Radiolabelled GLP‐1 analogues for in vivo targeting of insulinomas

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non‐internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon‐like peptide‐1 receptor (GLP‐1R) antagonist exendin(9–39) can be used for in vivo targeting of GLP‐1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP‐1R agonists exendin‐3 and exendin‐4. The binding and internalization kinetics of labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) were determined in vitro using INS‐1 cells. The in vivo targeting properties of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) were examined in BALB/c nude mice with subcutaneous INS‐1 tumours. ^natIn‐labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) exhibited similar IC₅₀ values (13.5, 14.4 and 13.4 n m , respectively) and bound to 26 × 10³, 41 × 10³ and 37 × 10³ receptors per cell, respectively. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed rapid in vitro binding and internalization kinetics, whereas [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 [25.0 ± 6.0% injected dose (ID) g^?1] in the tumour was observed at 0.5 h post‐injection (p.i.) with similar uptake up to 4 h p.i. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g^?1, respectively). Remarkably, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g^?1), rapidly decreasing over time. In conclusion, the GLP‐1R agonists [Lys⁴⁰(DTPA)]exendin‐3 and [Lys⁴⁰(DTPA)]exendin‐4 labelled with ¹¹¹In could be useful for in vivo GLP‐1R targeting, whereas [Lys⁴⁰(DTPA)]exendin(9–39) is not suited for in vivo targeting of the GLP‐1R. Copyright © 2012 John Wiley & Sons, Ltd.     

Pharmacokinetics of DTPA entrapped in conventional and long-circulating liposomes of different size for plutonium decorporation.

The aim of the present study was to develop an efficient DTPA liposome formulation designed for plutonium decorporation. DTPA was encapsulated in conventional (CL) and polyethylene glycol-coated stealth liposomes (SL) prepared by extrusion followed by the freeze-thawing method and sizing from around 100 to 800 nm. DTPA encapsulation percentages were approximately 30% in CL of any size but dropped from 48% to 7% as the diameter of SL was reduced. The pharmacokinetics of [(14)C]-DTPA encapsulated in large and small vesicles was evaluated in rats after a single intravenous administration. Both liposomal composition and size reduction had a significant impact on pharmacokinetic parameters, inducing a marked increased in exposure of the body to DTPA and its delayed excretion. DTPA distribution was moderate in liver but enhanced in spleen and bone and was dose-dependent, especially when SL of 100 nm were given. In conclusion, small and stealth(R) vesicles have interesting properties in delivering DTPA to contaminated tissues.     

A time-resolved fluoroimmunoassay for the determination of prostaglandin F2 alpha

We describe a solid-phase immunoassay based on fluorescence detection for Prostaglandin F2 alpha (PGF2 alpha). Europium was used as the fluorescence marker. Prostaglandin F2 alpha was covalently bound to poly-L-lysine (PL) and labelled by coupling Eu3+ via the complexonate diethylenetriaminepentaacetic acid (DTPA). The labelled PGF2 alpha was tested in a competitive immunoassay. The detection limit for PGF2 alpha was about 2.2 pg per microtiter well. The intraassay-variation was 17.2% at 1.1 pg, 4.1% at 10 pg and 7.6% at 90 pg. The precision was determined by measuring various amounts of unlabelled PGF2 alpha (2.5 to 2,400 pg) in the assay buffer. The regression line for these studies resulted in a good correlation (y = 1.01x +/- 0.03; r = 0.996; n = 21). The results of a radioimmunoassay and of the fluoroimmunoassay were similar (y = 0.983 +/- 0.04; r = 0.988; n = 15). The new method enables PGF2 alpha analyses at concentrations in the fmolar range.     

pH-dependent receptor/ligand dissociation as a determining factor for intracellular sorting of ligands for epidermal growth factor receptors in rat hepatocytes.

Both epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) bind to EGF receptors and TGF alpha has been reported to be more potent than EGF as far as many biological effects are concerned. One possible reason for this is thought to be the difference in their dissociation from the receptors in intracellular acidic compartments, which may affect the final pathway (lysosomal degradation or recycling to cell-surface) of endocytosed ligands. This study was aimed at clarifying the relationship between intracellular dissociation from the receptors and the fate of the endocytosed ligands. First, (125)I-human EGF (hEGF), mouse EGF (mEGF), or human TGF alpha (hTGF alpha) was prebound to cell-surface receptors or intracellularly preloaded, followed by further incubation at 37 degrees C in primary cultured rat hepatocytes. In these experiments, the magnitude of the dissociation rate constant (k(off)) of each ligand at pH 6.0, which is similar to that inside early endosomes, was found to be in the following order: hTGF alpha>mEGF>hEGF. The recycled portion of endocytosed ligands was also in the order: hTGF alpha>mEGF>hEGF. Digitonin treatment of preloaded cells revealed that the intracellular dissociation of hTGF alpha was more rapid than that of hEGF. Moreover, several histidine-inserted or -deleted hEGF mutants were prebound to rat liver sinusoidal membrane vesicle, followed by further incubation at 37 degrees C. The dissociation rate of histidine-deleted hEGF mutants was less rapid than that of hEGF itself. These results suggested that efficient dissociation in the earlier intracellular compartment leads the endocytosed ligands to be recycled to the cell surface whereas late dissociation results in intracellular degradation in hepatocytes. Thus, one possible strategy to improve their stability in the circulation may be a change in intracellular ligand/receptor dissociation with a minimal effect on the affinity for receptors on the cell-surface and histidine residues may partly contribute to the pH-sensitive dissociation.     

Pharmacokinetics,safety, and efficacy of bosentan in pediatric patients with pulmonary arterial hypertension

BACKGROUND: Bosentan, a dual endothelin-receptor antagonist, is registered for the treatment of pulmonary arterial hypertension. Little is known about the effects of bosentan in children. This study was conducted to investigate the pharmacokinetics, safety, and efficacy of bosentan in pediatric patients with pulmonary arterial hypertension. METHODS: In this 2-center, open-label study, 19 pediatric patients with pulmonary arterial hypertension were enrolled and stratified for body weight and epoprostenol use. Patients weighing between 10 and 20 kg, between 20 and 40 kg, or greater than 40 kg received a single dose of 31.25, 62.5, or 125 mg, respectively, on day 1, followed by 4 weeks of treatment with the initial dose. The dose was then up-titrated to the target dose (31.25, 62.5, or 125 mg twice daily). Pharmacokinetic and hemodynamic parameters were obtained at baseline and after 12 weeks of treatment. Six-minute walk distance and cardiopulmonary exercise testing results were measured at baseline and at week 12 in children aged 8 years or older. RESULTS: The variability in exposure among the 3 groups was less than 2-fold after single- and multiple-dose administration. The exposure to bosentan decreased over time in all groups. The covariates body weight, gender, age, and the use of epoprostenol had no significant effect on the pharmacokinetics of bosentan. Bosentan produced hemodynamic improvement and was well tolerated. The mean change from baseline in mean pulmonary artery pressure was -8.0 mm Hg (95% confidence interval, -12.2 to -3.7 mm Hg), and that in pulmonary vascular resistance index was -300 dyne x s x m(2)/cm(5) (95% confidence interval, -576 to -24 dyne x s x m(2)/cm(5)). CONCLUSIONS: The pharmacokinetics of bosentan in pediatric patients with pulmonary arterial hypertension and healthy adults are similar, and treatment with bosentan resulted in hemodynamic improvement. These results suggest that the applied dosing regimens may be appropriate to treat pediatric patients.     

Preclinical evaluation of [111In]MICA‐401, an activity‐based probe for SPECT imaging of in vivo uPA activity

Urokinase‐type plasminogen activator (uPA) and its inhibitor PAI‐1 are key players in cancer invasion and metastasis. Both uPA and PAI‐1 have been described as prognostic biomarkers; however, non‐invasive methods measuring uPA activity are lacking. We developed an indium‐111 (¹¹¹In)‐labelled activity‐based probe to image uPA activity in vivo by single photon emission computed tomography (SPECT). A DOTA‐conjugated uPA inhibitor was synthesized and radiolabelled with ¹¹¹In ([¹¹¹In]MICA‐401), together with its inactive, hydrolysed form ([¹¹¹In]MICA‐402). A biodistribution study was performed in mice (healthy and tumour‐bearing), and tumour‐targeting properties were evaluated in two different cancer xenografts (MDA‐MB‐231 and HT29) with respectively high and low levels of uPA expression in vitro, with either the active or hydrolysed radiotracer. MicroSPECT was performed 95 h post injection followed by ex vivo biodistribution. Tumour uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC). Biodistribution data with the hydrolysed probe [¹¹¹In]MICA‐402 showed almost complete clearance 95 h post injection. The ex vivo biodistribution and SPECT data with [¹¹¹In]MICA‐401 demonstrated similar tumour uptakes in the two models: ex vivo 5.68 ± 1.41%ID/g versus 5.43 ± 1.29%ID/g and in vivo 4.33 ± 0.80 versus 4.86 ± 1.18 for MDA‐MB‐231 and HT‐29 respectively. Human uPA ELISA and IHC showed significantly higher uPA expression in the MDA‐MB‐231 tumours, while mouse uPA staining revealed similar staining intensities of the two tumours. Our data demonstrate non‐invasive imaging of uPA activity in vivo, although the moderate tumour uptake and hence potential clinical translation of the radiotracer warrants further investigation. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and characterization of new low‐molecular‐weight lysine‐conjugated Gd‐DTPA contrast agents

Various blood pool contrast agents (CAs), characterized by intravascular distribution, have been developed to assist contrast enhanced magnetic resonance angiography (MRA). Among these CAs, the DTPA derivatives conjugated to synthetic polypeptides, such as polylysine, represent attractive candidates for blood pool imaging. However, due to the presence of charged residues located on their backbone, these agents are retained in the kidneys and this compromises their long blood half‐life. In order to overcome this major drawback of the polylysine compounds, two new low‐molecular‐weight CAs were synthesized in the present work by conjugating four or six 1‐p‐isothiocyanatobenzyl‐DTPA moieties to tri‐ or penta‐Lys peptides [(Gd‐DTPA)₄Lys₃ and (Gd‐DTPA)₆Lys₅], respectively. All the –NH₂ groups of Lys were thus blocked by covalent conjugation to DTPA. The stability and relaxometric properties of these compounds, as well as their pharmacokinetic and biodistribution characteristics, were then evaluated. The half‐life in blood of these new polylysine derivatives, as determined in rats, is twofold longer than that of Gd‐DTPA. The compounds could thus be optimal blood pool markers for MRA, which typically uses fast acquisition times. The absence of positive molecular charge did not limit their retention in kidneys 2 h after administration. On the other hand, (Gd‐DTPA)₄Lys₃ is retained in kidneys to a lesser extent than (Gd‐DTPA)₆Lys₅. Their moderate retention in blood and their higher stability and relaxivity in comparison with Gd‐DTPA highlight these polylysine derivatives as optimal compared with previously developed polylysine compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

A mathematical model of the hemoglobin-oxygen dissociation curve of human blood and of the oxygen partial pressure as a function of temperature

A mathematical model is described giving the oxygen saturation fraction (s) as a function of the oxygen partial pressure (p): y - y0 = x - x0 + h X tanh [k X (x - x0)], where y = kn[s/(1-s)] and x = ln(p/kPa). The parameters are: y0 = 1.875; x0 = 1.946 + a + b; h = 3.5 + a; k = 0.5343; b = 0.055 X [T/(K - 310.15)]; a = 1.04 X (7.4 - pH) + 0.005 X Cbase/(mmol/L) + 0.07 X [[CDPG/(mmol/L)] - 5], where Cbase is the base excess of the blood and CDPG is the concentration of 2,3-diphosphoglycerate in the erythrocytes. The Hill slope, n = dy/dx, is given by n = 1 + h X k X [1 - tanh2[k X (x - x0)]]. n attains a maximum of 2.87 for x = x0, and n----1 for x----+/- infinity. The model gives a very good fit to the Severinghaus standard oxygen dissociation curve and the parameters may easily be fitted to other oxygen dissociation curves as well. Applications of the model are described including the solution of the inverse function (p as a function of s) by a Newton-Raphson iteration method. The po2-temperature coefficient is given by dlnp/dT = [A X alpha X p + CHb X n X S X (1 - s) X B]/[alpha X p + CHB X n X s X (1 - s)], where A = -dln alpha/dT approximately equal to 0.012 K-1; B = (lnp/T)s = 0.073 K-1 for y = y0; alpha = the solubility coefficient of O2 in blood = 0.0105 mmol X L-1 X kPa-1 at 37 degrees C; CHb = concentration of hemoglobin iron in the blood. Approximate equations currently in use do not take the variations of the po2-temperature coefficient with p50 and CHb into account.     

Direct 99mTc labeling of pegylated liposomal doxorubicin (Doxil) for pharmacokinetic and non-invasive imaging studies

Pharmacokinetic and organ distribution studies of liposomal drugs in humans are a challenge. A direct labeling method using (99m)Tc-N,N-bis(2-mercaptoethyl)-N',N'-diethyl-ethylenediamine (BMEDA) complex to label the commercially available pegylated liposomal doxorubicin, Doxil, has been introduced. Biodistributions of (99m)Tc-Doxil in normal rats were performed to evaluate the feasibility of using it for monitoring the pharmacokinetics of liposomes encapsulating drugs. Labeling efficiency of (99m)Tc-Doxil was 70.6 +/- 0.8% (n = 3). In vitro incubation of (99m)Tc-Doxil in 50% fetal bovine serum or 50% human serum at 37 degrees C showed good labeling stability with 72.3 +/- 3.6% or 78.6 +/- 1.8% of activity associated with Doxil at 24 h, respectively (n = 3). There was a two-phase blood clearance with half-clearance times of 2.2 and 26.2 h after bolus intravenous injection in normal rats. Distribution of (99m)Tc-Doxil at 44 h after injection had 19.8 +/- 1.3% of injected dose in blood, 14.1 +/- 1.7% in liver, 2.6 +/- 0.3% in spleen, 9.0 +/- 0.8% in bone with marrow, 6.0 +/- 0.5% in skin, and 15.3 +/- 4.3% in bowel (n = 5). Unencapsulated (99m)Tc-BMEDA had a very rapid blood clearance with a half-clearance time of only 0.12 h (n = 4). By using this (99m)Tc labeling method, biodistribution and pharmacokinetics of ammonium gradient liposomes encapsulating drugs can be determined by noninvasive scintigraphic imaging. This labeling method may be extended to (186)Re and (188)Re labeling to combine chemotherapy and radionuclide therapy for tumor treatment.     

Replacing 99mTc with 111In Improves MORF/cMORF Pretargeting by Reducing Intestinal Accumulation

Purpose  To reduce accumulation in the abdomen by MORF/cMORF pretargeting, ¹¹¹In was compared to ^99mTc as the radiolabel. Procedures  After receiving either ^99mTc (MAG3)-cMORF or ¹¹¹In (DTPA)-cMORF, normal mice were imaged and killed for pharmacokinetics. Thereafter, tumored mice were pretargeted with MORF-antibody, 48 h later were given an injection of ^99mTc- or ¹¹¹In-cMORF, and finally were imaged repeatedly. Results  The cMORF biodistribution in both normal and pretargeted tumored mice was influenced by its radiolabel. While excretion of both ^99mTc-cMORF and ¹¹¹In-cMORF was rapid and mainly through the kidneys, about 2% of ^99mTc accumulated in the intestines compared to essentially no intestinal accumulation for ¹¹¹In at any time. Tumor accumulation was unchanged. Conclusion  In applications of MORF/cMORF pretargeting intended to image organs deep within the abdomen such as the pancreas, radiolabeling with ¹¹¹In may be superior to ^99mTc.     

Compartmental pharmacokinetics and tissue distribution of the antifungal triazole ravuconazole following intravenous administration of its di-lysine phosphoester prodrug (BMS-379224) in rabbits

OBJECTIVES: Ravuconazole is a broad-spectrum antifungal triazole in clinical development. We investigated the compartmental plasma pharmacokinetics and tissue distribution of ravuconazole following administration of its novel intravenous (i.v.) di-lysine phosphoester prodrug, BMS-379224. METHODS: Normal catheterized rabbits received the prodrug at 1.25, 2.5, 5, 10, 20 and 40 mg/kg once daily as 5 min i.v. bolus for 8 days. Serial plasma levels were collected at days 1 and 7, and tissues were obtained 30 min after the eighth dose. Concentrations of ravuconazole were determined by a validated HPLC method. Plasma concentration data were fitted to a three-compartment pharmacokinetic model. Pharmacokinetic parameters were estimated by weighted non-linear least squares regression analysis using the WinNonlin computer program. RESULTS: Following single dosing, ravuconazole demonstrated linear plasma pharmacokinetics across the investigated dosage range. Cmax, AUC(0-infinity), V(ss), CL and terminal half-life (means +/- SEM) ranged from 2.03 to 58.82 mg/L, 5.80 to 234.21 mg x h/L, 5.16 to 6.43 L/kg, 0.25 to 0.18 L/h/kg and 20.55 to 26.34 h, respectively. Plasma data after multiple dosing revealed non-linear disposition at the 20 and 40 mg/kg dosage levels as evidenced by a dose-dependent decrease in CL (from 0.104-0.147 to 0.030 and 0.022 L/h/kg; P = 0.1053) and an increase in the dose-normalized AUC(0-infinity) (from 2.40-3.01 up to 11.90 and 14.56 mg x h/L; P = 0.0382). Tissue concentrations 30 min after the last dose were highest in the liver (12.91-562.68 microg/g), adipose tissue (10.57-938.55 microg/g), lung (5.46-219.12 microg/g), kidney (3.95-252.44 microg/g) and brain tissue (2.37-144.85 microg/g). CONCLUSIONS: The pharmacokinetics of ravuconazole fitted best to a three-compartment pharmacokinetic model. The compound revealed non-linear pharmacokinetics at higher dosages, indicating saturable clearance and/or protein binding. Ravuconazole displayed a long elimination half-life and achieved substantial plasma and tissue concentrations including in the brain.     

Lopromide one-sample clearance as a measure of glomerular filtration rate

Objective: The pharmacokinetics of iopromide were analysed using a two‐compartment model. The optimal point of time for blood withdrawal for calculation of a one‐sample clearance was determined. Methods: Plasma concentration of iodine was measured up to 8 h postinjection (p.i.) in 62 adult patients who received 120 ml iopromide for computed tomography (CT). A two exponential function was fitted by a weighted least error square method. As reference method, clearance was calculated from this function and the injected amount of iodine. Empirical parameters for calculation of one‐sample clearance were determined. This one‐sample clearance was compared with one‐sample clearance calculated according to formulas developed for Tc99m‐DTPA by Jacobsson as well as a two sample method. Results: Total distribution volume of iopromide was calculated as 0·242 Lkg^?1± 5·9%. A high correlation of all one‐sample method and the two‐sample method with reference to clearance was found. Best estimation of iopromide plasma clearance was achieved by determining one‐sample clearance 270 or 285 min p. i. with SD_y.x of 5·8 ml min^–1. Conclusions: After administration of 120 ml iopromide, one‐sample plasma clearance can be calculated with low estimation error taking one blood sample at an appropriate time point. Late phase pharmacokinetics of iopromide found in the present study showed to be virtually identical to results published for iohexol and Tc99m‐DTPA.     

In Vivo Bacteriophage Peptide Display to Tailor Pharmacokinetics of Biological Nanoparticles

Purpose

Clinical use of most radiolabeled targeting agents has been limited because of the uptake and retention in kidney and/or liver. We hypothesized that bacteriophage (phage) display could be exploited to select for peptide sequences with fast clearance and low kidney uptake with the added ability to redirect phage clearance away from the reticuloendothelial system towards the kidney possessing rapid kidney clearance.

Procedures

In vivo phage display was performed to identify peptides displayed on phage that were excreted rapidly into the urine of mice. A novel in vitro assay using kidney cells, developed to predict in vivo kidney retention, and in vivo pharmacokinetic analyses were performed to characterize selected peptides/phage clones.

Results

Forty-three renal clearance clones (RCC) were identified. In vivo mixing experiments and in vitro kidney cell assays identified RCC1-02 as the lead compound. In vivo analysis of fluorescently labeled phage clones demonstrated the ability of RCC1-02 peptide to redirect the biodistribution of the large phage particle towards excretion via the kidney. Pharmacokinetic analysis of [¹¹¹In]-radiolabeled peptides revealed that kidney retention of the control ErBB-2-avid peptide, [¹¹¹In]DOTA-KCCYSL, at 2-h postinjection was 5.7?±?0.7 %ID/g. In comparison, [¹¹¹In]DOTA-RCC1-02 had kidney retention values of 1.66?±?0.43 %ID/g, respectively.

Conclusions

In vivo phage display can identify phage and corresponding peptides that rapidly clear the renal system. In the future, these peptides may be used to impart favorable pharmacokinetics onto a wide range of radioimaging or therapeutic macromolecules.     

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging

Purpose

Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with ¹¹¹indium, via the chelate diethylene triamine pentaacetic acid (DTPA).

Procedures

The necrosis avid properties of the radiotracer [¹¹¹In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo.

Results

Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [¹¹¹In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis.

Conclusions

The radiotracer [¹¹¹In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy.

     

Preclinical and clinical evidence for disappearance of long-circulating characteristics of polyethylene glycol liposomes at low lipid dose

This study describes the effect of the lipid dose of (99m)Tc-polyethylene glycol (PEG) liposomes in the low-dose range (0. 02-1.0 micromol/kg) on the pharmacokinetics and biodistribution in rats, rabbits, and humans. The biodistribution and pharmacokinetics of (99m)Tc-PEG liposomes at various dose levels were studied in rats and rabbits with a focal Escherichia coli infection. Scintigraphic images were recorded on a gamma camera. In addition, the role of macrophages in the biodistribution of a low-dose PEG liposome injection was studied. Finally, the pharmacokinetics of (99m)Tc-PEG liposomes at two lipid dose levels was studied in four patients. At a dose level of 0.03 micromol/kg, the blood level in rats at 4 h postinjection was significantly lower than at the highest dose level (1.1 micromol/kg). The same effect was observed in rabbits where enhanced clearance was observed at a dose level of 0.02 micromol/kg. The circulatory half-life decreased from 10.4 to 3.5 h (at 1.0 and 0. 02 micromol/kg, respectively). At the lowest dose level, liposomes were mainly taken up by the liver and to a lesser extent by the spleen. Injection of a low dose of PEG liposomes in macrophage-depleted rabbits resulted in normal pharmacokinetics, suggesting involvement of macrophages in the effectuation of the rapid elimination of the liposomes from the circulation. Most importantly, the rapid clearance of low-dose PEG liposomes was also observed in humans when relatively low lipid doses were administered. This study showed that at very low lipid doses the biodistribution of PEG liposomes is dramatically altered.     

Comparison of solution hybridization efficiencies using alkaline phosphatase-labelled and 32P-labelled oligodeoxynucleotide probes

The hybridization efficiencies of oligonucleotide probes directly labelled with alkaline phosphatase and probes labelled with 32P were compared by quantitating the enzyme activity or radioactivity associated with hybridization targets over time. The targets tested included both synthetic oligonucleotides (53 bases in length) and single-stranded and double-stranded cloned M13 DNA (7350 bases long). Hybrid molecules were separated from unhybridized probes using size exclusion FPLC. This system allowed quantitative analysis of the time course and efficiency of hybridization for both probes and targets in complex hybridization media containing protein blocking agents, formamide, and carrier DNA. Similar maximum hybridization efficiencies were attained for probes labelled with either radioactivity or alkaline phosphatase as a marker. The reaction rate constant for oligonucleotide hybridization to long M13 targets was 3.6 x 10(5) mol-1 s-1 for a probe labelled with alkaline phosphatase, and 5.8 x 10(5) mol-1 s-1 for the same probe labelled with 32P.     

Centrifugal analysis with automated sequential reagent addition: measurement of serum calcium

We describe an automated method for calcium assay, for use with the Cobas-Bio centrifugal analyzer. Calcium is reacted with cresolphthalein complexone and the absorbance of the calcium--dye complex at 575 nm is measured. EDTA is then added to break up the calcium--dye complex and the absorbance at 575 nm is re-measured, to correct for endogenous color and turbidity. Day-to-day precision data, determined over four months, were as follows: mean = 92.9 mg/L, CV = 1.47%; n = 216; mean = 128.7 mg/L, CV = 1.72%; n = 216. Comparison of the Cobas-Bio method (y) with an atomic absorption spectrometric method (x) gave the following results: y = 1.012x--2.05, r = 0.991, Sy/x = 1.2, mean x = 92.63 mg/L, mean y = 91.69 mg/L, n = 74. Hemoglobin, bilirubin, or turbidity does not interfere. At the medical decision value (110 mg/L), the overall analytical error is 4.6 mg/L, which is less than the 5 mg/L allowable (95% confidence limit) error.     

The application of the Reaction Rate Analyser LKB 8600 as an automatic coagulometer

The application of the Reaction Rate Analyser LKB 8600 (RRA) as an automatic coagulometer is described. The RRA was slightly modified without interfering with its function as an enzyme reaction rate analyser. The endpoint of coagulation was recorded when the increase in absorbance exceeded 0.047 at lambda = 340 nm. The coagulation time was monitored by a counter with automatic print-out or by a recorder. The analytical dispersion, s(x)x, ranged between 0.01 and 0.05. Results (x, y) of the determination of the quantity of plasma coagulation factors (II + VII + X) by RRA and manually by visual recording could be expressed by y = 0.93x + 0.04, r = 0.96, n = 66 (method of Owren & Aas) or by y = 1.00x + 0.02, r = 0.98, n = 49 (Normotest). Similarly, plasma activated partial thromboplastin time (Activated Thrombofax) could be expressed by y = 0.99x + 5.00, r = 0.99, n = 23.     

Tumor targeting using anti-her2 immunoliposomes.

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.     

Serum-stable and long-circulating, PEGylated, pH-sensitive liposomes.

pH sensitive liposomes were prepared using a terminally-alkylated copolymer of N-isopropylacrylamide (NIPAM) and methacrylic acid (MAA) and poly(ethylene glycol) phospholipid derivative. The pH-triggered content release was evaluated before and after incubation in serum. Pharmacokinetic and biodistribution profiles of the formulations were established in rats. This study showed that a pH-sensitive, serum-stable and long-circulating liposomal formulation can be produced.