The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 microg/micromol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.     

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 μg/μmol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.     

Quantification of cytotoxic hemocytes of Mytilus edulis using a cytotoxicity assay in agar

When a thin layer of agar containing a mixture of erythrocytes and Mytilus hemocytes is prepared on slides, the occurrence of plaques of lysed target cells can be observed around a limited number of hemocytes. These hemocytes remain completely intact cells and are viable as evidenced by their ability to phagocytose target cells and/or to form pseudopods. The number of hemocytes releasing cytotoxic molecules has been shown to vary greatly between different animals. The same holds true for the total number of circulating hemocytes, although no correlation exists between the number of hemocytes in the circulation and the percentage of cytotoxic blood cells.     

Stochastic Simulation of Human Pulmonary Blood Flow and Transit Time Frequency Distribution Based on Anatomic and Elasticity Data

The objective of our study was to develop a computing program for computing the transit time frequency distributions of red blood cell in human pulmonary circulation, based on our anatomic and elasticity data of blood vessels in human lung. A stochastic simulation model was introduced to simulate blood flow in human pulmonary circulation. In the stochastic simulation model, the connectivity data of pulmonary blood vessels in human lung was converted into a probability matrix. Based on this model, the transit time of red blood cell in human pulmonary circulation and the output blood pressure were studied. Additionally, the stochastic simulation model can be used to predict the changes of blood flow in human pulmonary circulation with the advantage of the lower computing cost and the higher flexibility. In conclusion, a stochastic simulation approach was introduced to simulate the blood flow in the hierarchical structure of a pulmonary circulation system, and to calculate the transit time distributions and the blood pressure outputs.     

Rates and impact of human antibody glycation in vivo

Glycation of immunoglobulin G (IgG) can result from incubation with a reducing sugar in vitro or during circulation in vivo. Upon injection of a recombinantly produced human therapeutic IgG into humans, changes in the glycation levels could be observed as a function of circulation time. Mass changes on the individual IgG polypeptide chains as the results of glycation were determined using reversed-phase liquid chromatography/mass spectrometry. Changes to the light and heavy chains were low but easily detectable at 0.00092 and 0.0021 glucose (Glc) additions per chain per day, respectively. Levels of glycation found on the Fc portion of IgG isolated from healthy subjects, using a similar analytical approach, were on average 0.045 Glc molecules per fragment. In vivo glycation rates could be approximated in vitro by modeling the physiological glycation reaction with a simplified incubation containing physiological Glc concentrations, pH and temperature but with a high concentration of a single purified IgG. To test the impact of glycation on IgG function, highly glycated IgG1 and IgG2 were prepared containing on average 42-49 Glc molecules per IgG. Binding to FcγIIIa receptors, neonatal Fc receptor or protein A was similar or identical to the non-glycated IgG controls. Although the modifications were well distributed throughout the protein sequence, and at high enough levels to affect the elution position by size-exclusion chromatography, no changes in the tested Fc functions were observed.     

Bioavailability of lutein in humans from intrinsically labeled vegetables determined by LC-APCI-MS

The aim of the investigation was to assess a stable isotope method for determining the relative bioavailability of food-derived lutein in humans. Subjects were administered a single dose of deuterium-labeled carotenoids from intrinsically labeled spinach or collard green; 10 mL blood samples were drawn at various time points over a 34 days period. The vegetables had been hydroponically grown using 25 atom-% deuterated water. Lutein molecules in the vegetables were partially deuterated with a highest abundance isotopomer at M(0) + 8 (unlabeled molecular mass, M(0,) plus 8 additional mass units from 8 deuterium atoms in the molecules). This allowed labeled lutein to be distinguished from endogenous lutein in serum samples after consuming the labeled meal. The presence of labeled lutein in the circulation was determined by liquid chromatography-mass spectrometry (LC/MS) equipped with an atmospheric pressure chemical ionization (APCI) interface. The quantification of the labeled lutein in serum samples enabled the calculation of the enrichment for each time point after the dose; these values were plotted vs. time to generate absorption-clearance curves for each of the subjects. Area under the curve analyses of four different subjects (integrated over 29 days) yielded serum lutein responses of 128, 145, 149, and 262 microg-day/mg dietary lutein, following an acute dose of spinach containing 15.4, 18.8, 18.8 and 9.8 mg labeled lutein, respectively. This technique will facilitate the study of lutein bioavailability from different foods of diverse carotenoid composition and/or following various food preparation procedures.     

长循环脂质体的研究

将ＰＥＧｓ（聚乙二醇单甲醚的衍生物）掺入脂质体,研究了该类脂质体在血清诱导下的体外稳定性及其经静脉注入鼠体后的体内分布。结果表明,该类脂质体在体外的稳定性明显提高,在血循环中的滞留时间也相应延长。也就是说,ＰＥＧｓ脂质体是一种长循环脂质体。     

Moments of physiological transit time distributions and the time course of drug disposition in the body

Characterizing the tissue distribution kinetics of drugs by physiological and physico-chemical parameters and using a circulatory model the time course of blood concentration after intravenous injection is predicted for linear pharmacokinetic systems. The interrelationships between the first three (zero to second) moments of the distribution functions of organ transfer times, circulation times and residence times of drug molecules in the body are described. Utilizing literature data the model is applied to the analysis of lidocain kinetics in humans.     

Fetal CO2 kinetics

Knowledge of CO2 kinetics in the fetus is important for the design and interpretation of fetal metabolic studies that use carbon-labelled tracers. To study fetal CO2 kinetics, four fetal sheep were infused at constant rate with NaH14CO3 to simulate a constant rate of fetal 14CO2 production from the metabolism of a 14C-labelled substrate. Uterine and umbilical blood flows, and concentrations of 14CO2 and total CO2 in umbilical arterial and venous blood and in uterine arterial and venous blood were measured. During steady state, the excretion of 14CO2 via the umbilical circulation was 99.6 +/- 1.0 (SEM)% of the NaH14CO3 infusion rate. The irreversible disposal rate of CO2 molecules from the fetal CO2 pool was approximately 5 times greater than the metabolic production of CO2 by the fetus. This evidence demonstrates that measurements of fetal 14CO2 excretion via the umbilical circulation can provide an accurate measurement of fetal 14CO2 production and that the exchange rate of CO2 molecules between placenta and fetal blood is much greater than the net rate of excretion of CO2 molecules from fetus to placenta.     

N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.     

Calcium-dependent translocation of calbindin-D28k from intestine to blood

Calbindin-D (vitamin D-induced calcium-binding protein; CaBP) is known to be present in blood at concentrations which vary directly with levels in the intestinal mucosa. Employing a sensitive radioimmunoassay and sampling mesentery venous blood, the present experiments demonstrated a direct relationship between intestinal calcium absorption and serum CaBP. Solutions containing 150 mM NaCl and 45Ca-labeled calcium chloride (5 or 20 mM) were placed in the lumen of ligated duodenal preparations in situ and mesentery venous blood sampled with time. The concentration of absorbed 45Ca in serum was maximal at 5 min, followed by a significant increase in mesentery CaBP maximizing at 15-20 min. Elevation of serum CaBP was not observed when calcium in the dosing solution was omitted or replaced by either glucose or glycine. The possible transfer of absorbed calcium from the enterocyte to the circulation as a CaBP complex was ruled out by calculations revealing that considerably more calcium was transferred than could be accounted for by the low and high affinity binding sites on the protein. It is proposed that vitamin D-dependent enhanced transcellular calcium transport constitutes a stimulus for the increased release of intestinal CaBP into the circulation.     

Study of dinitrosyl-iron complexes pharmacokinetics and accumulation in depot in rat organs

Protein-bound dinitrosyl-iron complexes (DNIC) in rat whole blood and organs were studied after intravenous injection of this substance with glutathione ligand (DNIC-GH). The effect of DNIC-GH injection on NO level (including NO physiological forms) in hydrophobic areas of rat tissues was also studied in normal physiological blood circulation condition. It has been shown, that after DNIC-GH injection the concentration of protein-bound DNICs in rat whole blood and organs rapidly reached maximum values, and then gradually decreased, that pointed to decomposition of DNIC molecules, coupled with NO release. At the beginning of the experiment the rates of DNIC decay in rat heart and lung were substantially higher, as compared with those in liver and kidney. By spin trappping it has been demonstrated that DNIC-GH, as a source of NO physiological forms (including S-nitrosothiols), in normal physiological blood circulation influence heart more selectively, as compared with the other organs.     

A portal vein cannulation technique for drug discovery in mice

One approach to understanding how orally administered drugs are absorbed and metabolized involves measuring compound concentrations in portal vein blood and in systemic circulation at various time points. In mice, blood samples are generally collected through terminal bleeding, a process that requires a large number of mice and is susceptible to variation between individuals. The authors developed a portal vein cannulation procedure for serial bleeding in the mouse, using a modified catheter containing a stainless steel stylet that is implanted directly in the portal vein. To demonstrate the technique, they orally administered two different compounds to mice and obtained blood samples from the tail vein and portal vein at different time points. They analyzed compound concentrations using liquid chromatography-tandem mass spectrometry. The technique refines existing methods for pharmacokinetic studies in the mouse and reduces the number of mice required.     

Oxidative cross-linking of ApoB100 and hemoglobin results in low density lipoprotein modification in blood. Relevance to atherogenesis caused by hemodialysis.

Human blood contains a form of minimally modified low density lipoprotein (LDL), termed LDL-, whose origin remains unknown. Exploring the mechanism of formation, we found that LDL- can be produced in plasma in the absence of oxygen following LDL incubation with oxidized hemoglobin species. A high degree of apolipoprotein B100 modification results from covalent association of hemoglobin with LDL involving dityrosine formation but not due to the malonaldehyde epitope formation. This was evidenced by the cross-reactivity of oxidized LDL with antibodies against hemoglobin that was accompanied by a 60-fold increase in dityrosine levels. In this study we found significantly higher LDL- levels in the blood of hemodialysis patients, perhaps contributing to their greatly increased risk of atherosclerosis. The mechanism of LDL- formation was studied during ex vivo blood circulation using a model system resembling clinical hemodialysis in terms of the induction of inflammatory responses. This circulation increased free hemoglobin and LDL- levels compared with non-circulated blood without appreciable lipid peroxidation. Pronounced increases in LDL- were found also during circulation of plasma supplemented with nanomolar hemoglobin levels. The increase in dityrosine content and presence of heme in LDL after blood circulation suggest that LDL is modified, in part, by hemoglobin-LDL conjugates containing heme. Thus, hemoglobin-mediated reactions leading to LDL oxidation in plasma can account for high LDL- levels in hemodialysis patients.     

Modulation of antibody pharmacokinetics by chemical polysialylation

Chemical coupling of a variety of polymers to therapeutic proteins has been studied as a way of improving their pharmacokinetics and pharmacodynamics in vivo. Conjugates have been shown to possess greater stability, lower immunogenicity, and a longer blood circulation time due to the chemicophysical properties of these hydrophilic long chain molecules. Naturally occurring colominic acid (polysialic acid, PSA) has been investigated as an alternative to synthetic polymers such as poly(ethylene glycol) (PEG) due to its lower toxicity and natural metabolism. Antibodies and their fragments are a good example of the types of proteins which benefit from pharmacokinetic engineering. Here, we chemically attached differing amounts and differing lengths of short (11 kDa) and longer (22 kDa) chain colominic acid molecules to the antitumor monoclonal antibody H17E2 Fab fragment. Different coupling ratios and lengths were seen to alter the electrophoretic mobility of the Fab fragment but have a minor effect on the antibody immunoreactivity toward the placental alkaline phosphatase (PLAP) antigen. Polysialylation generally increased Fab fragment blood half-life resulting in higher tumor uptake in a KB human tumor xenograft mouse model. One H17E2 Fab-PSA conjugate had over a 5-fold increase in blood exposure and over a 3-fold higher tumor uptake with only a marginal decrease in tumor/blood selectivity ratio compared to the unconjugated Fab. This conjugate also had a blood bioavailability approaching that of a whole immunoglobulin.     

Ganglioside GM1 and Hydrophilic Polymers Increase Liposome Circulation Times by Inhibiting the Association of Blood Proteins

Abstract

Several agents have been shown to prolong the circulation lifetime of liposomes. These agents, such as ganglioside G_M1 or phosphatidylethanolamine-derivatives of monomethoxypolyethyleneglycols, provide insight into the mechanism(s) by which liposomes are cleared from the circulation. It is suggested here that the primary mechanism by which these molecules alter the biodistribution of liposomes in vivo involves an inhibition of the association of blood proteins to liposomes, resulting in a diminished rate of clearance of liposomes from the circulation.     

A Titin mutation defines roles for circulation in endothelial morphogenesis

Morphogenesis of the developing vascular network requires coordinated regulation of an extensive array of endothelial cell behaviors. Precisely regulated signaling molecules such as vascular endothelial growth factor (VEGF) direct some of these endothelial behaviors. Newly forming blood vessels also become subjected to novel biomechanical forces upon initiation of cardiac contractions. We report here the identification of a recessive mouse mutation termed shrunken-head (shru) that disrupts function of the Titin gene. Titin was found to be required for the initiation of proper heart contractions as well as for maintaining the correct overall shape and orientation of individual cardiomyocytes. Cardiac dysfunction in shrunken-head mutant embryos provided an opportunity to study the effects of lack of blood circulation on the morphogenesis of endothelial cells. Without blood flow, differentiating endothelial cells display defects in their shapes and patterns of cell-cell contact. These endothelial cells, without exposure to blood circulation, have an abnormal distribution within vasculogenic vessels. Further effects of absent blood flow include abnormal spatial regulation of angiogenesis and elevated VEGF signaling. The shrunken-head mutation has provided an in vivo model to precisely define the roles of circulation on cellular and network aspects of vascular morphogenesis.     

Bronchial blood flow affects recovery from constriction in dog lung periphery

We investigated the effect of eliminating the bronchial circulation on recovery time from intravenous histamine challenge in canine lung periphery. Results from animals with intact bronchial circulations were compared with a second group in which the left lower lobe was isolated in situ. The pulmonary artery to this lobe was perfused and a bronchoscope was wedged in a small airway, which provided an index of resistance to airflow through the collateral system. The lobe was challenged with intravenous histamine, and the time constant of recovery (tau) from bronchoconstriction was measured. With or without pulmonary blood flow, elimination of the bronchial circulation increased tau 44.4 and 48.5%, respectively. This increase was similar to that found by stopping pulmonary blood flow alone (56.5%). Histamine challenges were also performed in sympathectomized or vagotomized animals with intact bronchial circulations. Neither of these conditions increased tau. We conclude that blood flow through the bronchial circulation affects the recovery time from intravenous histamine challenge in the lung periphery to a degree similar to that of the pulmonary circulation.