Liposome disposition in vivo: effects of pre-dosing with lipsomes

The effect of a high, intravenous dose of extruded multilamellar liposomes (1.1 g lipid/kg body weight) upon the subsequent ability of mouse tissues to take-up or bind a second intravenous dose of similar liposomes encapsulating 14C-inulin has been studied in vivo. The first and second doses were separated by either 1,5 or 24 hours. All tissue levels were measured one hour after the second dose. Controls received only the second dose. When the two doses were separated by one hours, 14C-levels in liver were depressed 6-fold and blood levels rose 29-fold relative to controls. However spleen uptake of lipsomes increased to three times control levels. When the two doses were separated by 24 hrs, the first dose had only a minimal effect on the disposition of the second dose. The results are consistent with a reversible blockade of hepatic, but not spleenic uptake and/or binding sites, by the first dose and indicate that adjusting a liposome dose (i.e. number of lipsomes) or use of a drug-free liposome pre-dose may be a useful technique for reducing hepatic uptake, increasing the circulation life time and/or modifying the tissue disposition properteries of therapeutic liposomes without changing liposome composition or size.     

In vivo tear-film thickness determination and implications for tear-film stability

PURPOSE: Previous measurements of tear-film thickness in vivo are limited and cannot be easily applied in a clinical setting. A novel technique to measure tear-film thickness indirectly is introduced here, requiring only a slit lamp, video camera, and computer. A recent fluid mechanical theory relates tear-film thickness h, to the tear meniscus radius R, tear surface tension or, tear viscosity mu, and upper lid velocity U. This theory yields the result that h/R = 2.12 (microU/sigma)2/3. All parameters except h/R are taken as known physical constants, and R was measured for each subject, allowing the above equation to establish h. Tear-film breakup was also evaluated and correlated with tear-film thickness. METHODS: A clinical study was performed in which aqueous tear-film thickness was determined for 45 subjects, including 24 non-lens subjects, 15 hydrogel contact lens wearers, and 6 RGP lens wearers. R was measured by instilling fluorescein dye in the form of an eyedrop and videotaping the tear meniscus in profile. Tear-film breakup was videotaped through the ocular port of the slit lamp and evaluated based on a severity scale. RESULTS: Aqueous tear-film measurements are in the same range as literature values, with most measured values falling between 6 and 12 microm. Average tear-film thicknesses for non-lens, hydrogel, and RGP subjects are 10.4, 6.5, and 5.8 microm, respectively. Tear-film breakup is most severe in subjects with thin tear films, especially in contact-lens wearers. CONCLUSIONS: Tear-film thickness is an important parameter that varies among individuals. These variations correlate with differences in tear-film stability.     

In vivo disposition and metabolism by liver and enterocyte microsomes of the antitubercular drug rifabutin in rats

The in vivo disposition and in vitro metabolism of rifabutin, a new spiropiperidylrifamycin, were studied in rats and in microsomes from rat liver and enterocytes, respectively. After i.v. doses of 1,5, 10 and 25 mg/kg the systemic clearance was 0.7 to 1.0 liters/hr/kg; the volume of distribution was 4.4 liters/kg for the 1 mg/kg dose and 7.4 to 7.7 liters/kg for the 5 to 25 mg/kg doses, and the half-life ranged from 4.4 to 9.1 hr. Urinary and fecal excretion over 0 to 96 hr after i.v. administration of 25 mg/kg [14C]rifabutin accounted for 40.1 and 52.2% of the dose, respectively. Exteriorization of the bile duct showed that approximately 24% of the dose was eliminated in bile, > or = 98% as metabolites. Bioavailability after oral administration of 25 and 1 mg/kg rifabutin was > 90% and 44%, respectively, suggesting significant first-pass metabolism of the lower dose. Concentrations of rifabutin in gastric juice were 10 to 17 times higher than in blood, indicating extensive secretion into the stomach. Experiments with the isolated small intestinal loop demonstrated direct exsorption of the drug into the lumen. The rate of rifabutin metabolism by enterocyte microsomes was > 10 times higher than that by liver microsomes, i.e., 84 and 8 pmol/min/mg protein, respectively. Biotransformation of rifabutin in vivo and in vitro was markedly induced by dexamethasone and inhibited by erythromycin, suggesting that CYP3A is involved in the metabolism of rifabutin. Several metabolites, including 20-OH-rifabutin and 27-O-demethyl-rifabutin, isolated from urine and microsomes were identified by mass spectrometry and nuclear magnetic resonance spectroscopy.     

Osteotropic drug delivery system (ODDS) based on bisphosphonic prodrug. V. Biological disposition and targeting characteristics of osteotropic estradiol

An osteotropic drug delivery system (ODDS) based on a bisphosphonic prodrug has been developed for 17 beta-estradiol (E2) to improve patient compliance in estrogen replacement therapy of postmenopausal osteoporosis. The biological disposition and the targeting efficiency of a bisphosphonic prodrug of E2, disodium [17 beta-(3'-hydroxy-1',3',5'-estratrienyloxy)carbonylpropyl carboxamidomethylene]bisphosphonate (E2-BP), was investigated in ovariectomized rats. After intravenous injection, E2-BP was rapidly taken up into the bone and subsequently cleared from the bone at a half-life of 13.5 d. The bone concentration of regenerated E2 was maintained throughout 28 d. In contrast, E2 injected intravenously showed extremely low bone distribution and rapid clearance from the bone, and E2 administered orally showed even lower bone distribution. Therapeutic availability (TA) and drug targeting index (DTI), which were calculated on the basis of the AUCs for E2 in the bone and plasma after injection of E2-BP and E2, were 64.6 and 451, respectively. These results suggest that ODDS has a potential to improve not only the apparent potency but also the therapeutic index of E2. As compared with the conventional estrogenic products, E2-BP should improve patient compliance with lower adverse effects and less frequent medication in long-term estrogen replacement therapy.     

CD4 dimerization and oligomerization: implications for T-cell function and structure-based drug design

Recent studies of CD4 structure and function have revealed possible mechanisms for CD4 self-association, with implications for its role in T-cell activation. Here, the authors discuss the formulation of a hypothetical three-dimensional model of CD4 oligomerization and how it impacts on the understanding of T-cell function and rational drug design targeting specific CD4 surface functional sites.     

Size constancy as a function of personal adjustment and disposition.

"A size-distance judging task was given to 40 men who were classified in 4 groups, respectively styled 'neurotic introverts,' 'neurotic extraverts,' 'normal introverts,' and 'normal extraverts.' Analysis of data from 4 distances under 2 conditions of judgment, i.e., objective and analytic, indicated that neuroticism was the major source of between-group variation. Under analytic conditions, neurotic persons tended to match the stimulus in terms of visual angle, and normals in terms of size." 15 references. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modified plasma and abomasal disposition of albendazole in nematode-infected sheep

The influence of gastrointestinal nematode infection on the kinetics of albendazole (ABZ) and its metabolites, albendazole sulphoxide (ABZSO) and sulphone (ABZSO2) in plasma and abomasal fluid was investigated in sheep. A micronised suspension of ABZ was administered intraruminally at 7.5 mg kg-1 to the following groups of sheep: (a) non-parasitised (control); (b) artificially infected with Haemonchus contortus; (c) naturally infected with Haemonchus contortus and other species of gastrointestinal nematodes. Plasma and abomasal fluid samples were obtained serially over 72 h post-treatment and they were analysed by HPLC for ABZ and its metabolites. The ABZ parent drug was not detected in plasma at any time post-treatment, however the metabolites ABZSO and ABZSO2 were recovered in the bloodstream. The active metabolite ABZSO was recovered in plasma between 0.5 and 48 (uninfected), 60 (H. contortus infected) or 72 h (naturally infected sheep) post-administration. The area under the plasma concentration vs time curve (AUC) values for ABZSO were higher in both artificially infected (64.0 micrograms h ml-1) and naturally infected (79.3 micrograms h ml-1) sheep as compared with non-infected animals (41.8 micrograms h ml-1). Peak plasma concentrations for ABZSO and ABZSO2 were higher in both artificially and naturally infected sheep than in non-parasitised animals. No changes in the half-lives and mean residence times for these metabolites were observed in infected sheep. ABZ and its metabolites were found in the abomasum between 0.5 and 48 (infected animals) or 72 h (uninfected) post-treatment. The availability (total AUCs) of ABZ and its metabolites in abomasal fluid were lower in H. contortus infected sheep than in the uninfected control animals. The increased abomasal pH induced by the presence of the H. contortus infection may reduce the plasma/abomasum pH gradient, which results in a decreased ionic-trapping of ABZ and its metabolites in the abomasum. Such a phenomenon correlates with: (a) the higher total AUC values obtained for ABZ metabolites in the bloodstream of the infected compared to the control sheep, (b) the lower concentration profiles of the ABZ parent drug and its metabolites found in the abomasal fluid of the infected animals.     

Evolution and thermal stability of Ni3V and Ni2V phases in a Ni-29 at. pct V alloy

Eutectoid decomposition of the disordered fcc Ni-V solid solution in the composition range of 25 to 33.3 at. pct V gives rise to a mixture of the ordered Ni₃V and Ni₂V phases. In the present work, the evolution and thermal stability of these phases were studied in a Ni-29 at. pct V alloy. Solution-treated and water-quenched specimens, when aged at 850 °C, were found to exhibit two types of microstructure. In the first, the Ni₂V phase precipitated in a lamellar Ni₃V matrix where a pair of conjugate lamellae corresponded to two variants of the Ni₃V phase. In the second morphology, the Ni₂V phase precipitated within a Ni₃V matrix comprising a single variant of the Ni₃V phase. The Ni₂V phase was observed to precipitate in a plate-shaped morphology, exhibiting {120}_fcc-type, habit planes. The precipitation of the Ni₂V plates in the Ni₃V lamellae resulted in zigzag interfaces between adjacent Ni₃V domains. Both the microstructures were found to be thermally quite stable and did not coarsen appreciably on prolonged aging. However, the prolonged aging caused the renucleation of the Ni₃V and the Ni₂V phases in the vicinity of the grain boundaries in a manner similar to “recrystallization.” The stability of the aged microstructure could be attributed to the nature of the interfaces between different domains of the Ni₃V and Ni₂V phases.     

Disclosure of traumas and immune function: Health implications for psychotherapy.

Can psychotherapy reduce the incidence of health problems? A general model of psychosomatics assumes that inhibiting or holding back one's thoughts, feelings, and behaviors is associated with long-term stress and disease. Actively confronting upsetting experiences—through writing or talking—is hypothesized to reduce the negative effects of inhibition. Fifty healthy undergraduates were assigned to write about either traumatic experiences or superficial topics for 4 consecutive days. Two measures of cellular immune-system function and health center visits suggested that confronting traumatic experiences was physically beneficial. The implications for psychotherapy as a preventive treatment for health problems are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The phosphate carrier from yeast mitochondria. Dimerization is a prerequisite for function

Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies. From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded. This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C12E8 and dialysis. Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo- and heterodimeric forms of PIC, which are all fully active after reconstitution. As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer. We investigated its dimeric nature in the functionally active state after reconstitution. When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence. Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport. In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier. These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers.     

The effect of hypothalamic peptide YY on hippocampal acetylcholine release in vivo: implications for limbic function in binge-eating behavior

Central injection of peptide YY (PYY) in sated rats produces the most powerful stimulating effect of food intake known to date. The neural mechanisms by which PYY regulates appetite are not clear but may be important because abnormal levels of PYY have been implicated in the neurobiology of bulimia nervosa. Interactions between brain acetylcholine (ACh) and PYY had not been studied. Therefore, the present experiments were designed to explore the in vivo release of ACh from the hippocampus (HPC) of rats in response to hypothalamic infusion of PYY. Hippocampal ACh release was found to increase 400% in response to 10 microg PYY. In a separate experiment, blockade of the same area of the HPC with bilateral intracerebral injections of 3.5 microg scopolamine did not affect intake stimulated by intrahypothalamic injection of 4 microg PYY. Furthermore, a third experiment showed, for the first time, that PYY (2.5-10.0 microg) can elicit robust feeding when infused directly into the HPC. The significance of these findings to the activation of limbic functions such as memory, reinforcement, and obsessional processes that accompany human binge-eating syndromes is discussed.     

Kinetic properties of saquinavir-resistant mutants of human immunodeficiency virus type 1 protease and their implications in drug resistance in vivo

In order to study the basis of resistance of human immunodeficiency virus, type 1 (HIV-1), to HIV-1 protease inhibitor saquinavir, the catalytic and inhibition properties of the wild-type HIV-1 protease and three saquinavir resistant mutants, G48V, L90M, and G48V/L90M, were compared. The kinetic parameter kcat/Km was determined for these proteases using eight peptide substrates whose sequences were derived from the natural processing site sequences of HIV-1. The kcat/Km values were determined using conventional steady-state kinetics as well as initial velocities of mixed substrate cleavages under the condition where the substrate concentrations [S]o < Km. The independently determined kcat and Km values for some of the substrates confirmed the accuracy of the mixed-substrate method and also permitted the calculation in all cases of true rather than relative kcat/Km values. The Ki values were also determined. Using a previously described kinetic model [Tang, J., & Hartsuck, J. A. (1995) FEBS Lett. 367, 112-116], the relative processing activities of HIV-1 protease variants were estimated in the saquinavir concentration range of 0-10(-7) M. Although the protease activity of G48V, L90M, and G48V/L90M are only about 10, 7, and 3% of that of the wild-type HIV-1 protease in the absence of inhibitor, the resistance tendencies of the three mutants are clearly manifest by relatively less activity loss as inhibitor concentration becomes higher. Also, the ratios of the activities of the four protease species at certain saquinavir concentrations appear to correlate with the population ratios of the four protease species at different time points of clinical trials. This correlation suggests that the population ratio of the protease species is driven by in vivo saquinavir concentration, which appears to be in the range 10(-10)-10(-9) M during the clinical trials.     

Macromolecule-macromolecule interaction in drug distribution. V. Effects of plasma proteins on uptake of fractionated [3H]heparin in isolated rat Kupffer cells

Effects of plasma proteins such as alpha-globulin on the uptake of high molecular weight (HMWFH: 23000 Da) and low molecular weight fractionated [3H]heparin (LMWFH: 10000 Da) were examined in isolated rat Kupffer cells. alpha-Globulin (8 mg/ml) affected neither surface binding nor internalization of LMWFH by Kupffer cells, while it reduced both surface binding and internalization of HMWFH without affecting the fraction internalized, which was a ratio of internalized amount to the total association. The total associations of HMWFH were about four times larger than that predicted assuming only the unbound fraction is available for uptake, suggesting the participation of protein-mediated transport in the uptake of HMWFH in Kupffer cells. Based on the same assumption, the saturable initial uptake of HMWFH versus concentration profile in the presence of alpha-globulin (8 mg/ml) was also analyzed to further examine the suggested protein-mediated transport. The estimated dissociation constant of 487 nM was three times larger than that in in vitro binding experiments (168 nM) and the binding capacity of 0.155 was one third of the value in vitro (0.5), suggesting apparent reductions in both binding affinity and capacity. Thus, we demonstrated the involvement of protein-mediated transport in the uptake of fractionated heparin in Kupffer cells and kinetically characterized it as the apparent enhancement of dissociation.     

Warfarin-fluconazole. II. A metabolically based drug interaction: in vivo studies

Consistent with expectations based on human in vitro microsomal experiments, administration of fluconazole (400 mg/day) for 6 days to six human volunteers significantly reduced the cytochrome P450 (P450)-dependent metabolic clearance of the warfarin enantiomers. In particular, P4502C9 catalyzed 6- and 7-hydroxylation of (S)-warfarin, the pathway primarily responsible for termination of warfarin's anticoagulant effect, was inhibited by approximately 70%. The change in (S)-warfarin pharmacokinetics caused by fluconazole dramatically increased the magnitude and duration of warfarin's hypoprothrombinemic effect. These observations indicate that co-administration of fluconazole and warfarin will result in a clinically significant metabolically based interaction The major P450-dependent, in vivo pathways of (R)-warfarin clearance were also strongly inhibited by fluconazole. 10-Hydroxylation, a metabolic pathway catalyzed exclusively by P4503A4, was inhibited by 45% whereas 6-, 7-, and 8-hydroxylations were inhibited by 61, 73, and 88%, respectively. The potent inhibition of the phenolic metabolites suggests that enzymes other than P4501A2 (weakly inhibited by fluconazole in vitro) are primarily responsible for the formation of these metabolites in vivo as predicted from in vitro kinetic studies. These data suggest that fluconazole can be expected to interact with any drug whose clearance is dominated by P450s 2C9, 3A4, and other as yet undefined isoforms. Overall, the results strongly support the hypothesis that metabolically based in vivo drug interactions may be predicted from human in vitro microsomal data.     

Physico-chemical characterization and application for drug carrier of soybean-derived sterols and their glucosides-containing liposomes

With a rapid demand to decrease the side effect of drug, a variety of drug delivery systems have been developed. This review will focus on the development of liposomes with soybean-derived sterols and their glucosides for drug carriers. Current status and further perspectives in this research field are reviewed mainly based on the results obtained in our laboratory. First we studied the different physicochemical properties of dipalmitoylphos-phatidylcholine (DPPC) liposomes with soybean-derived sterols (SS) and their glucosides (SG). SS rigidifies the liposomal membrane but SG fluidizes it. SS stabilizes liposomes more than cholesterol that is conventionally used as stabilizing agents in liposomes. On the basis of this information, we developed liposomes with SS and SG for a drug carrier. Secondly we studied the stability of liposomes in the blood and biodistribution and found that liposomes with SS were stable as expected in vitro results. In particular, DPPC: SS (7:4, molar ratio) size 0.2 micron showed long circulation. Thirdly successful targeting of the drugs to the liver was achieved by liposomes with SG. Finally, we succeeded in developing liposomal erythropoietin and doxorubicin using liposomes with SS for sustained release of drugs. Liposomal drugs increased the pharmacological effect compared with free drugs, suggesting a decrease of side effect and long circulation. The attempt for oral administration using liposomes of peptide drugs was carried out successfully. We have established that the study of physicochemical properties of liposomes is needed rationally as the distribution of drugs in liposomes and the rigidity of liposomal membrane, prior to the development of the drug carrier of liposomes. SS is useful to stabilize liposomes and SG to targeting to the liver parenchymal cells. This information can be useful and practical for the development of liposomes for drug carriers.     

Molecular basis of P450 inhibition and activation: implications for drug development and drug therapy

In previous studies many benzimidazole, imidazole and benzothiazole derivatives had been synthesized and their antimicrobial activities were tested in vitro conditions. Four of these compounds showed minimal inhibitory concentrations (MIC) of 5-25 micrograms/ml against standard strains and clinical isolates. In order to determine whether these four compounds can be used for therapeutic purpose, their serum MIC values and side effects on hepatic and renal functions were determined. Different concentrations of the compounds were tested on Wistar rats. Compound 1 was administered orally, intramuscularly and intravenously; compounds 2, 3 and 4 were given orally and intramuscularly. Blood samples were taken 4 and 24 h after administration of the compounds. Serum MIC values were investigated by bioassay and serum levels of biochemical parameters by autoanalyzer. None of the tested compounds showed antimicrobial activity at their serum concentrations. Although creatinine activity was found at normal levels in all experiments, compounds 1 and 2 caused a significant increase in blood urea nitrogen (BUN) level. The values of aspartate aminotransferase and/or alanine aminotransferase and/or alkaline phosphatase which are characteristic for liver function were generally found at high levels. According to these results, it can be concluded that the tested compounds caused damage in liver and biliary tracts without antimicrobial activity by their serum concentrations.