 2 h to 4.4 at 8 h in 99mTc images. In 111In images, T/BG of 5.5 at 8 h increased to 10.5 by 48 h. The 24-h spleen uptake of 111In- and 99mTc-liposomes was 24.14%ID and 8.91%ID. In femur, 99mTc-liposomes remained at approximately 10.5%ID, but 111In-liposomes increased from approximately 11%ID at 4 h to approximately 25.5%ID at 24 h. The simultaneous presence of 99mTc and 111In in the liposomes resulted in good early (2-8 h) as well as delayed (24-48 h) images delineating the infection site.

Providing cost data to physicians helps contain costs.

Findings of a survey of 1,200 physicians suggest that healthcare organizations that provide physicians with pricing information can reduce resource utilization and control costs more effectively than organizations that do not provide such information. These findings also suggest that healthcare financial managers who are aware of physicians' responses to cost information--and who furnish them with effective cost information--may gain a timely competitive advantage.

99mTc-insulin: labeling, biodistribution and scintiimaging in animals.

Porcine insulin was labeled with 99mTc by direct tin reduction. More than 95% labeling efficiency was obtained on paper chromatography in saline and methyl ethyl ketone. The stability of the labeled compound was confirmed by paper chromatography at 3 h post-labeling and by human serum albumin (HSA) challenge. PAGE pattern indicated no change in the electrophoretic behavior and the molecular size of insulin after the labeling procedure. Biodistribution in rats shows that kidney took up the maximum amount of 99mTc-insulin; maxima being maintained throughout 24 h post-injection. Liver and intestine were the other organs with significant uptake; the rest localizing little or negligible radioactivity. Most of the radioactivity was excreted via the renal pathway into urine. Scintiimages conformed to the biodistribution data. The results of this study present the potential of 99mTc-labeling of insulin by a simple method.

Clinical studies of employees in a sheet-forming process at a paper mill.

Formaldehyde resins are used to improve the wet strength of paper. During the sheet-forming process of paper manufacture, formaldehyde fumes are liberated. Twenty-two male subjects having such exposures in a paper mill and 27 unexposed subjects were clinically evaluated to determine the effect of low level formaldehyde exposure in a tropical country. The workers were exposed to 0.03 mg formaldehyde/m3 air as an 8-h TWA. Formic acid excretion in urine was 37.2 +/- 18.9 and 20.3 +/- 4.2 ug/L among the exposed and the unexposed subjects, respectively. Significantly more respiratory problems (31.8%) were observed among the exposed subjects as compared to controls. Complaints pertaining to gastrointestinal, musculoskeletal and cardiovascular systems were also more frequent in exposed subjects. In spite of formaldehyde concentrations being well within the prescribed ACGIH limits of 1 ppm, the high rates of sickness emphasise the need for detailed studies on formaldehyde-exposed subjects in tropical countries.

Biochemical alterations in rat fetal liver following in utero exposure to di(2-ethylhexyl)phthalate (DEHP).

Oral administration of DEHP, 1000 mg/kg body weight, to rats daily from 6 to 15 day of gestation resulted in retardation of fetal growth and increase in fetal liver weight which contained significant quantities of DEHP. The activities of mitochondrial succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and adenosine triphosphatase were decreased in fetal liver. The data indicate that exposure of mothers to DEHP during pregnancy could adversely affect the fetal livers by interfering with bioenergetics of the cell.

An analytical study of the neurosecretory cells of the brain of the mature larvae and diapause pupae of Amsacta collaris Hampson (Lepidoptera: Arctiidae) under different temperature regimes.

The neurosecretory cells (NSCs) of the brain of the mature larvae and 3 months old diapause pupae of Amsacta collaris Hampson (Lepidopera: arctiidae) have been studied under different temperature regimes (0, 7, 18, 37, and 42 degrees C), using paraldehyde fuchsin and performic acid resorcin fuchsin staining techniques. The 0 degree C is fatal to the larvae, and at 7 degrees C, within 24 h the quantity of NSM reduced considerably in all the subtypes of A-cells; activity started 3rd day onwards, and on 5th day, the A1-cells appeared loaded with NSM, the A2-cells moderately filled, but no change in A3-cells was observed. At 0 degree C, within 48 h or chilling, the NSCs of diapause pupae (DP) revealed a moderate amount of NSM with numerous, large vacuoles in the A1-cells and in some insects, the cells appeared gigantic in form; the A2- and A3-cells were moderately filled with NSM. After 1 week of chilling, at 0 degree C, the A1-cells appeared almost empty. The A1-cells of DP-kept at 7 degrees C and 18 degrees C respectively, discharged quickly all the previously stored NSM, within 2 days, hence appeared poorly filled. 3rd day onwards, a gradual increase in NSM was observed. After 15 days, a moderate amount of NSM in A1-, a large amount in A3-, and increased amount in A2-cells of the medial and lateral groups was observed. The 37 degrees C appeared to be the most suitable temperature at which all the cells show maximum activity in mature larvae and DP both, and contain a moderate amount of NSM in them. At 42 degrees C, the A1-cells in larvae and DP contain a moderate amount of NSM, A2-cells of the medial group a poor, and A2-cells of the lateral group, a depleted amount of NSM, the A3-cells, however, appeared heavily loaded. Later, the NSM decreased gradually in all the subtypes of A-cells. On 15th day, at 42 degrees C the A1-cells in DP revealed depletion of NSM, the A2-cells become totally inactive and lack NSM; the A3-cells a poor amount of NSM in them. The significance of these changes at different temperature regimes is discussed.