[Study on pharmacokinetics of ginkgolide B injection in Beagle dogs].

OBJECTIVE: To study the pharmacokinetics of ginkgolide B injection in Beagle dogs. METHODS: Determined the serum concentration of ginkgolide B by LC-MS and calculated its parameter of pharmacokinetics via DAS 2.0 software. RESULTS: After intravenous drips of 0.62, 2.07 and 10.35 mg/kg ginkgolide B, parameters of pharmacokinetics of ginkgolide B were as follows: Tmax were 0.444, 1, 1 h; Cmax were 0.764, 3.024, 11.013 mg/L; AUC(0-1) were 1.007, 3.644, 16.646 mg x h/Lo. CONCLUSION: Ginkgolide B has two compartment model in Beagle dogs.

Mitogen-activated protein kinases regulate platelet-activating factor-induced hyperpermeability.

OBJECTIVE: The authors tested the hypothesis that p42/44- (ERK-1/2) and/or p38-mitogen-activated protein kinases (MAPK) are in vivo regulatory elements in the platelet-activating factor (PAF) activated signaling cascade that stimulates microvascular hyperpermeability. METHODS: FITC-dextran 70 was used as the macromolecular tracer for microvascular permeability in the mouse mesenteric fat tissue. Interstitial integrated optical intensity (IOI) was used as an index of permeability. RESULTS: An application of 10(-7) M PAF increased IOI from 23.1 +/- 3.6 to 70.8 +/- 7.4 (mean +/- SEM). Inhibition of ERK-1/2 with 3 microM and 30 microM AG126 reduced IOI to 32.3 +/- 2.5. Similarly, inhibition of p38-MAPK with 6 nM, 60 nM and 600 nM SB203580 lowered IOI to 29.1 +/- 2.4. CONCLUSIONS: The results demonstrate that ERK-1/2 and p38MAPK participate in the signaling cascade that regulates PAF-induced microvascular hyperpermeability in vivo.

A pharmacokinetic/pharmacodynamic model for a platelet activating factor antagonist based on data arising from Phase I studies.

A nonlinear mixed-effects modelling approach was used to analyse pharmacokinetic and pharmacodynamic data from two Phase I studies of a platelet activating factor (PAF) antagonist under development for the treatment of seasonal allergic rhinitis. Data for single-dose (8 subjects) and multiple-dose (9 subjects) administration were available for analysis with a program based on an EM algorithm. Pharmacokinetic analyses of plasma drug concentrations were performed using a biexponential model with first-order absorption. PAF response data were modelled with a hyperbolic Emax model. The drug showed nonlinear pharmacokinetics, with the clearance decreasing from 46.0 to 27.1 L h(-1) over a dose range of 160-480 mg. There was an apparent dose dependency within the C50 (concentration producing 50% of the maximum effect) but at higher doses most of the data was above the estimated C50 and when the data was analysed simultaneously a value of 17.57 ng mL(-1) was obtained for C50, with considerable intersubject variability (103%). Consistent results were obtained from the two studies and the population and individual pharmacodynamic parameter estimates from the analyses provided predicted responses that were in good agreement with the observed data. The results were used to simulate a 320-mg twice-daily dosing regimen.

Sperm-immobilizing antibodies suppress an increase in the plasma membrane fluidity of human spermatozoa.

OBJECTIVE: To clarify the mechanism by which capacitation is blocked by sperm-immobilizing antibodies, changes in the plasma membrane fluidity of human spermatozoa exposed to sperm-immobilizing antibodies were evaluated. DESIGN: In vitro cell culture study using human spermatozoa. SETTING: Department of Obstetrics and Gynecology, School of Medicine, The University of Tokushima. PATIENT(S): Semen samples were obtained from four healthy, fertile volunteers. INTERVENTION(S): The internalization of [3H]lyso-platelet activating factor (lyso-PAF) across the plasma membranes of human spermatozoa, which were exposed to sperm-immobilizing antibodies (antisperm group) or not exposed (control group), was measured at 20 and 60 minutes after the addition of a phospholipid probe using the modified albumin-back extraction method. MAIN OUTCOME MEASURE(S): The percentage of internalization of [3H]lyso-PAF across the plasma membrane of human spermatozoa. RESULT(S): Although the percentages of internalization of [3H]lyso-PAF (mean +/- SE) in the antisperm and control groups 20 minutes after addition of [3H]lyso-PAF were not significantly different (6.6% +/- 1.5% and 9.2% +/- 2.1%, respectively), at 60 minutes after the addition, the percentage in the antisperm group (9.0% +/- 1.3%) was significantly lower than that in the control group (13.4% +/- 1.3%). This inhibitory effect was diminished when spermatozoa exposed to sperm-immobilizing antibodies were incubated in an antibody-free medium. CONCLUSION(S): Sperm-immobilizing antibodies suppress the increase in internalization of an alkyl ester lysophospholipid probe in plasma membranes of human spermatozoa, and this inhibitory effect is reversible. Therefore, sperm-immobilizing antibodies suppress the fluidity of the plasma membranes of human spermatozoa, thus blocking capacitation.

Human spermatozoa capacitated with progesterone or a long incubation show accelerated internalization by an alkyl ether lysophospholipid.

OBJECTIVE: To evaluate changes that occur in sperm plasma membranes during capacitation, the internalization of [(3)H]lyso-platelet activating factor ([(3)H]lyso-PAF) across the plasma membrane of human spermatozoa was measured as a function of incubation time or exposure to progesterone (P). DESIGN: In vitro cell culture study using human spermatozoa. SETTING: Department of Obstetrics and Gynecology, School of Medicine, the University of Tokushima, Japan. PATIENT(S): Semen were obtained from three fertile healthy volunteers. INTERVENTION(S): The internalization of [(3)H]lyso-PAF across the plasma membranes of human spermatozoa that were incubated for an extended period or exposed to P was measured at 5, 20, 60, and 120 minutes after the addition of the phospholipid probe using the modified albumin back-exchange method. MAIN OUTCOME MEASURE(S): The percentage of capacitated and acrosome-reacted sperm and the proportion of internalization of lyso-PAF across the plasma membrane. RESULT(S): A 6-hour incubation period significantly increased the percentage of capacitated spermatozoa and the proportion of internalization of [(3)H]lyso-PAF across the plasma membrane of human spermatozoa compared with controls (capacitated spermatozoa, 20.3 +/- 10.6% vs. 8.5 +/- 1.8%; internalization 120 minutes after the addition of the phospholipid probe, 25.6 +/- 2.5% vs. 11.6 +/- 3.0%) (mean +/- SEM). Exposure to P significantly increased the percentage of capacitated spermatozoa compared with controls (19.6 +/- 6.8% vs. 11.0 +/- 2.4%) and also significantly accelerated the internalization of [(3)H]lyso-PAF compared with controls (internalization 120 minutes after the addition of the phospholipid probe, 26.2 +/- 1.8% vs. 21.4 +/- 1.1%). CONCLUSION(S): The administration of P or a long incubation increased the proportion of internalization and consequently induced capacitation in human spermatozoa.

Sponge-induced angiogenesis and inflammation in PAF receptor-deficient mice (PAFR-KO).

1. To determine biological functions of platelet-activating factor (PAF) in chronic inflammation, we have investigated the kinetics of angiogenesis, inflammatory cells recruitment and cytokine production in sponge-induced granuloma in wild type and PAF receptor-deficient mice (PAFR-KO). 2. Angiogenesis as determined by morphometric analysis and hemoglobin content was significantly higher in the implants of PAFR-KO mice at all time points. Treatment with PAF receptor antagonist UK74505 (30 mg kg(-1)) also increased angiogenesis in sponge implants. 3. Neutrophils and macrophages accumulation, as determined by myeloperoxidase and N-acetylglucosaminidase activities in the supernatant of implanted sponges were markedly decreased in PAFR-KO mice. Surprisingly, the levels of the proinflammatory chemokines, keratinocyte-derived chemokine and chemokine monocyte chemoattractant protein 1 were higher in the implants of the transgenic animals. 4. We have shown that angiogenesis was stimulated in PAFR-KO mice whereas inflammation was decreased, indicating that PAF is an endogenous regulator of new blood vessels formation in the inflammatory microenvironment induced by the sponge implant.

Modulation of microvascular hydraulic permeability by platelet-activating factor.

BACKGROUND: Platelet-activating factor (PAF) is a modulator of the inflammatory response to shock. Edema formation and intravascular fluid loss have been associated with PAF. The increase in microvessel permeability caused by PAF may be related to direct endothelial cell activation and leukocyte activation. We hypothesized that PAF increases hydraulic permeability by means of the direct activation of endothelial cells. METHODS: Hydraulic permeability (Lp) was measured in rat mesenteric venules using the modified Landis micro-occlusion technique. After baseline Lp measurements, paired measures of Lp were obtained during PAF perfusion at doses of 0.1 nmol/L (n = 6), 1.0 nmol/L (n = 6), 10 nmol/L (n = 6), and 50 nmol/L (n = 6). The temporal effects of pulse administration of PAF and repeated exposures to PAF were also assessed. RESULTS: Compared with baseline values (Lp = 1.16 +/- 0.11), the Lp of the microvessels significantly increased at PAF doses of 0.1 nmol/L (Lp = 1.46 +/- 0.1) (p < 0.002), 1 nmol/L (Lp = 2.0 +/- 0.11) (p < 0.004), 10 nmol/L (Lp = 4.09 +/- 0.09) (p < 0.005), and 50 nmol/L (Lp = 5.13 +/- 0.07) (p < 0.0001). All units for Lp are given as +/- SE x 10 -7 cm s-1. cm H2O-1. CONCLUSION: PAF increased microvessel permeability in a dose-dependent manner. The permeability-increasing effect of PAF was transient even with continuous endothelial exposure to PAF. This study emphasizes the ability of PAF to directly modulate microvascular permeability and increase venular permeability.

Inhibition of prostaglandin G/H synthase unveils a potent effect of platelet activating factor on the permeability of bovine aortic endothelial cells to albumin.

Platelet Activating Factor (PAF) is a very potent stimulant of various cell functions but little is known about the mechanisms responsible for its marked effect on endothelial permeability. An in vitro assay system was used to assess the direct effect of PAF on the permeability of a bovine aortic endothelial cell (BAEC) monolayer to albumin. PAF produced a small but not significant increase of the permeability of BAEC monolayer to albumin. However, pre-treatment of the monolayer with indomethacin (10 microM) resulted in a significant increase of BAEC permeability following PAF administration. This increase was concentration-dependent up to a maximal effect of 105% above basal value (for 0.1 microM PAF). Addition of the PAF antagonist SRI 63 441ZI (5 microM) abolished this effect. Exogenous administration of PGE2 (10(-7) M) inhibited the effect of PAF on the BAEC permeability suggesting that prostaglandins synthesized by the endothelium behave as a negative autoregulatory factor. Compound SRI 63 441ZI also partially inhibited bradykinin-induced permeability to albumin but did not significantly modify the activity of thrombin. These findings show that PAF can increase endothelial permeability to albumin when the synthesis of prostaglandins is inhibited. Our results also show that PAF might have an autocrine activity by mediating part of BK-induced permeability.

Kinetics of PAF transfer, distribution and metabolism in human blood in vitro.

The transfer kinetics of PAF (0.1-100 nM), deposited as a thin film on a plastic surface, to human blood were studied under conditions of physiological significance. Almost all (83-87%) available PAF was solubilized in two waves. Initially, 40-50% of the available PAF was transferred to blood very fast in less than 15 seconds while the rest, 30-45%, obeyed first order kinetics, 0.0226 < k(app) < 0.0319 sec(-1). Blood cells following a parallel to whole blood binding time course bound 20-25% of the solubilized PAF. Dilution of blood up to 1:9 with 0.15M NaCl did not affect PAF transfer parameters favouring an aqueous phase diffusion mechanism. Blood-bound PAF was allocated mainly to plasma, 67 +/- 4%, erythrocytes, 18 +/- 1% and platelets, 5 +/- 1%. These data indicated the role of the high affinity binding sites in human platelets versus the low affinity binding by erythrocytes, although the latter, due to their number, dominated cell bound PAF. Even at 100 nM there were no saturation signs for the transfer of PAF to blood or blood cells. PAF hydrolysis did not affect its binding to the blood elements. Given infinite time only 62 +/- 1% of the blood bound PAF would be metabolised by the rather slow acting, 11.5 > t(1/2) > 6.5 min, PAF-acetylhydrolase.

A distribution study of 11C platelet-activating factor (PAF) analogs in normal and tumor-bearing mice.

As a preliminary study to image platelet-activating factor (PAF) receptors in vivo, comparative study of biodistribution between 1-O-hexadecy1-2-O-N, N-dimethylcarbamoyl-sn-glycero-3-phosphocholine [choline-methyl-11C](L-[11C]dimethylcarbamoyl-PAF) and nonspecific PAF analog, 3-O-hexadecyl-2-O-N,N-dimethylcarbamoyl-sn-glycero-1-phosphocholine [choline-methyl-11C](D-[11C]-dimethylcarbamoyl-PAF) was carried out in both normal and tumor-bearing mice. Higher accumulation of L-[11C]dimethylcarbamoyl-PAF than D-[11C]dimethylcarbamoyl-PAF was observed in normal mice spleen. The co-administration of PAF antagonists dose-dependently reduced the radioactivity level of the L-isomer only in the spleen. In mice bearing Ehrlich tumors and Sarcoma 180, more L-than the D-[11C]-isomer was accumulated in the tumor and spleen. We found that specific accumulation sites for L-[11C]dimethylcarbamoyl-PAF exist in the spleen and tumors than in other tissues. Moreover, the comparison of accumulation between L- and D-[11C] dimethylcarbamoyl-PAF would be a useful procedure for estimation of PAF receptors in vivo.

Differential metabolism of exogenous platelet-activating factor by glandular epithelial and stromal cells of rabbit endometrium.

Significant changes in platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) concentration have been observed in rabbit endometrium during the preimplantation period, but, under in vitro conditions, constitutive PAF biosynthesis by isolated endometrial tissues was not easily demonstrable. Relative changes in enzymes involved in the synthesis and metabolism of PAF in the tissues may account for this disparity. In addition, during this period of preimplantation, marked changes in PAF receptor concentration have been noted. The present study examines the factors that may modulate the metabolism of exogenous [3H]PAF in the endometrium of rabbits on day 6 of pregnancy. Since preferential [3H]PAF binding in situ by the glandular epithelial, but not by the stromal, cells was demonstrated, their cell-specific metabolism of exogenous [3H]PAF was also examined. After entry into the endometrial cell, [3H]PAF was rapidly metabolized by the sequential action of cytosolic Ca(2+)-independent acetylhydrolase to [3H]lyso-PAF and this was in turn acylated by membrane-associated transacylase to [3H]alkylacyl-glycerylphosphorylcholine. PAF resynthesis was not observed and, in stromal cells, there was a significant build-up of [3H]lyso-PAF, suggesting that lyso-PAF:acetyl-CoA acetyl-transferase may be a limiting factor. In the glandular epithelial cells, however, there was a significant accumulation of a neutral lipid without a significant build-up of [3H]lyso-PAF or [3H]PAF. The neutral lipid co-migrated with the product of phospholipase C-catalysed metabolism of PAF and authentic 1-O-hexadecyl-2-acetyl-glycerol. In addition, the elution times of phospholipase C digestion of C18 PAF and the neutral lipid produced by cellular metabolism of [3H]PAF, determined by gas chromatography/flame ionization detection, were similar. It seems that it is the synthesis of the neutral lipid from reacetylated [3H]lyso-PAF that prevented [3H]PAF accumulation under in vitro conditions. This is the first documentation of the synthesis of this lipid in the mammalian uterus. The lipid may serve as the precursor for de novo PAF synthesis in the glandular epithelial cells during endometrial proliferation.

Effects of liposome-entrapped platelet-activating factor in the isolated rat trachea.

The effects of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-filled liposomes upon rat tracheal rings in vitro were examined. The capture of liposomes by the smooth muscle cells of the isolated tracheal rings as well as the release of their content into the cytoplasm was shown by using Evans blue (5 x 10(-4) M)-loaded liposomes. Administration of PAF (10(-3) M)-filled liposomes contracted the preparations, in contrast with extracellular administration of PAF and control liposomes, which had no effect. Administration during the plateau or pretreatment with liposomes containing BN 52021 (3-t-butylhexahydro-4,7b-trihydroxy-8-methyl-9H-1,7a-epoxymethano- 1H,6aH- cyclopenta[c]furo(2,3-b)furo[3',2':3,4]cyclopental [1,2-d]furan-5,9,12(4H)-trione) ((10(-3) M, a selective PAF receptor antagonist) or heparin (5 x 10(-5) M) blocked this contraction. BN 52021 and heparin, not entrapped in liposomes, had no such effect. Our data suggest an intervention of PAF in the mechanisms of contraction of tracheal smooth muscle, involving a direct or indirect intervention (intracellular receptors for PAF cannot be excluded). At the same time, the rat trachea contraction induced by PAF-loaded liposomes could be linked to the PtdIns(1,4,5)P3-dependent Ca2+ channels from the endoplasmic reticulum and/or to the interaction with G proteins, as shown by the blocking effects of heparin-containing liposomes.

Role of neutrophils in breakdown of the blood-retinal barrier following intravitreal injection of platelet-activating factor.

Breakdown of the blood-retinal barrier occurs in inflammatory conditions and in ischemic retinal diseases such as diabetic retinopathy. Platelet-activating factor (PAF) is a potent inflammatory mediator which increases vascular permeability. The purpose of this study was to determine if intravitreally-injected PAF would cause breakdown of the blood-retinal barrier and, if so, by what mechanism. Fluorescein angiography was performed before and at 0.5, 1, 2, 3 and 4 hr after PAF injection into the vitreous cavity of rabbit eyes and the eyes were enucleated immediately for light and electron microscopy. Slow flowing thrombi were observed in all PAF-injected eyes. Complete vascular occlusion was observed in 10 of 16 eyes after 3 and 4 hr. There was no fluorescein leakage in any of eyes before or at 0.5 or 1 hr after PAF injection. Fourteen of 20 eyes had fluorescein leakage at 2, 3 and 4 hr after PAF injection. The extent of fluorescein leakage correlated with the degree of polymorphonuclear leukocyte (PMN) margination, disruption of the endothelial cell layer, infiltration into vascular walls and migration into the vitreous cavity. PMNs appeared to migrate by both intercellular and transcellular routes across the endothelium. Pretreatment of rabbits with a PAF inhibitor, BN52021, prevented most of the abnormal findings.

Changes in [3H]PAF binding and PAF concentrations in gerbil brain after bilateral common carotid artery occlusion: a quantitative autoradiographic study.

This study investigated the distribution of platelet activating factor (PAF) binding sites in the brain and their involvement in global ischemia in a model of bilateral common carotid occlusion in the gerbil. In sagittal sections of gerbil brain, labeling with [3H]PAF was mainly located in the cortex, hippocampus and cerebellum. The corpus striatum, the superior and inferior colliculi showed lower binding, while the thalamus was only weakly labeled. Scatchard analysis of the data obtained from displacement curves with unlabeled PAF revealed the presence of one or two populations of binding sites with different affinity constant values depending on the brain structures. When the gerbils were submitted to 10 min ischemia, similar autoradiography with [3H]PAF demonstrated a dramatic reduction of labeling in all brain structures, particularly in the hippocampus. Immunoreactive endogenous PAF concentrations in brain tissues showed a marked increase in ischemic animals: (8977.3 +/- 1113 pg/g wet weight) as compared to sham-operated control: (997.7 +/- 77 pg/g wet weight). Endogenous PAF levels returned to basal values following 30 min reperfusion. These results indicate that PAF may be involved in the early stages of brain ischemia in the gerbil and suggest that endogenous PAF produced during ischemia may contribute to the down-regulation of [3H]PAF binding sites in the brain.

Transfer and metabolism of platelet-activating factor by fetal membranes, amnion and chorio-decidua.

OBJECTIVE: To determine the metabolism of 3H-Platelet-activating factor (3H-PAF) during transfer through human fetal membranes. DESIGN: 3H-PAF was added to the fetal side of cultured intact fetal membranes, amnion and chorio-decidua obtained from three pregnancies ending in elective caesarean section. MAIN OUTCOME MEASURES: Radioactivity was measured on both sides of the tissue, and in the tissue itself. In some experiments, the metabolism of 3H-PAF was assessed by thin-layer chromatography. RESULTS: Very little 3H-PAF crossed the intact fetal membrane (< 2%) during 24 h of culture. Most of the 3H-PAF which accumulated in the membrane was converted to a range of metabolites in the chorio-decidua. CONCLUSIONS: These results suggest that PAF in amniotic fluid may not reach the decidua, and therefore is unlikely to be involved in the control of prostaglandin production from this tissue.

PAF-acether and the acute inflammatory reaction

PAF-aceter é um fosfolipídio que exerce um papel importante na reaçäo inflamatória aguda. No modelo experimental do edema da para de rato e pleurisia, a reaçäo induzida pelo PAF era acompanhada de hemoconcentraçäo, embora esses processos pareçam ser mediados por mecanismos distintos. Antagonistas específicos do PAF, como BN 52021 e WEB 2086, inibem 60% do edema induzido pelo PAF. Leucotrienos e mecanismos histaminérgicos envolvendo receptores H2 parecem participar no processo. A participaçäo do sistema adrenérgico e de metabólitos da ciclooxigenase é controversa. Infiltraçäo de leucócitos, observada após a injeçäo de PAF, e plaquetas näo säo necessárias para a formaçäo de edema. Auto-dessensibilizaçäo induzida pelo PAF pôde ser observada, sendo seletiva e dependente a interaçäo do PAF com receptores específicos em um processo de regulaçäo depressiva ("dow-regulation"). Nenhuma participaçäo deo PAF foi observada na reaçäo inflamatória induzida pela carregenina, contrariamente áquela provocada pelo zimosan, que é principalmente dependente do PAF-aceter

Comparison of three paf-acether receptor antagonist ginkgolides.

The aggregation of washed human platelets with paf-acether (paf) and [3H]paf binding were inhibited in a concentration- and time-dependent manner by three chemically defined ginkgolides (BN 52020, BN 52021, BN 52022) and their mixture, BN 52063 (molar ratio: 2:2:1). The IC50 values for aggregation correlated well with the IC50 values for [3H]paf binding (R-square 0.971). BN 52021, BN 52020 and BN 52063 (6 microM), as well as unlabelled paf (50 nM), displaced platelet-bound [3H]paf. Specific binding (total minus non-specific binding) in the presence of BN 52020 and BN 52063 was saturated but that observed in the presence of BN 52022 was not. However all ginkgolides shifted the dose-dependent paf-induced platelet aggregation curve rightwards. BN 52063 failed to modulate paf metabolism either in plasma or in the presence of platelets. The present results suggest that the ginkgolide mixture, BN 52063, acts at the paf binding level and the close correlation between ginkgolide effects on aggregation vs. paf binding illustrates the functional relevance of the putative paf platelet receptor.