The effect of divalent cations on the membrane properties and pharmacokinetics in rat of the lipid A analogue E5531

To obtain information on the effects of Ca2+ on the membrane properties of the lipid A analogue E5531, we have determined the aggregate size, zeta potential, membrane fluidity, micropolarity and permeability of the E5531 membrane as a function of Ca2+ levels. Within the molar ratios of [Ca2+]/[E5531] = 1 and 3, Ca2+ increased the zeta potential of the E5531 membrane but had no effect on aggregate size (approximately 20 nm). Within the above ratios, Ca2+ decreased the membrane fluidity, as measured by micropolarity of E5531 and increased the phase transition temperature. The pharmacokinetics in rats for these samples with different membrane fluidity, prepared by changing the pre-dose formulation concentration of Ca2+, was determined and a correlation between membrane fluidity and pharmacokinetics was clearly observed. It thus appears that Ca2+ effects the membrane fluidity of E5531 as well as its pharmacokinetics in rats.     

E5531, a synthetic non-toxic lipid A derivative blocks the immunobiological activities of lipopolysaccharide.

1. The major pathological responses to Gram-negative bacterial sepsis are triggered by endotoxin or lipopolysaccharide. As endotoxin is shed from the bacterial outer membrane, it induces immunological responses that lead to release of a variety of cytokines and other cellular mediators. As part of a program aimed at developing a therapeutic agent for septic shock, we have developed E5531, a novel synthetic lipopolysaccharide antagonist. 2. As measured by release by tumour necrosis factor-alpha, human monocytes or whole blood can be activated by lipopolysaccharide, lipid A, and lipoteichoic acid (from Gram-positive bacteria). E5531 potently antagonizes activation by all these agents while itself being devoid of agonistic activity. 3. The inhibitory activity of E5531 was dependent on time of addition. When 10 nM E5531 was added simultaneously with lipopolysaccharide or 1 - 3 h before addition of lipopolysaccharide, production of tumour necrosis factor-alpha was inhibited by more than 98%. The addition of E5531 1 h after lipopolysaccharide reduced the efficacy of E5531 by 47%. 4. Antagonistic activity of E5531 was specific for lipopolysaccharide as it was ineffective at inhibiting interferon-gamma mediated NO release of RAW 264.7 cells, phorbor 12-myristate 13-acetate stimulated superoxide anion production in human neutrophils, concanavalin A stimulated mitogenic activity in murine thymocytes and tumor necrosis factor-alpha induced E-selectin expression in human umbilical vein endothelial cells. 5. E5531 as well as MY4, an anti-CD14 antibody, inhibited radiolabelled lipopolysaccharide binding in human monocytes. 6. These results support our contention that E5531 is a potent antagonist of lipopolysaccharide-induced release of tumour necrosis factor-alpha and other cellular mediators and may be an effective therapeutic agent for human septic shock due to Gram-negative bacteria.     

The effect of the lipid A analog, E5531 on fever induced by endotoxin from Escherichia coli.

E5531 is a specific lipid A antagonist and is expected to be a drug for the treatment of septic shock. The LAL (limulus amebocyte lysate) activity and pyrogenicity of E5531 were determined. The LAL activity of E5531 is large and confirmed that E5531 had a high affinity to the lipopolysaccharide receptor. The pyrogenic activity of E5531 is weak and the pyrogenic profile of E5531 is different from that of USP (United States Pharmacopoeia) reference standard endotoxin (ETX). E5531 suppressed the pyrogenicity of ETX in rabbits, indicating that E5531 is expected to be useful for the treatment of fever induced by ETX.     

Development of an injectable formulation for the novel lipid A analog E5531 using a 'pH-jump method'

In order to design an injectable formulation of E5531, a novel synthetic disaccharide analog of novel lipid A, for the treatment of septic shock, a 'pH-jump method' was developed. In this method, E5531 was dispersed in 0.003 mol/l NaOH (pH 11.0, above pK(a2)) at 50 degrees C (above phase transition temperature) and then mixed with a buffer to neutralize the pH to 7.3. E5531 was dispersed as particles, and the size was approximately 20 nm. The structure of the particles was vesicular. After dispersal, the solution was sterilized using a filter, filled aseptically into vials, and lyophilized. The size of the particles did not change before and after lyophilization. The relationship between the physicochemical properties of the particles and the pharmacokinetics in rats after intravenous administration was investigated. The membrane fluidity of the particles was affected by the dispersal methods, the dispersal time in 0.003 mol/l NaOH in the pH-jump method, and the addition of Ca(2+) to the solution. The membrane fluidity was correlated with the pharmacokinetics in rats.     

Disposition of a synthetic analogue of lipid A (E5564) in rats

1. E5564, a lipid A analogue that potently antagonises lipopolysaccharide, is being developed to treat sepsis caused by Gram-negative bacterial infections. The pharmacokinetic profile of E5564 is independent of dose between 0.1 and 1?mg kg ? 1. The distribution volume of E5564 is slightly larger than the total plasma volume, and the terminal elimination half-life is about 5?h. 2. Following 14 C-E5564 administration (0.5?mg kg ? 1), radioactivity rapidly accumulates in the liver and spleen. The half-life of E5564 in the liver is 5.1?h, which is similar to that in the plasma. At 48 weeks after dosing, 35.27% of the administered radioactivity was still present in the liver. Cumulative urinary and faecal excretion of radioactivity for up to 48 weeks after administration were 3.86 and 67.17% of the dose, respectively. 3. The results of mass spectroscopy and nuclear magnetic resonance analysis reveal that the main hepatic metabolite is di-dephosphorylated E5564. The half-life of di-dephosphorylated E5564 in the liver is 87.4 days, which is similar to that for the hepatic radioactivity. 4. The results indicate that E5564 is rapidly taken up by the liver, is metabolized via dephosphorylation pathways to form dephosphorylated E5564 and is mainly excreted in the faeces.     

Disposition of a synthetic analogue of lipid A (E5564) in rats

1. E5564, a lipid A analogue that potently antagonises lipopolysaccharide, is being developed to treat sepsis caused by Gram-negative bacterial infections. The pharmacokinetic profile of E5564 is independent of dose between 0.1 and 1 mg kg(-1). The distribution volume of E5564 is slightly larger than the total plasma volume, and the terminal elimination half-life is about 5 h. 2. Following (14)C-E5564 administration (0.5 mg kg(-1)), radioactivity rapidly accumulates in the liver and spleen. The half-life of E5564 in the liver is 5.1 h, which is similar to that in the plasma. At 48 weeks after dosing, 35.27% of the administered radioactivity was still present in the liver. Cumulative urinary and faecal excretion of radioactivity for up to 48 weeks after administration were 3.86 and 67.17% of the dose, respectively. 3. The results of mass spectroscopy and nuclear magnetic resonance analysis reveal that the main hepatic metabolite is di-dephosphorylated E5564. The half-life of di-dephosphorylated E5564 in the liver is 87.4 days, which is similar to that for the hepatic radioactivity. 4. The results indicate that E5564 is rapidly taken up by the liver, is metabolized via dephosphorylation pathways to form dephosphorylated E5564 and is mainly excreted in the faeces.     

Cryoprotective effects of saccharides on the freeze-drying of sonicated vesicles of the novel lipid A analog, E5531

A sonicated dispersion of the novel lipid A analog, E5531, was feeze-dried in the presence of various additives such as saccharides and polyalcohols, and their cryoprotective effects were investigated. Fusion of the vesicles was examined by measuring fluorescence energy transfer and size distribution. Cryoprotective ability differed among the addtive species. The addition of polyalcohols led to considerable fusion. Although monosaccharides, similar to disaccharides, completely prevented the fusion of the vesicles during lyophilization, they showed far less ability to retain the entrapped calcein in the vesicles compared to disaccharides. Differential scanning calorimetry heating profiles of vesicles that had been lyophilized with various additives were obtained. Disaccharides and monosaccharides again resulted in markedly different thermal properties of the vesicles. This variety in cryoprotective ability of saccharide species can be attributed to differences in their interaction with the E5531 head group.     

重组人甲状旁腺素类似物PTH（1-34）在大鼠体内药动学研究

目的：研究大鼠scPTH（1-34）的药动学特征。方法：用125I标记PTH（1-34）示踪法对大鼠scPTH（1-34）后的血清药物浓度进行了测定,并用3P97程序拟和分析并计算药动学参数,采用超滤离心的方法进行药物与血浆蛋白结合率的研究。结果：大鼠scPTH（1-34）10、20和40μg/kg后,药物消除符合一房室模型。平均t1/2ke为（0.92±0.04）h;平均CL为（0.56±0.05）L/（kg.h）;Cmax和AUC0-8h均与剂量呈线性正相关,不同剂量药物与血浆蛋白平均结合率为13.4%。结论：大鼠scPTH（1-34）后,药物消除符合线性动力学特征。     

A lipid microsphere vehicle for vinorelbine: Stability, safety and pharmacokinetics

A lipid microsphere vehicle for vinorelbine (VRL) was designed to reduce the severe venous irritation caused by the aqueous intravenous formulation of VRL. Lipid microspheres (LMs) were prepared by high pressure homogenization. The physical stability was monitored by the appearance, particle size and zeta potential changes while the chemical stability was achieved by using effective antioxidants and monitored by long-term investigations. Safety tests were performed by testing rabbit ear vein irritation and a guinea pig hypersensitivity reaction. A pharmacokinetic study was performed by determining the drug levels in plasma up to 24h after intravenous administration of VRL-loaded LMs and conventional VRL aqueous injection separately. The VRL-loaded LMs had a particle size of 180.5+/-35.2nm with a 90% cumulative distribution less than 244.1nm, while the drug entrapment efficiency was 96.8%, and it remained stable for 12 months at 6+/-2 degrees C. The VRL-loaded LMs were less irritating and toxic than the conventional VRL aqueous injection. The pharmacokinetic profiles were similar and the values of AUC(0-t) were very close for the two formulations. A stable and easily mass-produced VRL-loaded LM preparation has been developed. It produces less venous irritation and is less toxic but has similar pharmacokinetics in vivo to the VRL aqueous injection currently commercially available.     

Tolerance and pharmacokinetics of A515U,an acyclovir analogue,in healthy volunteers

Summary A515U (6-deoxyacyclovir) is an analogue of acyclovir devoid of antiviral activity in vitro but which is well absorbed and undergoes conversion to acyclovir after oral administration to rats. The tolerance and pharmacokinetics of various doses of A515U have been studied in 8 healthy volunteers. Single oral doses of 25, 50, 100, 200 and 400 mg A515U and 400 mg acyclovir for comparison were administered to the volunteers at weekly intervals. Concentrations of the parent drug and acyclovir were determined in plasma and urine. The prodrug was well tolerated and did not cause adverse reactions or changes in haematological or biochemical variables. It was well absorbed and conversion to acyclovir was rapid and extensive at all doses. Plasma concentrations of acyclovir achieved with 50 mg A515U orally were comparable to and less variable than those produced by 400 mg acyclovir. A515U was rapidly cleared with a short plasma elimination half life of approximately 0.5 h. The attainment of high plasma concentrations of acyclovir by oral administration of a prodrug may represent an important advance in antiviral chemotherapy.     

Cryoprotective Effects of Saccharides on the Freeze-Drying of Sonicated Vesicles of the Novel Lipid A Analog,E5531

A sonicated dispersion of the novel lipid A analog, E5531, was feeze-dried in the presence of various additives such as saccharides and polyalcohols, and their cryoprotective effects were investigated. Fusion of the vesicles was examined by measuring fluorescence energy transfer and size distribution. Cryoprotective ability differed among the addtive species. The addition of polyalcohols led to considerable fusion. Although monosaccharides, similar to disaccharides, completely prevented the fusion of the vesicles during lyophilization, they showed far less ability to retain the entrapped calcein in the vesicles compared to disaccharides. Differential scanning calorimetry heating profiles of vesicles that had been lyophilized with various additives were obtained. Disaccharides and monosaccharides again resulted in markedly different thermal properties of the vesicles. This variety in cryoprotective ability of saccharide species can be attributed to differences in their interaction with the E5531 head group.     

Safety,pharmacokinetics, and pharmacodynamics of E5564, a lipid A antagonist,during an ascending single-dose clinical study

E5564, a structural analog of the lipid A portion of lipopolysaccharide (LPS), is a potent antagonist of the biochemical and physiologic effects of LPS in several in vitro and in vivo models and is currently under clinical development as a possible therapeutic for the treatment of sepsis and septic shock. The objectives of this study were to (1) assess the safety and tolerability of E5564 following a 30-minute intravenous (i.v.) infusion, (2) evaluate the pharmacokinetic profile of E5564, and (3) measure the ability of E5564 to block LPS stimulation ex vivo in blood taken from subjects up to 8 hours after ending the infusion. Healthy male volunteers (n = 7/dose group) were randomly assigned to each of four dose levels (350, 1000, 2000, or 3500 micrograms). Within each dose group, 5 subjects received drug and 2 received placebo. E5564 or matching placebo was administered by a 30-minute infusion, and blood samples were collected at predetermined time points. All doses of E5564 were demonstrated to be safe and well tolerated. E5564 plasma concentrations were determined using a validated LC/MS/MS method. The Cmax and AUC of E5564 increased in a dose-proportional manner. E5564 pharma-cokinetics were characterized by a slow clearance (0.67-0.95 mL/h/kg), a small volume of distribution (41-54 mL/kg), and a relatively long elimination half-life (42-51 h). As measured in the ex vivo assay, E5564 inhibited LPS-induced tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner, and at the higher doses (2 and 3.5 mg), antagonistic activity was measurable up to 8 hours postinfusion. E5564 lacked LPS-like agonist activity at doses up to 3.5 mg. Taken together, we believe that E5564 is a safe, potent antagonist of LPS in blood and will likely benefit patients in the treatment of LPS-related diseases.     

Effects of prostaglandin E1 analogue,misoprostol, on the development of adjuvant arthritis in rats

Prostaglandins (PG) E , E and the PGE analogue, misoprostol, have been shown to inhibit T-cell functions and the production by activated monocytes or macrophages of interleukin-1, indicating that these PGs may have potential anti-arthritic activity by suppressing T-cell and monocyte activity. In view of this the potential anti-arthritic effects of the long half-life PG, misoprostol (MPL), were examined in adjuvant arthritic rats under prophylactic and therapeutic treatment regimes. Transcutaneous or subcutaneous MPL given at 200 Μg/kg/day but not at 50 or 5 Μg/kg/day when given 0 to +5 or 0 to + 14 days post-induction inhibited the development of the disease whereas the orally administered drug was without effects. MPL given transcutaneously with oral indomethacin (1 or 2 mg/kg/day) on days +17 to + 30 post-induction produced greater anti-inflammatory effects than with this NSAID alone. MPL given orally in combination with this NSAID did not enhance the anti-inflammatory effects of the latter. MPL 200 Μg/kg given transcutaneously exhibited anti-ulcer activity against indomethacin (30 mg/kg p.o.), naproxen (10 mg/kg i.p.) or piroxicam (5 mg/kg i.p.) induced gastric damage in arthritic rats and this was comparable with that from 100 Μg/kg MPL given orally. These results show that MPL has both unique anti-arthritic effects only when given transcutaneously or parenterally as well as anti-ulcer activity.     

酮洛芬异丙酯脂质微球的镇痛作用及大鼠体内药动学研究

目的:研究酮洛芬异丙酯脂质微球的镇痛作用及其在大鼠体内的药动学。方法:采用小鼠热板法考察酮洛芬异丙酯脂质微球的镇痛作用。大鼠静脉给药后,分别于0,0．083,0．25,0．5,1,1．5,2,4,6,8,12和24 h取血。以高效液相色谱法测定血浆中的酮洛芬浓度。结果:酮洛芬异丙酯脂质微球(6．5,13．0,26．0 mg·kg-1)可剂量依赖性延长小鼠热板痛阈时间,且效果好于等剂量酮洛芬溶液剂组。酮洛芬异丙酯脂质微球及酮洛芬溶液剂静脉注射后的消除半衰期(t1/2)分别为(8．40±1．15)和(5．91±1．27)h,清除率(CL)分别为(0．062±0．008)和(0．030±0．008)L·h-1,稳态表观分布容积(Vss)分别为(0．539±0．299)和(0．219±0．097)L。结论:酮洛芬异丙酯脂质微球较酮洛芬溶液剂镇痛作用增强,静脉注射后在体内消除较慢。     

淫羊藿苷固体脂质纳米粒的大鼠体内药动学研究

目的:考察淫羊藿苷固体脂质纳米粒(ICA-SLN)在大鼠体内的药动学行为,以及利用固体脂质纳米粒技术提高ICA口服生物利用度的可行性。方法:对2组大鼠分别灌胃ICA混悬液和ICA-SLN混悬液后,采用HPLC-MS/MS法测定大鼠体内ICA的血药浓度,比较ICA混悬液和ICA-SLN混悬液在大鼠体内的吸收情况。结果:ICA在体内的药-时曲线呈现双峰,tmax为1 h,t1/2为3 h。载药纳米粒组与对照组的AUC0-∞分别为(233.6±71.2)ng.h.mL-1和(107.4±15.7)ng.h.mL-(1P<0.05)。结论:与单纯口服ICA相比较,ICA-SLN在大鼠体内的生物利用度更高。     

Inhibition of lipid peroxidation by IRFI 042, a vitamin E analogue, decreases monensin cardiotoxicity in chicks

Monensin, a well-known ionophore antibiotic, may cause severe damage in myocardial cells. We investigated whether IRFI 042, a new analogue of vitamin E, may block lipid peroxidation in myocardial cells and in turn protect against monensin toxicity. Monensin toxicity was induced by repeated daily administration of the ionophore antibiotic (150 mg/kg/day for 7 days). Sham animals received by oral gavages only a saline solution and were used as controls. All animals were randomized to receive concomitantly by oral gavages IRFI 042 (20 mg/kg) or its vehicle. The experiment lasted 8 days. Survival rate, heart lipid peroxidation, studied by means of thiobarbituric acid-reactive substances (TBARs) levels, cardiac expression of endothelial nitric oxide (e-NOS) and histological analysis of the heart were performed. Monensin administration caused a decrease in survival rate. Mortality appeared following the second monensin injection and at day 7 caused a survival rate of 20%. Thereafter, no further mortality was observed. IRFI 042 administration improved survival rate. Injection of the ionophore antibiotic resulted in a marked cardiac lipid peroxidation and in a significant reduction in cardiac e-NOS message and protein expression. IRFI 042 decreased heart TBARs levels (Monensin + vehicle = 6.5 +/- 0.8 nmol/mg; Monensin + IRFI 042 = 3.2 +/- 1.1 nmol/mg; P < 0.001) and increased e-NOS message and protein expression. Histological analysis showed that IRFI 042 improved myocardial cells damage and enhanced the depressed e-NOS expression in chick heart samples following monensin administration. Our data suggest that IRFI 042 is a promising drug to reduce monensin cardio-toxicity in chicks.     

Pharmacodynamics and pharmacokinetics of the basic triamterene analogue dimethylaminohydroxypropoxytriamterene

The diuretic, natriuretic and potassium retaining effects of dimethylaminohydroxypropoxytriamterene (RPH 2823), a basic triamterene derivative, were studied in male Wistar rats. Compared to triamterene (TA) RPH 2823 has a pronounced effect on urine volume and on the excretion of sodium; in addition it possesses a higher antikaliuretic potency than TA. In combination with furosemide, 2.5 mumol/kg RPH 2823 can avoid the kaliuresis of 25 mumol/kg furosemide. The pharmacokinetics of RPH 2823 after i.v. application of 1 mg/kg and 5 mg/kg were studied in female rats. The substance is not metabolized. RPH 2823 has a terminal elimination half-life of 3 h. About 47% of the given dose are excreted unchanged with urine. Furthermore, the volume of distribution VD beta and the total body clearance were calculated.     

Baicalin-loaded PEGylated lipid nanoparticles: characterization,pharmacokinetics, and protective effects on acute myocardial ischemia in rats

Context: Baicalin has many pharmacological activities, including protective function against myocardial ischemia by antioxidant effects and free radical scavenging activity. However, its rapid elimination half-life in plasma and poor water solubility limits its clinical efficacy.

Objective: Novel baicalin-loaded PEGylated nanostructured lipid carriers (BN-PEG-NLC) were developed to improve bioavailability of BN, to prolong retention time in vivo and to enhance its protective effect.

Methods: In this study, BN-PEG-NLC were prepared by the emulsion-evaporation and low temperature-solidification method using a mixture of glycerol monostearate and polyethylene glycol monostearate as solid lipids, and oleic acid as the liquid lipid. The physicochemical properties of NLC were characterized. The pharmacokinetic and pharmacodynamic behaviors of BN-PEG-NLC or BN-NLC were evaluated in acute MI rats.

Results and discussion: The particle size, zeta potential, and entrapment efficiency for BN-PEG-NLC were observed as 83.9?nm, ?32.1?mV, and 83.5%, respectively. The release profiles of BN from both BN-PEG-NLC and BN-NLC were fitted to the Ritger–Peppas modal, which presented burst release initially and prolonged release afterwards. Pharmacokinetics results indicated that BN-PEG-NLC exhibited a 7.2-fold increase in AUC in comparison to BN solution, while a 3-fold increase in comparison to BN-NLC. Biodistribution results revealed that BN-PEG-NLC exhibited higher heart drug concentration compared with BN-NLC as well as BN solution. In the present study, BN-PEG-NLC significantly ameliorated infarct size.

Conclusion: The results of the present study imply that PEG-NLC could be the biocompatible carriers for heart-targeted drug delivery to improve myocardial ischemia.     

Preclinical evaluation of the pharmacokinetics, brain uptake and metabolism of E121, an antiepileptic enaminone ester, in rats

The present study describes the brain uptake, pharmacokinetics and metabolism of an anticonvulsant enaminone ester E121, which belongs to a new and active series of compounds with potential in vivo anticonvulsant activity in rodent models, in rats. A single dose of E121 was administered i.p. to male Sprague Dawley rats at 10 mg E121/kg body weight. Cohorts of animals (n=3) were killed at varying times over 0-24 h to collect plasma and brain samples. Urinary excretion of E121 was studied in a separate group of five rats at the same dose. A validated HPLC method was used to quantify E121 and its metabolites in plasma, brain and urine. LC-MS/MS was used to characterize the metabolites. The plasma and brain Cmax of 11.0+/-3.0 mg/l and 10.4+/-1.4 mg/kg, respectively, were observed for E121 at 15 min post dose and they declined in a mono-exponential fashion. The plasma Cl/F and t1/2 were 0.57 l/h/kg and 0.75 h, respectively. The brain uptake ratio of E121 was 0.9. Mass spectral analysis of urine showed two major metabolites (m/z 280) and one minor metabolite (m/z 236) that were consistent with initial hydrolysis of the compound to the acid followed by further decarboxylation and appears to be the major route of elimination of E121. The rapid and moderate brain uptake of E121 correlates well with its potential anticonvulsant activity (ED50 3.0 mg/kg p.o. in rats). The brain uptake, pharmacokinetic and metabolic profile of E121 supports the need to further evaluate this compound for its potential as an antiepileptic.     

石杉碱甲,E2020和他克林对大鼠胆碱酯酶的抑制作用

AIM: To compare the anticholinesterase effects of huperzine A (Hup A), E2020, and tacrine in rats. METHODS: Spectrophotometry was used to determine AChE activity in brain and BuChE activity in serum. RESULTS: Following intragastric gavage, Hup A, E2020, and tacrine all produced dose-dependent inhibitions of brain AChE. Oral Hup A exhibited a higher inhibition than E2020 and tacrine. Tacrine was more effective in inhibiting serum BuChE correlated with severe peripheral adverse effects. The BuChE activity was less affected by Hup A and E2020. After a single oral dose of Hup A, a relatively steady state of AChE inhibition produced, which was longer than that after E2020 and tacrine. No change in the cholinesterase inhibition was seen for the 3 drugs following repeated i.g. medications. CONCLUSION: Hup A i.g. exhibited a higher efficacy, a longer duration of action, and a more selective inhibition on AChE than E2020 and tacrine.