Prediction of the Effects of Genetic Polymorphism on the Pharmacokinetics of CYP2C9 Substrates from <Emphasis Type="BoldItalic">In Vitro</Emphasis> Data

Purpose  The *2 and *3 alleles of CYP2C9, with decreased enzymatic activity, are highly polymorphic and contribute to inter-individual differences in pharmacotherapy of CYP2C9 substrates. Here, we sought for a simplified theoretical method to predict the pharmacokinetic changes with minimal in vivo data. Methods  The changes in clearances of CYP2C9 substrates in subjects with these alleles were quantitatively estimated by parameters from literature data: intrinsic metabolic clearance and the enzyme expression level of mutated CYP2C9, contribution of CYP2C9 to the CYP-mediated clearance (f _m2C9), and the contribution of the dominant metabolic pathways to the total clearance (f _h). To validate the accuracy of our prediction, the changes were compared to reported in vivo values. Results  Sufficient data were available for nine substrates: celecoxib, diclofenac, S-flurbiprofen, losartan, S-phenprocoumon, phenytoin, tolbutamide, torsemide, and S-warfarin. These predicted values, either using the intrinsic clearance specific to each substrate, or the averaged values (*2: 0.66, *3: 0.13, (ratio to *1)), correlated well with observed values (r ² = 0.812, 0.786, respectively). Conclusions  This theoretical method well estimated the quantitative changes in pharmacokinetics of CYP2C9 substrates in subjects with mutated alleles of CYP2C9. This can be applied to drug development even from the early clinical phases. Makiko Kusama and Kazuya Maeda contributed equally to this work.     

In Vivo ESR Studies on Pharmacokinetics and Metabolism of Parenteral Lipid Emulsion in Living Mice

Purpose. We applied non-invasive and real-time method with in vivo ESR spectroscopy to determining pharmacokinetics and metabolism of lipid emulsion as a drug carrier in living mice. Methods. A spin-labeled triglyceride (SL-TG) was newly synthesized and lipid emulsion containing SL-TG was prepared. In vivo ESR spectra in mice were observed after intravenous administration of the lipid emulsion. Results. In vivo ESR spectra consisted of three components, coinciding with the in vitro spectra of SL-TG particles, free and immobilized fatty acids. The amount of the components depended on both the observing domain and the period after administration. In the chest, all three components were observed, while SL-TG particle was lacking in the abdomen. The half-life of the lipid particles in the chest was 2 hr. Conclusions. Non-invasive and real-time analysis of drug carriers in living animal is successfully accomplished using an in vivo ESR method.     

In Vivo Evaluation of Acyclovir Prodrug Penetration and Metabolism Through Rat Skin Using a Diffusion/Bioconversion Model

Purpose. In order to evaluate the in vivo penetration of prodrugs which undergo metabolism in skin, we analyzed thein vivo penetration profiles of acyclovir prodrugs based on a two-layer skin diffusion model in consideration of metabolic process. Methods. Acyclovir prodrugs (e.g., valerate, isovalerate and pivarate) were used as model prodrugs and the amounts excreted in urine were measured after percutaneous application. In vivo penetration profiles were then estimated by employing a deconvolution method and the penetration of acyclovir prodrugs was analyzed using a diffusion model. Subsequently, diffusion, partitioning and metabolic parameters were compared under in vitro and in vivo conditions. Results. Although total penetration amounts at the end of the experiment were similar for the three prodrugs, the ratio of intact prodrug to total penetration amount differed significantly. Moreover, the excretion and absorption profiles were also very different for each prodrug. Enzymatic hydrolysis rate constants calculated under in vivo conditions were considerably larger than those obtained in the skin homogenate and in vitro penetration experiments. Conclusions. The present skin diffusion/bioconversion model combined with computer analysis enables us to comprehensively account for diffusion, partitioning and metabolism during in vivo percutaneous absorption. Nevertheless, different enzymatic hydrolysis rate constants obtained under bothin vivo and in vitro conditions demonstrate the difficulty of obtaining accurate values for in vivo enzymatic activity from related in vitro experiments.     

Effect of pregnancy on hepatic microsomal drug metabolism in rabbits and rats

In Dutch-belted rabbits, pregnancy caused several-fold decrease of in vitro hepatic microsomal aminopyrine, benzphetamine, and hexobarbital biotransformations. In pregnant Sprague-Dawley rats, various kinds of expressing the in vitro rates of hexobarbital biotransformation (per mg of microsomal protein, g of liver, 100 g of body weight) indicated unchanged or slightly elevated microsomal enzyme activity. In vivo, the course of hexobarbital blood levels after i. p. hexobarbital sodium, 100 mg/kg, indicated that the fate of hexobarbital was not primarily determined by the small changes of microsomal enzyme activity but, rather, by changed hexobarbital distribution. Different ways of expressing in vitro rates of aniline biotransformation showed decreased or unchanged enzyme activity during pregnancy and in vivo experiments indicated that these changes did not affect aniline metabolism in living rats. The results pointed out marked species differences in the effect of pregnancy on drug metabolism. Interpretation of in vitro biotransformation data for living animals suggested that with different substrates, microsomal enzyme activity and distribution, respectively, may exert different effects playing either significant or apparently minor role in drug disposition.Supported in part by U.S. Public Health Grant NIGMS 12,675.     

Brain Penetration and In Vivo Recovery of NMDA Receptor Antagonists Amantadine and Memantine: A Quantitative Microdialysis Study

Purpose. To determine free brain concentrations of the clinically used uncompetitive NMDA antagonists memantine and amantadine using microdialysis corrected for in vivo recovery in relations to serum, CSF and brain tissue levels and their in vitro potency at NMDA receptors. Methods. Microdialysis corrected for in vivo recovery was used to determine brain ECF concentrations after steady-state administration of either memantine or amantadine. Additionally CSF, serum, and brain tissue were analyzed. Results. Following 7 days of infusion of memantine or amantadine (20 and 100 mg/kg/day respectively) whole brain concentrations were 44-and 16-fold higher than free concentrations in serum respectively. The free brain ECF concentration of memantine (0.83 ± 0.05 M) was comparable to free serum and CSF concentrations. In case of amantadine, it was lower. A higher in vivo than in vitro recovery was found for memantine. Conclusions. At clinically relevant doses memantine reaches a brain ECF concentration in range of its affinity for the NMDA receptor and close to its free serum concentration. This is not the case for amantadine and different mechanisms of action may be operational.     

Percutaneous Absorption of Benzoic Acid Across Human Skin. II. Prediction of an in Vivo,Skin-Flap System Using in Vitro Parameters

The possibility of predicting the behavior of in vivo systems based on physical and chemical parameters determined by in vitro experiments is examined using benzoic acid. The physical and chemical parameters governing percutaneous absorption of benzoic acid—permeability, partition coefficient, and skin thickness—were determined by in vitro experiments as described in Ref. 1. These parameters were used, in combination with benzoic acid elimination kinetics, to predict the results of in vivo experiments using a comprehensive mathematical model. The in vivo system consists of a congenitally athymic (nude) rat with a surgically constructed human skin sandwich (HSSF) flap on which a donor cell is placed. To apply the in vitro parameters to an in vivo system requires a suitable pharmacokinetic model describing distribution and elimination for benzoic acid in the nude rat. Blood concentrations of benzoic acid following a bolus intravenous injection are closely described by a two-compartment open pharmacokinetic model with elimination occurring from only one compartment. The mathematical model of the rat-donor cell system combines this two-compartment model of the rat with a percutaneous absorption model to provide useful estimates of the measured in vivo blood levels. Comparisons of predicted and measured results suggest that the parameters determined by in vitro experimentation can be used to predict the behavior of complex in vivo systems, if a suitable mathematical model is available.     

Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of a high purity sample of single-wall carbon nanotubes (SWCNTs) was evaluated using a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation test (Ames test), an in vitro chromosomal aberration test, and an in vivo mouse bone marrow micronucleus test. The SWCNTs exerted no genotoxicity in Salmonella typhimurium TA97, TA98, TA100, and TA1535, or in Escherichia coli WP2 uvrA/pKM101, whether in the absence or presence of metabolic activation and at concentrations of 12.5–500 μg/plate. In the chromosomal aberration test, at 300–1000 μg/mL, the SWCNTs did not increase the number of structural or numerical chromosomal aberrations, whether the test was conducted with or without metabolic activation. In the in vivo bone marrow micronucleus test, doses of 60 mg/kg and 200 mg/kg SWCNTs did not affect the proportions of immature and total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of mice. The results of these studies show that the high purity and well-dispersed sample of SWCNTs are not genotoxic under the conditions of the in vitro bacterial reverse mutation assay, chromosomal aberration assay, or in vivo bone marrow micronucleus test, and thus appear not to pose a genotoxic risk to human health in vivo.     

The Use of Human Hepatocytes to Select Compounds Based on Their Expected Hepatic Extraction Ratios in Humans

Purpose. The present investigation retrospectively evaluates the use of human hepatocytes to classify compounds into low, intermediate or high hepatic extraction ratio in man. Methods. A simple approach was used to correlate the in vivo hepatic extraction ratio of a number of compounds in man (literature and in-house data) with the corresponding in vitro clearance which was determined in human hepatocytes. The present approach assumes that, for compounds eliminated mainly through liver metabolism, intrinsic clearance is the major determinant for their in vivo hepatic extraction ratio and subsequently their bioavailability in man. The test compounds were selected to represent a broad range of extraction ratios and a variety of metabolic pathways. Results. The present data show that in vitro clearances in human hepatocytes are predictive for the hepatic extraction ratios in vivo in man. Most of the test compounds (n = 19) were successfully classified based upon human hepatocyte data into low, intermediate or high hepatic extraction compounds, i.e. compounds with potential for high, intermediate or low bioavailabilities in humans. Conclusions. The present approach, validated so far with 19 test compounds, appears to be a valuable tool to screen for compounds with respect to liver first-pass metabolism at an early phase of drug discovery.     

硝苯地平控释片体内外相关性溶出方法研究

目的 开发相应的能区分和反映参比制剂与自研制剂之间释放差异的溶出方法，使自研制剂与参比制剂体外释放达到最大程度的匹配，从而为生物等效性试验提供体内外更具相关性的研究。方法 采用美国药典(USP)溶出2法(桨法)作为质量控制方法；另采用流池法作为溶出区分性方法，评估不同实验参数下的溶出结果。结果 优化并确立方法为开环系统，流速3.0 mL·min^-1，加1/2勺玻璃珠。该方法可以有效区分处方和生产工艺变化对产品溶出的影响。以此法为基础所得到的体外溶出速率和溶出度与体内药动学研究结果一致。结论 本研究所开发的流池法适合作为硝苯地平控释片的溶出区分性方法，并具备一定的体内体外相关性。     

天然化合物-O-甲基转移酶的研究进展

天然化合物-O-甲基转移酶（NPOMT）是一种以S-腺苷甲硫氨酸（SAM）为供体，靶向天然化合物中氧原子的甲基转移酶，在体内主要以两种形式存在：儿茶酚-O-甲基转移酶（COMT）、羟基吲哚-O-甲基转移酶（HIOMT）。COMT、HIOMT代谢多种内源性物质（主要是邻苯二酚类以及羟基吲哚类化合物），参与体内信号转导、生长增殖、能量代谢、节律调控等一系列生理学过程。对NPOMT的体内分布和表达、底物、功能、在体内的表达调控和抑制剂进行综述，以期为用于治疗相关代谢性疾病的NPOMT特异性激动剂/抑制剂的研发以及相关疾病的诊断和预后评估提供参考。     

Characteristics of Choline Transport Across the Blood-Brain Barrier in Mice: Correlation with In Vitro Data

Purpose. We examined the functional properties of choline transport across the blood-brain barrier (BBB) in mice. We compared the kinetic parameters and transport properties with those found in our in vitro uptake experiments using mouse brain capillary endothelial cells (MBEC4). Methods. The permeability coefficient-surface area product (PS) values of [³H]choline at the BBB were estimated by means of anin situ brain perfusion technique in mice.Results. [³H]Choline uptake was well described by a two-component model: a saturable component and a nonsaturable linear component. The [³H]choline uptake was independent of pH and Na⁺, but was significantly decreased by the replacement of Na⁺ with K⁺. Various basic drugs, including substrates and inhibitors of the organic cation transporter, significantly inhibited the [³H]choline uptake. These in situ (in vivo) results corresponded well to the in vitro results and suggest that the choline transporter at the BBB is a member of the organic cation transporter (OCT) family. Conclusion. The choline transport mechanism at the BBB is retained in MBEC4.     

Effect of Concentration and Degree of Saturation of Topical Fluocinonide Formulations on In Vitro Membrane Transport and In Vivo Availability on Human Skin

Purpose. The thermodynamic acitvity of drugs in topical vehicles is considered to significantly influence topical delivery. In vitro diffusion across a synthetic membrane was shown to be correlated to the degree of saturation of the drug in the applied vehicle and therefore offers a potential for increased topical drug delivery. Fluocinonide a topical corticosteroid, was chosen as a model compound to investigate in vitro and in vivo availability from formulations with different degrees of saturation. Methods. Sub-, as well as, supersaturated drug solutions were prepared using PVP as an antinucleant agent. In vitro membrane diffusion experiments across silicone membrane and in vivo pharmacodynamic activity assessments, using the human skin blanching assay, were carried out. Results. Over the concentration range studied, the in vitro membrane transport of fluocinonide was proportional to the degree of saturation of the respective formulations. The in vivo pharmacodynamic response in the human skin blanching assay was related to the concentration of the drug in the vehicle irrespective of the degree of saturation. Conclusions. From the membrane permeation experiment it can be concluded, that the drug flux might be increased supra-proportionally with increasing donor concentration, drug (super-)saturation (proportional), beyond what would be anticipated based on ideal donor concentration and partition coefficient considerations only. These findings could not be confirmed in the in vivo investigation, probably due to additional vehicle effects (e.g., enhancement, irritation, drug binding) which have to be expected and could have altered the integrity of the stratum corneum and therewith topical bioavailability of the drug.     

京尼平苷体内外微透析回收率的研究

目的 探究京尼平苷在自制线性微透析探针上的体内外回收率与灌流速度、药物浓度、透析膜长之间的关系。方法 采用HPLC测定微透析样品浓度,考察不同灌流速度、不同药物浓度和不同透析膜长对体外正向和体外反向回收率、体内反向回收率的影响。结果 自制探针性质稳定;探针回收率与京尼平苷的药物浓度无关,与灌流速度成反比,随透析膜长增加而增加;体外的正向与反向探针回收率有显著性差异（P<0.01）,而体内反向回收率低于体外反向回收率,与体外正向回收率无显著性差异;新探针体内回收率大于使用过1次的探针体内回收率（P<0.05）。结论 在京尼平苷的皮肤药动学研究中,宜采用体内反向回收率或体外正向回收率作为药物的探针回收率来校正。     

Development of an in Vitro Rat Intestine Segmental Perfusion Model to Investigate Permeability and Predict Oral Fraction Absorbed

Purpose The aims of the study are to develop and evaluate an in vitro rat intestine segmental perfusion model for the prediction of the oral fraction absorbed of compounds and to assess the ability of the model to study intestinal metabolism. Methods The system consisted of a perfusion cell with a rat intestinal segment and three perfusion circulations (donor, receiver, and rinsing circulation). Lucifer yellow (LY) was applied as internal standard together with test compounds in the donor circulation. To validate the model, the permeability of eight noncongeneric passively absorbed drugs was determined. Intestinal N-demethylation of verapamil into norverapamil was followed in the donor and receiver circulations by high-performance liquid chromatography analysis. Results The in vitro model allowed ranking of the tested compounds according to their in vivo absorption potential. The Spearman's correlation coefficient between the oral fraction absorbed in humans and the ratio of permeation coefficient of test compound to the permeation coefficient of LY within the same experiment was 0.98 (P < 0.01). Moreover, intestinal N-demethylation of verapamil, its permeation, and the permeation of its metabolite norverapamil could be assessed in parallel. Conclusions Up to six permeation kinetics can be obtained per rat, and the method has shown to be a valuable tool to estimate human oral absorption.     

姜黄素类化合物体内代谢途径及其代谢产物的研究进展

姜黄素类化合物具有明确而广泛的药理作用,但因其溶解度差、结构不稳定,生物利用度极低。主要通过对姜黄素类化合物体内代谢途径及其相关代谢产物进行综述,以明确姜黄素生物利用度低的原因,并提出姜黄素发挥药理活性的物质基础可能与其通过自身氧化和催化氧化过程产生的双环乙酰丙酮结构有关。因此对姜黄素代谢产物进行药动学及相关药效研究,能够为临床应用提供更为关键的基础性数据,也具有更为重要的研究价值。     

Drug metabolism by CYP2C8.3 is determined by substrate dependent interactions with cytochrome P450 reductase and cytochrome b5

Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and Escherichia coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe substrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3's higher k_cat seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR (K_D and K_m,red) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher k_cat. In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in k_cat between the two enzymes, and may provide an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level.     

Evaluation of Human Liver Slices and Reporter Gene Assays as Systems for Predicting the Cytochrome P450 Induction Potential of Drugs in Vivo in Humans

Purpose The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. Methods The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). Results In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUC_tot to PXR EC₅₀ values. Conclusions Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.     

In Vitro/in Vivo Correlation of the Bioadhesive Properties of a Buccal Bioadhesive Miconazole Slow-Release Tablet

An in vitro–in vivo correlation study was performed on the bioadhesive properties of three buccal formulations based on modified starch (drum-dried waxy maize)/polyacrylic acid mixtures. Mixtures containing 10 mg miconazole nitrate, characterized by a different in vitro detachment force and work of adhesion, were evaluated for their bioadhesive properties in human volunteers. The results obtained showed that no significant difference could be seen among the formulations in vivo. The in vitro method showed no significant influence of miconazole nitrate on the bioadhesion properties of the polymers, while the in vivo adhesion time of the pure polymer mixtures was significantly higher than for the polymers containing miconazole. The results from the in vitro method thus did not correlate well with the in vivo data. The in vitro method provided information only on the initial bioadhesion and no correlation could be made with the residence time of the tablet in the oral cavity.     

Lyophilized Paclitaxel Magnetoliposomes as a Potential Drug Delivery System for Breast Carcinoma via Parenteral Administration: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">in Vivo</Emphasis> Studies

No HeadingPurpose. The study reports in vitro and biological evaluation of lyophilized negatively charged paclitaxel magnetic liposomes as a potential carrier for breast carcinoma via parenteral administration.Methods. Paclitaxel in magnetoliposomes were extracted by centrifugation and quantified by high-performance liquid chromatography (HPLC). Biological properties were studied using pharmacokinetics, in vivo distribution and cytotoxicity assays, as well as a mouse model of EMT-6 breast cancer.Methods. Pharmacokinetic studies showed that encapsulation of paclitaxel in magnetoliposomes produced marked difference over the drug in Cremophor EL/ethanol pharmacokinetics, with an increased t_1/2 19.37 h against 4.11 h. For in vivo distribution, paclitaxel concentration of lyophilized magnetoliposomes in the tumor was much higher than that of lyophilized conventional liposomes or Cremophor EL/ethanol, whereas in heart it was much lower than the latter two formulations via s.c. and i.v. administration. Lyophilized paclitaxel magnetic liposomes showed more potency on the therapy of breast cancer than other formulations via s.c. and i.p. administration.Conclusions. The current study demonstrates that paclitaxel magnetoliposomes can effectively be delivered to tumor and exert a significant anticancer activity with fewer side effects in the xenograft model.     

An in Vitro/in Vivo Correlation for the Disintegration and Onset of Drug Release from Enteric-Coated Pellets

An empirical mass-transfer model for enteric-coating dissolution that uses in vitro dissolution data to characterize the pH-dependent solubility properties of the polymer film and a mass-transfer coefficient determined from in vivo dissolution or disintegration studies is developed. Once the in vivo mass- transfer coefficient has been evaluated, it can be used in conjunction with in vitro dissolution data from other formulations to predict the in vivo time to disintegration and onset of drug release. Results of in vitro dissolution experiments using the USP basket dissolution apparatus and in vivo disintegration experiments using gamma scintigraphy with four enteric-coated pellet formulations are presented. The good agreement among the in vivo mass-transfer coefficients that were determined supports the validity of the model.