Intestinal Drug Absorption and Metabolism in Cell Cultures: Caco-2 and Beyond

Pharmaceutical Research -     

Investigations on the Percutaneous Absorption of the Antidepressant Rolipram in Vitro and in Vivo

In vitro experiments using full-thickness human skin showed that it was feasible to deliver therapeutic amounts of the new antidepressant drug rolipram. Simple transdermal devices were constructed, and the presence of isopropyl myristate (IPM) in a silicone adhesive (Dow Corning X7-2920) enhanced the flux across excised human skin. The steady-state fluxes from adhesive mixtures containing 0, 5, and 10% IPM were 3, 5.2, and 6 µg/cm²/hr, respectively. The in vitro experiments were confirmed in a clinical study involving six healthy male volunteers. The formulations tested were an alcoholic solution and adhesive patches containing 5 and 10% IPM. The dose of drug administered was 0.5 mg/cm² and the device size 25 cm². Blood samples were withdrawn over a 24-hr period and analyzed using radioimmunoassay. The topical applications were well tolerated, with only mild or no side effects. A lag time of approximately 2 hr was found for the detection of rolipram in the plasma (detection limit, 50 pg/ml). Interindividual variations both for the peak drug levels and throughout the delivery were quite high but this magnitude of variation has been observed in many other transdermal studies. Plasma levels between 1 and 2 ng/ml were found for all formulations and the AUC_0–30hr was significantly higher for the patch containing 5% IPM.     

抗体偶联药物临床前安全性评价考虑要点

抗体偶联药物（Antibody Drug Conjugate,ADC）,是通过连接子将高细胞毒性化合物连接到靶向肿瘤抗原的抗体上,利用抗体靶向识别将化合物递呈至肿瘤细胞表面,杀死肿瘤细胞。ADC药物同时具有抗体和化学药物属性,是将两者的优势结合,极大地提高了药物安全有效性。ADC药物结构复杂,在抗原识别表位、连接位点、连接子以及小分子药物各组分均存在特异性,所以在开展安全性评价研究时有一定的特殊性。本文将从动物种属选择、一般毒性研究、毒代动力学研究、组织交叉反应、免疫原性检测、安全药理研究、遗传毒性研究等方面,阐述ADC药物临床前安全性评价的考虑要点和研究策略。     

The Development of USP Dissolution and Drug Release Standards

Dissolution tests have been in use in the pharmaceutical industry for over 20 years, and they are official in The United States Pharmacopeia since the early 1960s. The dissolution test, reviewed primarily as a quality control tool, replaced the use of disintegration tests which had been official in The United States Pharmacopeia since 1950. Refinements in the dissolution test equipment and methodology have occurred over the years in order to enhance its relevance. The Subcommittees of the USP Committee of Revision dealing with these issues have developed and refined compendial dissolution standards and policies for conventional solid-oral dosage forms and modified-release dosage forms.     

Modification of the Dialysis Membrane Method for Drug Release from Suppositories

Pharmaceutical Research -     

Correlation Between Oral Drug Absorption in Humans,and Apparent Drug Permeability in TC-7 Cells,A Human Epithelial Intestinal Cell Line: Comparison with the Parental Caco-2 Cell Line

Purpose. To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (P _app ) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. Methods. Both cell lines were grown for 5–35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. P _app were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. Results. The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The P_app and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and P_app values, allowing to determine a threshold value for P_appof 2 10^–6 cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. Conclusions. On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.     

In Vivo Bioavailability and Metabolism of Topical Diclofenac Lotion in Human Volunteers

Purpose. The primary objective of this study was to determine the rate and extent of transdermal absorption for systemic delivery of diclofenac from Pennsaid (Dimethaid Research, Inc.) topical lotion into the systemic circulation after the lotion was applied to human volunteers, in an open treatment, non-blinded, non-vehicle controlled study. In addition, the in vivo metabolism of this topical diclofenac lotion has also been studied. Methods. Human volunteers were dosed with topical [¹⁴C]-diclofenac sodium 1.5% lotion on the knee for 24 h. Sequential time blood and urine samples were taken to determine pharmacokinetics, bioavailability and metabolism. Results. Topical absorption was 6.6% of applied dose. Peak plasma ¹⁴C occurred at 30 h after dosing, and peak urinary ¹⁴C excretion was at 24–48 h. The urinary ¹⁴C excretion pattern exhibits more elimination towards 24 h and beyond, as opposed to early urinary ¹⁴C excretion. This suggests a continuous delivery of [¹⁴C]-diclofenac sodium from the lotion into and through skin which only ceased when the dosing site was washed. Skin surface residue at 24 h was 26 ± 9.5% dose (remainder assumed lost to clothing and bedding). Extraction of metabolites from urine amounted to 7.4–22.7% in untreated urine, suggesting substantial diclofenac metabolism to more water soluble metabolites, probably conjugates, which could not be extracted by the method employed. Two Dimensional TLC analysis of untreated urine showed minimal or no diclofenac, again emphasizing the extensive in vivo metabolism of this drug. Treatment of the same urine samples with the enzymes sulfatase and (-glucuronidase showed a substantial increase in the extractable material. Three spots were consistently present in each sample run, namely diclofenac, 3hydroxy diclofenac and an intermediate polar metabolite (probably a hydroxylated metabolite). Therefore, there was significant sulfation and glucuronidation of both diclofenac and numerous hydroxy metabolites of diclofenac, but many of the metabolites/conjugates remain unidentified. Conclusions. There was a continuous delivery of diclofenac sodium from the lotion into and through the skin, which ceased after the dosing site was washed. The majority of the material excreted in the urine were conjugates of hydroxylated metabolites, and not the parent chemical, although further identification is required.     

Microdialysis: An Alternative for in Vitro and in Vivo Protein Binding Studies

The aim of the present study was to compare the performance of conventional equilibrium dialysis method with a microdialysis method in studying drug protein binding. The two methods were assessed by comparing the measured mean unbound drug fraction in different plasma species in vitro in plasma of four different species and at two concentrations of the non-indolic melatonin analog S 20098. For the microdialysis study, the unbound drug fraction was calculated after correction for membrane recovery. Plasma protein binding of S 20098 ranged from 75 to 95%. In humans, rabbits and rats (10 ng/ml), equal unbound percentages were found between equilibrium dialysis and microdialysis. Microdialysis gave slightly but significantly higher values in rat (2000 ng/ml), and in monkey plasma independent of the drug concentration. Microdialysis was also performed in vivo in freely moving rats under steady-state conditions, yielding similar unbound fraction values (26.0 ± 0.9%) to those obtained using microdialysis probes in rat plasma in vitro (24.4 ± 1.6%). These results support the use of in vivo microdialysis in pharmacokinetic studies in freely moving animals.     

Comparative bioavailability: A new type ofin vitro-in vivo correlation exemplified by prednisone

The average time to reach half-maximal plasma concentration of prednisolone and the average plasma concentrations of prednisolone at 0.5 and 1 hr obtained from three crossover bioavailability studies, involving testing of commercially available 5-mg prednisone tablets, were highly correlated (r0.88) with parameters derived from in vitrotablet dissolution rates performed in the spin filter apparatus of Shah. The in vitroparameters were the times to dissolve 16% or 50% of the labeled amount of prednisone or the percent of the labeled amount of prednisone dissolved in 20 min in water at 37C. Such correlations may be useful in the setting of in vitrodissolution rate specifications for commencal prednisone tablets.Supported by Contract FDA 69-22, Food and Drug Administration, Washington, D.C., and in part by Public Health Service Grant 5-P11-GM15559.     

A Population Growth Model of Dissolution

Purpose. To develop a new approach for describing drug dissolution which does not require the presuppositions of time continuity and Fick's law of diffusion and which can be applied to both homogeneous and heterogeneous media. Methods. The mass dissolved is considered to be a function of a discrete time index specifying successive 'generations' (n). The recurrence equation: _n+1 = _n + r(l – _n)(1 – _n X ₀/) was derived for the fractions of dose dissolved _n and _{n +1}, between generations n and n + 1, where r is a dimensionless proportionality constant, X ₀ is the dose and is the amount of drug corresponding to the drug's solubility in the dissolution medium. Results. The equation has two steady state solutions, _ss = 1 when (X ₀/) 1 and _ss = /X ₀ when (X ₀/) > 1 and the usual behavior encountered in dissolution studies, i.e, a monotonic exponential increase of _n reaching asymptotically the steady state when either r < /X ₀ < 1 or r < 1 < /X ₀. Good fits were obtained when the model equation was applied to danazol data after appropriate transformation of the time scale to 'generations'. The dissolution process is controlled by the two dimensionless parameters /X ₀ and r, which were found to be analogous to the fundamental parameters dose anddissolution number, respectively. The model was also used for the prediction of fraction of dose absorbed for highly permeable drugs. Conclusions. The model does not rely on diffusion principles and therefore it can be applied under both homogeneous and non-homogeneous conditions. This feature will facilitate the correlation of in vitro dissolution data obtained under homogeneous conditions and in vivo observations adhering to the heterogeneous milieu of the GI tract.     

A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability

A biopharmaceutics drug classification scheme for correlating in vitro drug product dissolution and in vivo bioavailability is proposed based on recognizing that drug dissolution and gastrointestinal permeability are the fundamental parameters controlling rate and extent of drug absorption. This analysis uses a transport model and human permeability results for estimating in vivo drug absorption to illustrate the primary importance of solubility and permeability on drug absorption. The fundamental parameters which define oral drug absorption in humans resulting from this analysis are discussed and used as a basis for this classification scheme. These Biopharmaceutic Drug Classes are defined as: Case 1. High solubility-high permeability drugs, Case 2. Low solubility-high permeability drugs, Case 3. High solubility-low permeability drugs, and Case 4. Low solubility-low permeability drugs. Based on this classification scheme, suggestions are made for setting standards for in vitro drug dissolution testing methodology which will correlate with the in vivo process. This methodology must be based on the physiological and physical chemical properties controlling drug absorption. This analysis points out conditions under which no in vitro-in vivo correlation may be expected e.g. rapidly dissolving low permeability drugs. Furthermore, it is suggested for example that for very rapidly dissolving high solubility drugs, e.g. 85% dissolution in less than 15 minutes, a simple one point dissolution test, is all that may be needed to insure bioavailability. For slowly dissolving drugs a dissolution profile is required with multiple time points in systems which would include low pH, physiological pH, and surfactants and the in vitro conditions should mimic the in vivo processes. This classification scheme provides a basis for establishing in vitro-in vivo correlations and for estimating the absorption of drugs based on the fundamental dissolution and permeability properties of physiologic importance.     

In Vitro System to Evaluate Oral Absorption of Poorly Water-Soluble Drugs: Simultaneous Analysis on Dissolution and Permeation of Drugs

Purpose. The aim of the present work was to develop a new in vitro system to evaluate oral absorption of poorly water-soluble drugs by utilizing Caco-2 monolayers. Methods. Caco-2 monolayer was mounted between side-by-side chambers, which enabled the simultaneous assay of dissolution and permeation of drugs (dissolution/permeation system; D/P system). Apical and basal sides of the chamber were filled with buffer solutions. Drugs were applied to the apical side as powder, suspension, or solution, and then, the permeated amounts into the basal side were monitored for 2 h. At the same time, dissolved amounts of drugs at the apical side were detected. The amount of drug applied to the D/P system was based on its in vivo clinical dose. Results. Sodium taurocholate (5 mM, apical side) and bovine serum albumin (4.5% w/v, basal side) increased the permeated amount of poorly water-soluble drugs. Both additives were considered to be effective at mimicking in vivo conditions of intestinal drug absorption. From the correlation between the permeated amount of 13 drugs (% dose/2 h) in the D/P system and their percentage dose absorbed in humans in vivo, this system was found to be useful in evaluating oral absorption of poorly water-soluble drugs. Conclusions. With attempts made to mimic the physiologic conditions of the human GI tract, in vivo oral absorption of drugs was quantitatively assessed in the D/P system in vitro. This system is quite useful to predict the oral absorption of poorly water-soluble drugs after administration as solid dosage forms.     

基于生物等效性分析呋塞米片体内外试验的相关性

目的 开展体内外相关性试验以探讨国内生产的呋塞米片的药物溶出度试验是否可以替代生物等效性试验。方法 选择呋塞米片受试与对照制剂,按照中国药典要求和光纤药物溶出度实时测定仪测试方法,监测呋塞米累积溶出百分率并计算f₂相似因子,测定2种制剂的体内血药浓度并计算药动学参数,测算其体内吸收分数、生物利用度和生物等效性,分析呋塞米片体内外试验的相关性。结果 经f₂相似因子方法评估,受试与对照制剂的呋塞米溶出曲线相似,2种制剂的主要药动学参数t_max、C_max、AUC_0-24和AUC_0-∞、生物利用度和生物等效性均差异明显,其体内百分吸收系数和体内外试验的相关性较差。结论 国内生产的呋塞米片的药物溶出度试验尚不能替代生物等效性试验。     

Analysis of Drug Permeation Across Caco-2 Monolayer: Implication for Predicting In Vivo Drug Absorption

Purpose. The aim of the present work is to characterize in vitro drug permeation processes across Caco-2 monolayer and to identify the advantages of this cultured cell system in predicting in vivo drug absorption after oral administration. Methods. The passive permeability of various drugs through Caco-2 monolayer was measured using Ussing-type chambers and compared with that of the isolated rat jejunum and colon. The in vivo drug permeability to the intestinal membrane was estimated by means of an intestinal perfusion study using the rat jejunum. Results. In Caco-2 monolayer, drug permeability increased with increasing drug lipophilicity and showed a good linear relationship with the in vivo permeability. In contrast, in the isolated jejunum and colon, the permeability of high lipophilic drugs was almost constant and, propranolol, a drug with the highest lipophilicity, hardly passed through the jejunal membrane in vitro. As a result, there was no significant relationship between in vitro and in vivo drug permeability in rat jejunum. However, the amount of drugs accumulated in the jejunal mucosa increased with increasing drug lipophilicity even under the in vitro condition. Conclusions. The permeation and the accumulation studies suggested that the rate-limiting process of in vitro permeation of lipophilic drugs through the intestinal membrane differs from that of in vivo drug absorption. On the other hand, drug permeation through Caco-2 monolayer, which consists of an epithelial cell layer and a supporting filter, is essentially the same process as that of in vivo drug absorption. We concluded that the simple monolayer structure of a cultured cell system provides a distinct advantage in predicting in vivo drug absorption.     

Release and Absorption Characteristics of Novel Theophylline Sustained-Release Formulations: In Vitro-in Vivo Correlation

Five new experimental sustained-release (SR) formulations of theophylline, T-l, T-l-A, T-2, T-2-A, and T-2-E, in a matrix tablet form with a protein were developed. The in vitro release of theophylline from these novel experimental formulations and two commercial (Theotrim and Theo-Dur) SR formulations, was studied for 2 hr immersed in simulated gastric fluid TS, followed by an additional 10 hr immersed in simulated intestinal fluid TS. Like Theotrim and Theo-Dur, theophylline release profiles from all the novel experimental formulations were smooth, controlled, and unaffected by changes in the pH and the proteolytic enzyme content of the incubation media. Pharmacokinetic evaluation of T-l, T-l-A, T-2-A, Theotrim, and Theo-Dur was carried out in five dogs and six healthy human volunteers under fasting conditions, using immediate-release aminophylline tablets as controls. Pharmacokinetic analysis by the Wagner–Nelson procedure revealed sustained-release absorption characteristics for all the formulations with the exception of the immediate release aminophylline tablet. For each of the formulations tested, the regression analysis results of the percentage of theophylline absorbed in dogs or humans against the mean percentage released in vitro, at the corresponding times, indicated a high correlation. These data imply that the in vivo release profiles under fasting conditions in the gastrointestinal tract of dogs and humans may be similar to those in the in vitro studies.     

Pharmacokinetic Analysis of an Oral Sustained-Release Diltiazem Preparation Using Multifraction Absorption Models

Application of multifraction absorption models to pharmacokinetic analysis of an oral sustained-release diltiazem preparation (HER-SR) was investigated. The plasma diltiazem concentrations after oral administration of the HER-SR preparation were analyzed using both the two-fraction absorption model and the two-step discontinuous absorption model. The two-fraction absorption model was suitable for the pharmacokinetic analysis of the HER-SR preparation, whereas the two-step discontinuous absorption model is often unsuitable for the analysis of sustained-release preparations which disintegrate into fractions with different release characteristics in the gastrointestinal tract. The two-step discontinuous absorption model is usually not applicable to plasma concentration data when the first peak is sharp. MFA-MULTI(V) was shown to be useful for the prediction of the bioavailability in each fraction of HER-SR. It was further demonstrated that a two-fraction absorption model is useful for the comparison of in vitro and in vivo release profiles or evaluating the influence of food on the absorption behavior of HER-SR. In addition, the application of a two-fraction absorption model to population pharmacokinetics of HER-SR was investigated.     

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.     

Pharmacodynamic analysis of contractile potentiation by cholinesterase inhibitors in rats

Pharmacological profiles of four cholinesterase (ChE) inhibitors: edrophonium, pyridostigmine, neostigmine, and ambenonium after iv administration to rats were analyzed. A pharmacodynamic model was developed by considering acetylcholinesterase (AChE) inhibition, direct antagonism to the nicotinic receptor, and desensitization of the nicotinic receptor. Pharmacokinetics of these drugs are dose-independent and have similar volumes of distribution at steady state (0.4–0.6 L/kg various doses). The t_1/2 increases in the order of neostigmine, edrophonium, pyridostigmine, and ambenonium. Inhibitory constants of ChE inhibitors to bovine erythrocyte AChE determinedin vitro were 2019, 276, 26, and 3.7 nM for edrophonium, pyridostigmine, neostigmine, and ambenonium, respectively. The effect of ChE inhibitors was monitored as the increase of developed tension of triceps muscle induced by sciatic nerve stimulation. The maximum value of contractile tension after iv administration decreased at high doses of each drug and the dose-response curves were biphasic. Time courses of plasma concentration and contractile muscle tension were modeled to estimate the association/dissociation rate constants to AChE and the nicotinic receptor, desensitization rate constant of receptor, and the dissociation constant of acetylcholine (ACh) to nicotinic receptor/basal acetylcholine level ratio (Kd/ACh ₀). The estimatedKd/ACh ₀ values were not dependent on the drug. A significant correlation between inhibitory constants of ChE inhibitors to AChE estimated byin vivo pharmacodynamic analysis and those determined by anin vitro enzyme kinetic study was shown, while the relationship between dissociation constants to nicotinic receptor estimated byin vivo pharmacodynamic analysis and those measured by anin vitro binding study was not clear. Other process such as desensitisization induced by endogenous ACh, diffusion rate of drugs into the synaptic cleft, action of presynaptic receptors, etc., might contribute to the dose-effect relationship of ChE inhibitors.     

Identification of Novel Urease Inhibitors by High-Throughput Virtual and in Vitro Screening

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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.