Conversion of spironolactone to canrenone and disposition kinetics of spironolactone and canrenoate-potassium in rats

Summary The metabolic conversion of spironolactone (I) to canrenone (II) was investigated using a chemical model reaction by treatment of spironolactone in aqueous medium at pH 13. The dethioacetylation proceeds in two steps, i.e., a fast hydrolysis to the 7-thiol function present in congeners VI and VI₀ (K ₁=0.42 min^–1), and a rate limiting step by elimination of H₂S yielding the 4,6-dien-3-one function present in canrenone (II) and canrenoate (III) (K ₂=0.13 h^–1). The metabolic conversion of I to II was evaluated by intravenously injecting equimolar amounts of I, the 7-thiol VI, II and III. Following injection of I, the only fluorigenic metabolites detectable were II, III, and an intermediate polar metabolite, which was identified by tlc and mass spectrometry as the 7-thiol-17-hydroxycarboxylic acid VI₀. The plasma t _1/2 of I was 4 and 5 min, respectively, in two rats. The t _1/2 of VI₀ amounted to 8 and 12 min, respectively, and roughly corresponded to the formation rate canrenone (II). Injection of the 7-thiol VI resulted in plasma concentrations of VI₀, II and III similar to those obtained from injection of I. It can be concluded that hydrolysis of the 7-thioacetyl I to the 7-thiol VI is a very rapid metabolic step, that the -lactone ring is in a rapid enzymatic equilibrium with the corresponding -hydroxylic acids, and that elimination of H₂S from VI yielding II is the overall rate limiting step in the metabolic conversion of I to II. The elimination of intravenous doses of ³H-I and ³H-III occurred predominantly by biliary excretion of polar conjugated metabolites (80 and 95%, respectively, of the dose over 12 h) followed by extensive enterohepatic cycling. Urinary excretion remained below 3% of the dose over 12 h in bile fistula rats.     

Influence of spironolactone pretreatment on pharmacokinetics and metabolism of digitoxin in rats

Summary The influence of pretreatment with spironolactone (84 mg/kg at 3 days, twice per day) on the tritium levels in plasma, urine and feces of female SD-rats (n=9) was investigated at various time periods after oral administration of 25 g/kg ³H-digitoxin. In plasma, the concentrations of total radioactivity are reduced in pretreated animals to about 20% of tritium levels in control rats, while the half-life of radioactivity in both groups is almost identical, 2.9 days in pretreated rats and 2.8 days in controls. The lower plasma levels of tritium in pretreated rats coincide with a six-fold decrease in the urinary ³H-elimination and a corresponding increase in the fecal excretion. This is due to a higher biliary clearance of tritiated products in the early phase of elimination. The separation of the excretion products by TLC shows that spironolactone pretreatment enhances the splitting of the glycosidic bonds of digitoxin. The amount of digitoxigenin-bis-digitoxoside and of digitoxigenin-mono-digitoxoside excreted in urine and feces within 96 hrs is four and ten times greater than that recovered in control animals, respectively. The formation of the hydroxylation products digoxin and digoxigenin-bis-digitoxoside is decreased from 50% of the total excreted radioactivity in control to 15% in pretreated rats. The conjugation reactions with glucuronic and sulfuric acid are increased after pretreatment with spironolactone. Thus, the effect of spironolactone on digitoxin kinetics is apparently related to an enhancement of the hepatic excretory mechanism as well as to an enhanced metabolism.Supported by the Deutsche Forschungsgemeinschaft.     

Identification of the hydroxylated derivatives of bufalin: phase I metabolites in rats

Bufalin was a typical bioactive bufadienolide, existed in the traditional Chinese medicine Chan Su with the high content of 1–5%. The in vivo metabolites (1–5) of bufalin were prepared by various chromatographic techniques from the bile samples of SD rats, which were administrated with bufalin orally. Their structures were determined on the basis of the widely spectroscopic data, including HRESIMS, 1D-, and 2D NMR. And 1–3, 5 were new compounds. In the in vitro cytotoxicity assay, metabolites (1–5) showed weaker cytotoxic effects than bufalin against human cancer cell lines A549 and H1299, which indicated that the metabolism was a significant pathway for the detoxification of bufalin. Structures analyses indicated that metabolites 1–5 were hydroxylated derivatives of bufalin. This study suggested that Phase I metabolism catalyzed by CYP450 enzymes was one of the metabolic ways of bufalin, which may promote the excretion of bufalin.     

Identification and human exposure prediction of two aldehyde oxidase-mediated metabolites of a methylquinoline-containing drug candidate

1. Aldehyde oxidase (AO enzymes)-mediated oxidation predominantly occurs at a carbon atom adjacent to the nitrogen on aromatic azaheterocycles. In the current report, we identified that AO enzymes oxidation took place at both the C-2 and C-4 positions of the methylquinoline moiety of Compound A based on data from mass spectrometric analysis, AO enzymes “litmus” test, and comparison with authentic standards.

2. To assess the potential for inadequate coverage for these two AO enzyme-mediated metabolites in nonclinical safety studies, given concerns due to differences in AO enzymes expression between preclinical species and humans, the human circulating levels of the two AO enzyme-mediated metabolites were predicted prospectively using in vitro and in vivo models. Both formation clearance and elimination clearance of the two metabolites were predicted based on in vitro to in vivo correlation and comparison with in vivo data from rats.

3. The result showed that the 4-OH metabolite of Compound A would account for less than 3% of the total drug-related exposure in human plasma, while the exposure to the 2-oxo metabolite would be relatively high (～70%).

4. The predicted human exposure levels for the two metabolites are in similar ranges as those observed in monkeys. These data taken together support the advancement to clinical development of Compound A.     

Identification of novel metabolites of pioglitazone in rat and dog

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [3 H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.     

美洛昔康片溶出度考察及其体内外相关性

目的:考察2种市售美洛昔康片的体外溶出度,评价其质量及其体内外相关性.方法:采用转篮法测定溶出度,计算累积溶出百分率并与体内吸收百分率进行相关性评价;用Weibull分布模型对溶出曲线进行拟合,提取溶出参数并进行统计分析.结果:2种美洛昔康片的溶出参数之间差异有非常显著意义(P＜0.01).t检验表明同一厂家3批产品的参数之间差异有时也有显著性(P＜0.05).2种片剂的体外溶出与体内吸收之间均具有显著相关性.结论:2个厂家产品的溶出度之间存在差异并且体内外具有相关性,提示在临床用药时应加以注意.     

Reliability of human cryopreserved hepatocytes and liver microsomes as in vitro systems to predict metabolic clearance

A total of 110 drugs, selected to cover a range of physicochemical and pharmacokinetic properties, were used to explore standard approaches to the prediction of in vivo metabolic clearance using drug-depletion profiles from human liver microsomes (HLMs) and cyropreserved hepatocytes. A total of 41 drugs (37% of the compounds tested) showed measurable depletion rates using HLMs (depletion by 20% or more over the time course). The most reliable correlations in terms of bias (average fold error (AFE) = 2.32) and precision (root mean square error (RMSE) = 3501) were observed by comparing in vivo intrinsic clearance (CL_int), calculated using the parallel-tube model and incorporating the fraction unbound in blood, with in vitro CL_int adjusted for microsomal binding. For these reference drugs, 29% of predictions were within two-fold of the observed values and 66% were within five-fold. Compared with HLMs, clearance predictions with cryopreserved hepatocytes (57 drugs) were of similar precision (RMSE = 3608) but showed more bias (AFE = 5.21) with 18% of predictions within two-fold of the observed values and 46% within five-fold. However, with a broad complement of drug-metabolizing enzymes, hepatocytes catalysed measurable CL_int values for a greater proportion (52%) of the reference compounds and were particularly proficient at defining metabolic rates for drugs with predominantly phase 2 metabolic routes.     

Isoniazid-induced intolerance to ethanol in rabbit,guinea pig and rat

Isoniazid (50 or 100 mg kg^?l p.o.) inhibited the elimination of ethanol (0·5, 1·0, and 2·5 g kg^?l p.o.) from the plasma of rabbit, guinea pig, and rat. The effect of isoniazid, isonicotinic acid, N-acetylisoniazid, N-acetylhydrazine, N,N-diacetylhydrazine, and hydrazine on the in vitro metabolism of alcohol by rabbit liver alcohol dehydrogenase was examined. Only isoniazid, hydrazine, and acetylisoniazid showed significant inhibitory activity with I₅₀'s of 17·7, 1·25, and 0·78 mmol 1^?1 respectively. Intolerance to ethanol following isoniazid treatment was attributed to isoniazid inhibition of liver alcohol dehydrogenase.     

Excretion,distribution and metabolism of 1,2,4-trichlorobenzene in rats

1,2,4-Trichlorobenzene (TCB) labeled with C-14 was given perorally to rats at a dosage of 50 mg/kg for excretion and distribution studies.About 66% and 17% of the oral dose was excreted in the urine and feces, respectively, within 7 days. Trapped radioactivity in the expired air amounted to 2.1% of the dose, but production of labeled carbon dioxide was negligible. Tissue residues were evenly distributed throughout the organs and tissues examined, except for the adipose tissue which consistently had a little higher concentration.The urinary, fecal and expiratory metabolites were identified. Free 2,4,5- and 2,3,5-trichlorophenol (TCP) and their conjugates were mainly detected in the urine. 5- or 6-Sulfhydryl, methylthio, methylsulfoxide and methylsulfone derivatives of TCB were also detected as minor metabolites. Dichlorobenzenes and unchanged TCB were confirmed in the expired air. Reductive dechlroination seems to be catalysed by intestinal microflora enzymes.     

Venoconstrictor responses to ergosine and ergosinine: Evidence for the isomerization of ergosinine

Ergosine and its D-isolysergic acid derivative ergosinine were investigated on canine saphenous veins both in vivo and in vitro. Following local i.v. infusion in vivo, about 5 times higher doses of ergosinine were necessary to produce the same venoconstrictor response as induced by ergosine. When administered orally, however, both ergot alkaloids were equi-effective. In vitro methiothepin, a 5-HT receptor blocker with high affinity for 5-HT₁ receptors, antagonized venoconstrictor responses to 5-HT and ergosine within the same concentration range, being significantly less potent when tested against norepinephrine. The reverse was true for the α₂-selective adrenoceptor blocker yohimbine, which was significantly more potent against norepinephrine and ergosine than against 5-HT, suggesting that ergosine has affinity to both 5-HT₁-like receptors and α₂-adrenoceptors. Concentration-response curves to norepinephrine were shifted to the right in a parallel fashion when ergosine or ergosinine were present in the organ baths, suggesting competitive antagonism. The blocking potency of ergosinine increased with increasing incubation times in Krebs-Henseleit solution becoming similar to that of ergosine when an incubation time of 2 hr was applied. It is suggested that the pharmacological activity of ergosinine is the consequence of an isomerization into its natural stereoisomer ergosine, which may occur both in vivo and in vitro.     

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in␣vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and β-oxidation (liver specific functions). Ethionine (0–30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10–30 mM ethionine and reduced after 20 h in cultured hepatocytes (18–30 mM). Protein synthesis was reduced and β-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in␣vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo. Received: 16 June 1998 / Accepted: 29 June 1998     

Physiologically based pharmacokinetic model for tetrachlorobenzyltoluenes in rat: comparison of in vitro and in vivo metabolic rates.

Ugilec 141 is a technical mixture of tetrachlorobenzyltoluenes (TCBTs). It was introduced in the early 1980s as a replacement for polychlorinated biphenyls (PCBs). Based on physicochemical properties and accumulation in the environment, the use of this mixture was prohibited. To gain more insight in the toxicokinetics of these compounds in mammals, rats were exposed to a single iv bolus injection of a mixture of 3 TCBTs. At different time points after dosing, the tissue and blood concentrations of the TCBTs were determined. The adipose tissue is the main storage compartment, followed by skin and muscle. The TCBTs were rapidly eliminated from the liver and the blood, with half lives ranging from 65 to 72 h. Additionally, the tissue concentration data for all 3 TCBTs were analyzed using a physiologically based pharmacokinetic (PB-PK) model. Sensitivity analysis illustrated that the elimination of the TCBTs was not influenced by metabolism only, but also by the blood flow through the liver. Furthermore, the metabolic rates derived from the model were compared to previously reported in vitro metabolic rates. The in vitro values for the TCBTs were only a factor 2 to 3 smaller than the in vivo metabolic rates, indicating the value of in vitro techniques for a priori parameterization of PB-PK models.     

Structural elucidation of in vitro metabolites of bavachinin in rat liver microsomes by LC-ESI-MSn and chemical synthesis

1.?Bavachinin isolated from Psoralea corylifolia has various activities, such as antimicrobial, antiallergic, antitumor and so on. Our previous study showed that natural bavachinin exhibits peroxisome proliferator-activated receptor γ-agonist activity.

2.?In vitro studies on bavachinin metabolism were conducted using rat liver microsomes incubated at 37?°C for 60?min.

3.?Structures of eight metabolites of the incubation mixtures were cautiously characterized using electrospray tandem mass spectra and three synthetic compounds. The results indicated that eight metabolites of bavachinin were biotransformed mainly through oxidation.

4.?The metabolic pathways of bavachinin were elucidated in vitro. These results contribute to the understanding of bavachinin’s in vivo metabolism.     

缓释制剂的体内外相关性试验及检验

目的：探讨缓释制剂体内外相关试验方法。方法：以植入用缓释依托泊苷为例，根据体内及体外预试验数据，求算对应时间点关联系数K，按中华人民共和国药典要求作对应时间点的点对点相关试验及检验。结果：成功进行植入用缓释依托泊苷的体内外相关试验并实现相关。结论：此法为长效缓释制剂提供了一种体内外相关试验的参考方法。     

藤茶总黄酮对小鼠的体内外抗氧化作用研究

目的:研究藤茶总黄酮(AGTF)的体内外抗氧化作用。方法:使用丙二醛(MDA)测定试剂盒测定AGTF对体内外MDA含量的影响;用分光光度法测定AGTF对H2O2诱导小鼠红细胞溶血和Fe2 -Vit C诱导的肝线粒体肿胀度的抑制作用。结果:2.00mg.mL-1AGTF对脂肪氧合酶的粗酶活性抑制率超过50%;AGTF在0.5~10.0mg.mL-1范围内,具有较强的体外抗活性氧能力,并能减少小鼠肝线粒体及肝匀浆MDA的生成,可抑制H2O2诱导的小鼠红细胞溶血和Fe2 -VitC诱导的肝线粒体肿胀;腹腔注射AGTF对小鼠体内肝组织MDA生成的抑制作用显著。结论:AGTF具有较强的体内外抗氧化活性。     

Effect of phenobarbital pretreatment on in vitro enzyme kinetics and in vivo biotransformation of benzene in the rat

Phenobarbital pretreatment (50 mg/kg/day for 3 days orally) of male Wistar rats increased V _max of benzene in vitro hepatic microsomal biotransformation about 6-fold without changing K _m . However, benzene blood levels after oral, intraperitoneal, or subcutaneous benzene administration (3–3.5 mmoles/kg) were not influenced by phenobarbital pretreatment. The phenol blood levels after oral or intraperitoneal benzene were increased by phenobarbital pretreatment, but less than expected from in vitro data and only 3 h after benzene administration. Phenol elimination in urine after subcutaneous benzene was not affected by phenobarbital. After oral or intraperitoneal benzene administration, phenol urine excretion closely followed the levels of phenol in blood, i.e., rate of phenol urine excretion was significantly, but shortly increased, and the cumulative urine excretion of phenol increased very little or remained unchanged. Differences between the in vitro and in vivo observations of the effect of phenolbarbital on benzene biotransformation may partly be explained by distribution of benzene, which apparently limited benzene availability for biotransformation (V _d =5.5) and caused rapid decrease of benzene concentrations in blood. Conditions for enzyme activity may have been substantially different in vitro vs. in vivo: in vitro concentrations of benzene were at least by an order of magnitude higher than phenol concentrations, while in vivo, an opposite relation prevailed making a competition for microsomal monooxygenase possible. Cofactor availability may be another rate-limiting step or factor of in vivo benzene biotransformation, as benzene ring hydroxylation requires high energy. The rate of in vitro hepatic microsomal benzene biotransformation proved to be of limited value when predicting benzene quantitative biotransformation in vivo in contradistinction to various substrates where the in vitro and in vivo biotransformation data are in good agreement     

舒乐洗剂治疗外阴阴道念珠菌病的体内外实验研究

目的：观察舒乐洗剂治疗外阴阴道念珠菌病（VVC）的效果。方法建立体外白念珠菌生物膜模型,采用 XTT 法检测生物膜抑制率;建立 VVC 小鼠模型,采用阴道涂片检查和病理组织检测法,比较给药前后炎症状况的变化。结果舒乐洗剂对体外白念珠菌生物膜的形成有明显抑制作用,并成一定的剂量依赖性;能够抑制体外白念珠菌菌落形成,其中原液稀释一倍的效果最为显著;能够明显减少小鼠体内白念珠菌菌落形成,并能够减少炎性细胞向阴道黏膜的浸润。结论舒乐洗剂具有体外抗生物膜形成的作用,并能降低体内小鼠白念珠菌的感染状况,减轻VVC 小鼠阴道黏膜炎症,从而达到治疗 VVC 的作用。     

Tissue distribution of decabrominated diphenyl ether (BDE-209) and its metabolites in sucking rat pups after prenatal and/or postnatal exposure

Growing evidence has shown that decabromodiphenyl ether (BDE-209) can disrupt thyroid hormones and induce neurological and developmental effects, especially for the fetuses and neonates after prenatal or postnatal exposure. The present study was carried out to examine the effects of in utero and lactational exposure to BDE-209 on the absorption and tissue distribution of BDE-209 and its metabolites in offspring. Pregnant Sprague-Dawley rats were given daily oral doses of 5 μmol/kg b.w. BDE-209 in peanut oil during gestational and lactational period or during lactational period only. BDE-209 and its debrominated congeners were analyzed in several maternal tissues, offspring carcass and neonatal tissues. The occurrence of polybrominated diphenyl ethers (PBDEs) and their time profiles in maternal blood, placenta and fetuses/sucking pups indicated that BDE-209 and its debrominated products can be transferred from mother to offspring via in utero or lactational exposure. Nona-BDEs were the predominant congeners in the analyzed pup tissues, and BDE-206 was the most abundant congener while BDE-197/204 was the major congener of octa-BDE. Then the contributions of transplacental and lactational transfer were compared for BDE-209 and its debrominated congeners. The levels of PBDEs in tissues of sucking pups of the in utero and lactational exposure group were much higher than those of only lactationally exposed group. BDE-197/204 was the debrominated congener with the most significant difference between these two groups and the pup brain was the tissue with the most significant difference of the levels of debrominated congeners. The results provide a basis for understanding the possible adverse effects caused by maternal transfer of BDE-209 during the critical periods of development of fetuses and sucking neonates.     

Bottom-up modeling and simulation of tacrolimus clearance: prospective investigation of blood cell distribution, sex and CYP3A5 expression as covariates and assessment of study power

The objectives were to investigate the ability of population-based in vitro-in vivo extrapolation (IVIVE) to reproduce the influence of haematocrit on the clearance of tacrolimus, observed previously, and to assess the power of clinical studies to detect the effects of covariates on the clearance of tacrolimus. A population-based pharmacokinetic simulator (Simcyp) was used to simulate tacrolimus clearance from in vitro metabolism data and demographic characteristics of Japanese liver transplant patients (JLTs). The relationship between haematocrit and dose-to-concentration (D/C) ratio was validated using seven JLTs, whose highly variable haematocrit and D/C ratio were previously analysed. This validation was used as a surrogate for establishing 'interindividual' variability and to assess the power of clinical studies to discern the effect of haematocrit, sex and CYP3A5 genotype on tacrolimus clearance in a virtual JLT population. The relationship between haematocrit and D/C ratio was reproducible by Simcyp and corresponded well to those observed in seven JLTs. The number of JLTs required to detect the influence of CYP3A5 genotype and sex were estimated to be about 50 and > 600, respectively, which was consistent with the results of previous population pharmacokinetic studies for tacrolimus. In conclusion, population-based IVIVE is considered to be a useful approach to assess the influence of covariates a priori before conducting clinical studies. This is also helpful with study design and assessment of the statistical power of clinical studies involving population-based pharmacokinetics to detect the effects of covariates.     

Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole

1.?Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects.

2.?Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC₅₀ data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole.

3.?Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1′-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2.

4.?The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.