HPLC Assay for FK 506 and Two Metabolites in Isolated Rat Hepatocytes and Rat Liver Microsomes

Despite the current use of a standard two-step enzyme immunoassay in the clinical monitoring of the immunosuppressant FK 506, the lack of specificity for the parent drug in this assay renders it unsuitable for drug metabolism studies. An HPLC assay has been developed for studying the metabolism of FK 506 in isolated hepatocytes and microsomal mixtures. This assay allows simultaneous measurement of the parent drug and two of its time dependent metabolites. Metabolism of this drug was studied in intact rat liver cells and rat liver microsomes. We have shown that the metabolites observed are products of phase 1 oxidation reactions. Correlation of the 6-testosterone hydroxylase activity with the FK 506 metabolite (Ml) initial formation rate is consistent with the belief that CYP 3A isozymes are involved in FK 506 metabolism in male rats.     

他可莫司血药浓度监测

目的：为临床肾移植患者他可莫司（FK506）的合理用药提供参考。方法：采用微粒子酶免分析法对206例肾移植患者进行血药浓度监测，并对其结果及疗效进行分析总结。结果：为206例肾移植患者监测了1732例次FK506血药浓度，其中有效血药浓度范围内的917例次，占52．9％；低于有效血药浓度的有649例次，占37．5％；高于有效血药浓度的有166例次，占9．6％。结论：为用药安全有效，应监测FK506血药浓度，实行个体化给药。     

肾移植后FK506的监测状况

目的:研究动态监测免疫抑制剂FK506的临床意义。方法:使用ELISA法进行全血浓度测定,监测42例使用FK506的患者在肾移植后第3天、第1周、第2周、第1个月～第4个月及第6个月的血药浓度。结果:服用FK506后测得第3天平均血药浓度为(15.3±2.2)ng/mL,第1周(16.4±2.8)ng/mL,第2周(16.1±2.5)ng/mL,第1个月(16.8±2.3)ng/mL,第2个月(11.3±1.7)ng/mL,第3个月(10.8±1.4)ng/mL,第4个月(6.8±0.9)ng/mL,第6个月(6.1±0.7)ng/mL。2例发生急性排斥反应的血药浓度明显低于正常平均浓度,3例发生肾中毒患者的血药浓度明显高于正常平均浓度,及时调整用药剂量,情况有所好转。结论:由于FK506个体差异大,治疗窗窄,极易造成急性排斥反应或肾中毒,因此,对于临床医生来说,要密切监测FK506的全血浓度,以便能够及时调整用药剂量,充分发挥免疫抑制作用,最大限度地减低毒副作用,取得肾移植的理想治疗效果,延长移植物的存活率。     

FK506 does not affect cardiac contractility and adrenergic response in vitro

FK506 (tacrolimus) is a new immunosuppressant being used in cardiac allograft transplantation. While cyclosporine A has been shown to exert an acute negative inotropic effect on isolated heart muscle preparations, little is known of the inotropic influence of FK506. The Ca²⁺ release channel of human skeletal muscle and cardiac muscle is associated with FK506 binding proteins (FKBP), FKBP12 and FKBP12.6, respectively. FKBPs can be dissociated by treatment with FK506. As a consequence of FK506 exposure, isolated skeletal muscle and cardiac muscle ryanodine receptors show altered gating characteristics. Therefore, we analyzed the direct inotropic effect of FK506 exposure to isolated, intact heart muscle preparations from the human and rabbits. Experiments were performed on isolated, electrically stimulated right atrial auricular muscle strips obtained from human myocardium during elective open heart surgery and on intact right ventricular trabeculae from rabbit hearts. The human preparations were exposed to concentrations of 8×10⁻⁹, 8×10⁻⁸ and 8×10⁻⁶ M FK506 followed by a cumulative dose–response curve with isoprenaline as a non-selective β-adrenoceptor agonist. Our data suggest that FK506 does not exert any positive or negative inotropic effect in either human or rabbit myocardium.     

The Rate and Extent of Oral Bioavailability versus the Rate and Extent of Oral Absorption: Clarification and Recommendation of Terminology

In the literature, the meanings of the terms oral absorption and oral bioavailability of drugs vary greatly. Absorption has been considered to take place at the mucosal membrane of the gastrointestinal (GI) tract. It has also been defined as the process from the site of drug administration to the site of measurement. In the latter definition, the extent of oral absorption depends on the extent of first-pass elimination in the gut wall and liver even though a drug may be completely absorbed from the GI tract. Moreover, these two terms have also been used interchangeably. Inconsistency in the definition of these two terms has led to varying interpretations of these terms among students, researchers and laymen, and such an inconsistency seems undesirable. Apparently because of these inconsistencies, improper correlations between the Caco-2 permeability or intestinal permeability and the oral bioavailability of drugs subject to extensive first-pass effect may have occurred. It is suggested that absorption be defined as movement of drug across the outer mucosal membranes of the GI tract, while bioavailability be defined as availability of drug to the general circulation or site of pharmacological actions. Since transit times (this may range from about 1 min to several hours) across enterocytes, liver, lungs, and the peripheral venous sampling tissue are virtually unknown for all drugs, this factor alone would favor the use of oral bioavailability rate rather than oral absorption rate in all routine studies.     

Detailed in vitro pharmacological analysis of FK506-induced neuroprotection

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.     

冷藏同种异体神经移植中应用FK506的最短有效时间

目的研究冷藏同种异体神经移植中FK506的最短有效时间。方法75只Winster大鼠分为A组,冷藏保存异体移植无免疫抑制;B组,自体原位移植;C组,冷藏保存异体移植应用FK506免疫抑制4周;D组,冷藏保存异体移植应用FK506免疫抑制6周;E组,冷藏保存异体移植应用FK506免疫抑制8周。各组分别于术后4周、6周和8周移植物行大体和光镜观察及轴突图像分析、比目鱼肌湿重比较、患肢坐骨神经电生理检查,于6周行移植神经电镜检查。结果4周时各组实验动物神经再生不完全,功能未恢复。6、8周时B、D、E组动物在功能学指标、电生理指标及光镜、电镜指标上均优于其它各组,而B、D、E组间在上述各指标上差异无显著性。结论1.0cm缺损的异体神经移植动物实验中,应用FK5062mg·kg-1·d-16周为最短有效疗程。     

以FK506结合蛋白52为靶点的高通量药物筛选模型的建立及应用#br#

目的 发现新的FKBP52抑制剂类药物,建立以人FKBP52为靶点的高通量药物筛选模型。方法 通过大肠埃希菌表达系统重组表达FKBP52蛋白,并进行纯化。基于FKBP52的肽基-脯氨酰顺反异构酶(peptidyl-prolyl cis-trans isomerase, PPIase)的活性特性,建立FKBP52高通量药物筛选体系。对本公司化合物库中的31558个放线菌及真菌次级代谢产物粗提物进行筛选。结果 成功构建重组表达菌株BL21(rosetta)/pET-30a/FKBP52。纯化后的重组表达人FKBP52蛋白纯度大于90%,并具有很好的PPIase活性。阳性药FK506能剂量依赖地抑制FKBP52活性,IC50为12.035ng/mL。筛选得到121个抑制率大于80%的样品,其中11株放线菌的次级代谢产物中含FK506,6株放线菌的次级代谢产物中含FK520,7株放线菌的次级代谢产物中含雷帕霉素。对随机抽取的100块96孔板筛选数据进行统计,模型的信号/本底比平均值为2.35±0.26,Z'因子平均值为0.78±0.1。结论 本研究成功建立了人FKBP52抑制剂高通量药物筛选模型,该方法具有快捷、灵敏度高、稳定性好的特点,不仅可以快速进行新FKBP52抑制剂的筛选,还可以实现对FKBP52已知抑制剂如FK506、FK520和雷帕霉素等产生菌的定向筛选。     

大孔吸附树脂吸附分离FK506的工艺研究

目的：研究并优化FK506提取工艺。方法：通过几种大孔吸附树脂的吸附容量及分离能力比较实验,筛选到一种吸附容量高又有分离能力的树脂;通过吸附等温线及柱分离实验,确定最佳吸附浓度、上样及洗脱流速等工艺参数。结果：FK506提取液通过HZ816树脂吸附分离掉杂质,中间体湿粉含量达到60.1%（折干含量81.3%）,吸附分离总收率达87.1%。结论：HZ816树脂可有效吸附分离FK506。     

Oral Absorption of Poorly Water-Soluble Drugs: Computer Simulation of Fraction Absorbed in Humans from a Miniscale Dissolution Test

Purpose The purpose of this study was to develop a new system for computer simulation to predict fraction absorbed (F_a) of Biopharmaceutical Classification System (BCS) class II (low solubility–high permeability) drugs after oral administration to humans, from a miniscale dissolution test. Methods Human oral absorption of 12 lipophilic drugs was simulated theoretically by using the dissolution and permeation parameters of the drugs. A miniscale dissolution test and a solubility study were carried out in a conventional buffer and a biorelevant medium (pH 6.5). A dissolution parameter, which can simulate in vivo dissolution, was obtained from the in vitro dissolution curve. Human intestinal permeability was estimated assuming that the permeation was limited by diffusion through the unstirred water layer. The F_a in humans was predicted and then compared with clinical data. Results The dissolution and solubility of most model drugs were faster and higher in a biorelevant medium than in a conventional buffer. The simulated absorption was limited by the drug dissolution rate and/or solubility. Predicted F_a was significantly correlated with clinical data (correlation coefficient r² = 0.82, p < 0.001) when the dissolution profiles in biorelevant medium were used for the simulation. Conclusions This new system quantitatively simulated human absorption and would be beneficial for the prediction of human F_a values for BCS class II drugs.     

Evaluation of Drug Absorption After Intrapulmonary Administration Using Xenopus Pulmonary Membranes: Correlation with In Vivo Pulmonary Absorption Studies in Rats

Pharmaceutical Research -     

Effect of FK506 (tacrolimus) in animal models of inflammation

Inflammation is the response of living tissue to damage. Cytokines play an important role in inflammatory processes. FK506 (tacrolimus), an immunosuppressant, is known to reduce the activation of microglia in vitro and affect the expression of various cytokines like interleukin-1, interleukin-6 and tumor necrosis factor. With this background the present study was designed to explore the effect of FK506 in animal models of acute inflammation and experimental pleurisy. Acute inflammation in rats was induced by intraplantar injection of carrageenan (1%, w/v). Experimental pleurisy was induced in rats by intrapleural injection of carrageenan (2%, w/v). Pretreatment with FK506 (0.5–3 mg/kg p.o.) significantly and dose-dependently reduced carrageenan-induced increase in paw volume, as well as carrageenan-induced inflammatory nociception. FK506 (1 and 3 mg/kg p.o.) inhibits exudate formation and migration of polymorhonuclear leukocytes and monocytes in carrageenan-induced experimental pleurisy. The myeloperoxidase enzyme level was significantly increased in carrageenan-treated animals, which was significantly reversed by FK506 treatment. The results of the present study suggest the potential anti-inflammatory properties of FK506 against carrageenan-induced acute inflammation and experimental pleurisy.     

The effects of FK506, a specific calcineurin inhibitor, on methamphetamine-induced behavioral change and its sensitization in rats

RATIONALE: FK506 inhibits calcineurin activity, resulting in the inhibition of calcium-dependent intracellular processes. Recent studies have suggested that intracellular calcium is likely to be involved in methamphetamine (MAP)-induced locomotor activity and stereotyped behavior, and in the development of sensitization to MAP. OBJECTIVES: We investigated the effects of FK506 on MAP-induced behavioral changes and the development of sensitization in rats. METHODS: In experiment 1, animals were administered IP 2 mg/kg FK506 or vehicle followed 10 min later by MAP (1, 2, 4 and 8 mg/kg, IP). Another set of animals were administered FK506 (0.1, 2, 5 and 10 mg/kg) followed by 2 mg/kg MAP. Locomotor activity and stereotyped behavior were assessed. In experiment 2, animals received repeated IP injections of 2 mg/kg MAP pretreated with 2 mg/kg FK506 or vehicle for 5 consecutive days. One week later, rats were challenged with 1 mg/kg MAP. RESULTS: Pretreatment with 2 mg/kg FK506 caused a rightward shift of the inverted U-shaped response curve of the locomotor activity induced by 1-8 mg/kg MAP. The same pretreatment significantly attenuated augmentation of the MAP-induced stereotyped behavior. FK506 at doses of 0.1-10 mg/kg dose-dependently inhibited the behavioral response induced by 2 mg/kg MAP. Coadministration of 2 mg/kg FK506 with 2 mg/kg MAP for 5 consecutive days resulted in significant suppression of the behavioral response to challenge with 1 mg/kg MAP. CONCLUSIONS: These results suggest that calcineurin plays an important role in MAP-induced behavioral changes and sensitization, especially the latter.     

Effect of telaprevir on the metabolism and hepatic uptake of tacrolimus (FK506)

Telaprevir, a chronic hepatitis C virus (HCV) protease inhibitor, is known to be a cytochrome P450 (CYP) 3A4/5 substrate and inhibitor. In the present study, the in vitro inhibitory effect of telaprevir on the metabolism of tacrolimus in human liver microsomes was investigated using 13‐O‐demethyltacrolimus (M‐I) as a monitor metabolite. Telaprevir inhibited M‐I formation in a time‐dependent fashion with rate of enzyme inactivation (k_inact) and concentration to reach 50% of k_inact (K_I) values of 0.113 min^‐1 and 0.511 µm , respectively. Using the inhibition parameters generated, in vitro–in vivo extrapolations were performed to evaluate the clinical relevance of the effect of telaprevir on the area under the curve versus time (AUC) of tacrolimus. When 750 mg of telaprevir was administered orally, the intestinal wall availability (F_g) of tacrolimus was estimated to be increased 3.7‐ to 7.0‐fold. The hepatic intrinsic clearance (CL_int) of tacrolimus was also estimated to be decreased 4.4‐ to 19‐fold. These results suggest that the increased AUC of tacrolimus in the presence of telaprevir was caused by intestinal and hepatic metabolism inhibition. In addition, the inhibitory effect of telaprevir on the hepatic uptake of tacrolimus was also examined using human cryopreserved hepatocytes. However, no significant inhibitory effect was noted, suggesting that the effect of telaprevir on hepatic transporters did not contribute to the increase in tacrolimus exposure. Copyright © 2014 John Wiley & Sons, Ltd.     

ELISA法监测肾移植患者FK506切换组血药浓度

目的　通过监测肾移植后FK5 0 6切换组患者全血谷浓度 ,观察并建立切换组患者治疗窗 ,为临床提供参考。方法　用ELISA法测定FK5 0 6全血谷浓度 ,对 4 5例患者的 4 11例次监测结果进行分析。结果　FK5 0 6全血谷浓度平均值随切换时间于移植后时间延长而逐渐下降。切换于肾移植后 1个月内、第 2～ 3个月、第 4～ 6个月和 >6个月时 ,用ELISA法监测FK5 0 6全血谷浓度的合理治疗窗范围应分别为 :8～ 14ng/ml,7～12ng/ml,6～ 10ng/ml和 4～ 8ng/ml。结论　常规监测FK5 0 6全血谷浓度 ,按推荐治疗窗调整给药方案 ,可获得满意的免疫抑制治疗效果。     

Rate-Limiting Steps of Oral Absorption for Poorly Water-Soluble Drugs in Dogs; Prediction from a Miniscale Dissolution Test and a Physiologically-Based Computer Simulation

PURPOSE: Nonlinear oral absorption due to poor solubility often impedes drug development. The purpose of this study was to elucidate the rate-limiting process in oral absorption of Biopharmaceutical Classification System (BCS) class II (low solubility-high permeability) drugs in order to predict nonlinear absorption of dose caused by solubility-limited absorption. METHODS: Oral absorption of danazol, griseofulvin, and aprepitant was predicted from a miniscale dissolution test and a physiologically-based model. The effect of particle size reduction and dose increase on absorption was investigated in vitro and in vivo to clarify the rate-limiting steps of dissolution-rate-limited and solubility-limited absorption. RESULTS: The rate-limiting steps of oral absorption were simulated and increase in the dissolution rate and administration dose showed a shift from dissolution rate-limited to solubility-limited absorption. In the study in dogs, particle size reduction improved the oral absorption of large particle drugs but had little effect on small particle drugs. Dose nonlinearity was observed with small particles at a high dose. Our model quantitatively predicted results observed in vivo, including but not exclusively, dissolution-rate-limited and solubility-limited absorption. CONCLUSION: The present study provides a powerful tool to predict dose nonlinearity and will aid in the success of BCS class II drug development.     

Kinetic Analysis of Molecular Interconversion of Immunosuppressant FK506 by High-Performance Liquid Chromatography

High-performance liquid chromatography of FK506, a macrolide immunosuppressant, was performed on a reversed-phase column. The peak was broad with the column kept at room temperature, which was accounted for by slow interconversion between the two forms of FK506. With the use of a heated column, a sharp peak was observed because of the rapid interconversion at high temperature. When the column was cooled to 0°C, two sharp peaks were observed because essentially no interconversion occurred at 0°C during elution. Analysis of the chromatograms obtained at various eluant flow rates indicated that the conversion of the two forms follows first-order kinetics, and the apparent activation energies for the conversions were calculated. The interconvertibility between two molecular forms may be related to the immunosuppressive activity.     

肾移植患者血液中他克莫司稳态谷浓度的影响因素分析

目的:探讨肾移植患者口服他克莫司(FK506)稳态谷浓度与体内病理生理因素以及联合用药的相关性,为临床合理使用FK506提供参考。方法:采用回顾性调查方法在空军总医院收集68例次因肾移植术后口服FK506的患者病历资料,记录患者性别、年龄、体重、单位体重剂量、稳态谷浓度、血红蛋白及相应生化检验值。应用SAS软件(6.04版)对数据进行多元线性回归分析。结果:FK506稳态谷浓度与总胆红素(TBIL)及血红蛋白(HB)具有显著正相关性(P<0.05)。结论:临床使用FK506时应重点考虑总胆红素及血红蛋白对FK506血药浓度的影响,定期监测其血药浓度。