Alternative and generalized approach to in vitro-in vivo correlation

Evaluation of in vitro-in vivo correlation (IVIVC) plays important role in securing therapeutic effect if a dosage form undergoes technological modifications. Similarity (closeness) of dissolution profiles of the original and modified dosage forms has been traditionally considered to be sufficient for similar in vivo responses. This may be true if the IVIVC model (dependence between the dissolution and corresponding absorption profiles) is given by a linear straight line with the unit slope. The paper presents an alternative and generalized approach to IVIVC evaluation. Influences of pre-systemic processes (disintegration, dissolution, absorption) on the system response (concentration time profile C(t), bioavailability BD and other) are analyzed and evaluated. Both the magnitude and sign of IVIVC are then derived from the magnitudes and signs of these influences. The underlining idea is that pre-systemic processes do not correlate with the system response, (e.g., plasmatic concentration) if small modifications of the former do not induce significant changes of the later. If this is so, the therapeutic effects of the modified and original dosage forms may be considered equal or at least similar. In this way the problem of IVIVC is not only exactly mathematically founded but modifications of pre-systemic processes are directly projected to the system output-- the time profile of plasmatic concentration. Moreover, the approach is applicable to virtually any dosage form. Its feasibility was validated in vivo.     

Modulation of tight junctions does not predict oral absorption of hydrophilic compounds: Use of caco-2 and calu-3 cells

Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway.     

Relevance of p-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation.

1. The interaction of cyclosporin A (CyA) with p-glycoprotein during intestinal uptake was investigated by a combination of in vitro experiments with human Caco-2 cells and an intubation study in healthy volunteers. 2. CyA uptake into the cells was not saturable and exhibited only a low temperature sensitivity, suggesting passive diffusion. When the permeation of CyA across Caco-2 monolayers from the apical to the basolateral side was determined, overall transport had an apparently saturable component up to a concentration of 1 microM. At higher concentrations permeation increased over-proportionally. Calculation of the kinetic parameters of apical to basolateral permeation suggested a diffusional process with a KD of 0.5 microliter min-1 per filter, which was overlayed by an active system in basolateral to apical direction with a KM of 3.8 microM and a Jmax of 6.5 picomol min-1 per filter. 3. CyA permeation was significantly higher when the drug was given from the basolateral side as compared to the permeation from the apical side. Apical to basolateral transport of CyA was increased in the presence of vinblastine, daunomycin and a non-immunosuppressive CyA-derivative. All compounds inhibit p-glycoprotein-mediated transport processes. Basolateral to apical permeation of CyA showed a dose-dependent decrease in the presence of vinblastine. Permeation of daunomycin across Caco-2 cell monolayers was also higher from the basolateral to the apical side than vice versa. Basolateral to apical permeation was decreased in the presence of SDZ PSC 833 and cyclosporin A. 4. Western blot analysis of Caco-2 cells with the monoclonal antibody C219 confirmed the presence of p-glycoprotein in the used cell system. 5. When the absorption of CyA in the gastrointestinal (GI)-tract of healthy volunteers was determined, a remarkable decrease of the plasma AUC could be observed dependent on the location of absorption in the rank order stomach > jejunum/ileum > colon. The decrease in absorption exhibited a marked correlation (r = 0.994) to the expression of mRNA for p-glycoprotein over the GI-tract (stomach < jejunum < colon). 6. All data provide evidence that CyA is a substrate of p-glycoprotein in the GI-tract, which might explain the local differences and the high variability in cyclosporin absorption found in vivo.     

Release and absorption characteristics of chlorphenesin carbamate sustained-release formulations: In vitro-in vivo and in vivo dog-human correlations

The bioavailability of chlorphenesin carbamate (CPC) in sustained-release (SR) formulations exhibiting different in vitro release characteristics in dogs and humans was examined using formulations tested previously in the in vitro dissolution method. The human-dog correlation of the bioavailability of SR formulations was examined under fasting conditions. Pharmacokinetic analysis using the Wagner-Nelson procedure revealed sustained-release absorption characteristics for the SR formulations, with the exception of the immediate-release (IR) formulation as control in dogs and humans. For each of the SR formulations tested, regression analysis results of the percentage of CPC absorbed in human against that in dogs, at corresponding times, indicated a high correlation. Moreover, the correlation of the dissolution rates and bioavailabilities of these formulations in humans was also examined. Although the in vitro CPC release profiles from the SR formulations were smooth and controlled, they were too rapid when compared with the in vivo human data. However, the rank order was exactly the same between in vivo and in vitro data, and a good relationship was found after time scaling of the release data. These data imply that the release characteristics of CPC after changing of the formulations could be evaluated using the in vitro dissolution method or dogs as an animal model in place of human studies.     

Pharmacokinetics and pharmacodynamics analysis of transdermal iontophoresis of 5-OH-DPAT in rats: in vitro-in vivo correlation

Pharmacokinetics and dopaminergic effect of dopamine agonist 5-OH-DPAT in vivo were determined following transdermal iontophoresis in rats based on drug concentration in plasma (C(p)) and dopamine levels in striatum (C(DA)). Correlation of the in vitro transport with the pharmacokinetic-pharmacodynamic (PK-PD) profiles was characterized in the transport in dermatomed rat skin (DRS) and rat stratum corneum (RSC). The integrated in vivo PK-PD and in vitro transport models successfully described time course of C(p), C(DA), and in vitro flux in DRS and RSC. Population value of steady-state flux (J(ss)) in vivo (31 nmol/cm(2) . h with 95% confidence interval (CI) = 20-41) is closer to J(ss) in vitro in DRS (61 nmol/cm(2) . h, CI = 54-67) than in vitro J(ss) in RSC (98 nmol/cm(2) . h, CI = 79-117). On the other hand, skin release rate constant (K(R)) in vivo was similar to the K(R) in RSC (4.8/h, CI = 2.4-7.1 vs. 2.6/h, CI = 2.5-2.6). Kinetic lag time (t(L)) in vivo was negligible, which is close to in vitro t(L) in RSC (0.0 h, CI = 0.0-0.1). Based on nonlinear mixed-effect modeling, profiles of C(p) and C(DA) were successfully predicted using in vitro values of J(ss) in DRS with K(R) and t(L) in RSC. A considerable dopaminergic effect was achieved, indicating the feasibility to reach therapeutically effective concentrations of 5-OH-DPAT upon transdermal iontophoresis.     

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-l-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H₂O₂, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H₂O₂. Furthermore, both arsenic and H₂O₂ were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation.     

非诺贝特缓释微球片体内外相关性评价

目的：用脱卷积法进行自制非诺贝特缓释微球片体内外相关性的评价。方法：以市售微粒化非诺贝特胶囊剂的大鼠体内血药浓度数据为权函数，根据自制非诺贝特缓释微球片制剂大鼠体内血药浓度数据，采用脱卷积法，将不同时间点的体内吸收分数与体外累计释放百分数做线性回归，考察体内外相关性。结果：采用脱卷积法计算体外累计释放百分数和体内吸收分数相关系数较好（r=0．9225），相关性方程为Y=0．4642R＋42．044。结论：脱卷积法适用于非诺贝特缓释微球片的体内外相关性研究。     

Comparative in vitro-in vivo percutaneous absorption of the pesticide propoxur.

In vitro and in vivo skin absorption of the pesticide propoxur (2-isopropoxyphenyl N-methyl carbamate, commercially Baygon(TM) and Unden (TM); log Po/w 1.56, MW 209.2) was investigated. In vivo studies were performed in rats and human volunteers, applying the test compound to the dorsal skin and the volar aspect of the forearm, respectively. In vitro experiments were carried out in static diffusion cells using viable full-thickness skin membranes (rat and human), non-viable epidermal membranes (rat and human) and a perfused-pig-ear model. Percutaneous penetration of propoxur in human volunteers was measured by analysis of its metabolite (2-isopropoxyphenol) in blood and urine; in all other studies radiolabeled propoxur ([ring-U-(14)C]propoxur) was used. In order to allow for direct comparison, experimental conditions were standardized with respect to dose (150 microg propoxur per cm(2)), vehicle (60% aqueous ethanol) and exposure time (4 h). In human volunteers, it was found that approximately 6% of the applied dose was excreted via the urine after 24 h, while the potential absorbed dose (amount applied minus amount washed off) was 23 microg/cm(2). In rats these values were 21% and 88 microg/cm(2), respectively. Data obtained in vitro were almost always higher than those obtained in human volunteers. The most accurate in vitro prediction of the human in vivo percutaneous absorption of propoxur was obtained on the basis of the potential absorbed dose. The absorbed dose and the maximal flux in viable full-thickness skin membranes correlated reasonably well with the human in vivo situation (maximal overestimation by a factor of 3). Epidermal membranes overestimated the human in vivo data up to a factor of 8, but the species-differences observed in vivo were reflected correctly in this model. The data generated in the perfused-pig-ear model were generally intermediate between viable skin membranes and epidermal membranes.     

注射用乳酸-羟基乙酸共聚物微球的体内外相关性研究进展

注射用乳酸-羟基乙酸共聚物(polylactide-polyglycolide, PLGA)微球作为一种储库型释药系统,自1989年第1个产品Lupron depot获准在美国上市起,已成功用于多种疾病的治疗,具备在体内几天到几个月长时间释药的能力,可显著改善用药安全性,提升患者顺应性。体内外相关性(in vitro-in vivo correlation, IVIVC)研究给微球制剂的发展带来更多可能。IVIVC可以通过微球的体外释放行为阐述体内释药的动态信息,在表征微球性能的同时减轻各阶段的工作量,对药物的研发、生产变更和监督管理等具有指导或支持作用。本文将注射用PLGA微球的释放机制、体内外释放测定涉及的常用方法和理论进行归纳总结,重点讨论了IVIVC尤其是A级IVIVC在微球制剂领域的建立及应用,为进一步的微球体内外相关性研究提供参考。     

Pharmacokinetics and metabolism of a RAS farnesyl transferase inhibitor in rats and dogs: in vitro-in vivo correlation.

Compound I (1-(3-chlorophenyl)-4-[(1-(4-cyanobenzyl)-1H-imidazol-5-yl)methyl]piperazin-2-one) is a potent and selective inhibitor of farnesyl-protein transferase (FPTase). The pharmacokinetics and metabolism of compound I displayed species differences in rats and dogs. After oral administration, the drug was well absorbed in dogs but less so in rats. Following i.v. administration, compound I was cleared rapidly in rats in a polyphasic manner with a terminal t(1/2) of 41 min. The plasma clearance (CL(p)) and volume of distribution (V(dss)) were 41.2 ml/min/kg and 1.2 l/kg, respectively. About 1% of the dose was excreted in rat bile and urine as unchanged drug over a period of 24 h, suggesting that biotransformation is the major route of elimination of compound I. Using liquid chromatography (LC)-tandem mass spectometry, nineteen metabolites of compound I were identified in urine and bile from dogs and rats. Structures of two major metabolites were confirmed by LC-NMR. N-Dealkylation and phase II metabolism were the major metabolic pathways. Animal and human liver microsomal intrinsic clearance values were scaled to predict hepatic clearance and half-life in humans, and the predicted values were in good agreement to the in vivo data.     

氯氮平漂浮缓释胶囊体内外相关性评价

目的建立氯氮平漂浮缓释胶囊体内外相关性评价模型 ,评价体内外相关性。方法利用Wanger Nelson方法和卷积模型分别评价氯氮平漂浮缓释胶囊体内外相关性。通过反卷积方法由体外释放数据得到了氯氮平漂浮缓释胶囊的体内输入函数。利用基本卷积模型和扩展卷积模型预测了氯氮平漂浮缓释胶囊体内血药浓度峰值 (Cmax)和血药浓度 时间曲线下面积值 (AUC)。结果Wanger Nelson方法的相关系数为0 9798;反卷积方法得到的体内输入值与体外释药量相关性良好 ,相关系数为 0 996。扩展卷积模型预测的Cmax和AUC值与实验值误差 <6 % ;而基本卷积模型预测值误差 >2 8%。结论氯氮平漂浮缓释胶囊体内外相关性良好。应用扩展的卷积模型能很好的预测氯氮平体内血药浓度 ,预测的体内情况可用来指导处方筛选     

Dependence of in vitro-in vivo correlation analysis acceptability on model selections

The objectives of this study were to assess the influence of pharmacokinetic and dissolution model selection on the success of in vitro-in vivo correlation (IVIVC) analysis of fast-, medium-, and slow-dissolving metoprolol tartrate immediate-release tablet formulations. Several different compartmental models were fit to the fast formulation plasma data. Three candidate models with the best fits were ranked 1, 2, and 3 and used to predict AUC and Cmax of the medium and slow formulations. Acceptability of each model to predict the medium and slow formulations was determined using +/- 20% as the limit for acceptable relative prediction error. When the best dissolution models were used, models 1 and 2 each failed to adequately predict Cmax for slow formulation (-26.8% error for model 1 and -20.4% error for model 2). However, the less appropriate model 3 adequately predicted Cmax for slow formulation (-15.1% error). The selection of the dissolution model also determined the outcome of IVIVC analysis, again with a less appropriate model resulting in successful prediction. When the Weibull function was used to characterize dissolution, model 2 failed to adequately predict Cmax for slow formulation (-20.4% error); however, model 2 adequately predicted Cmax for slow formulation when dissolution was characterized using the poorer fitting first-order model (-14.4% error). These results indicate that the success or failure of external validation of these metoprolol tartrate tablets was dependent on the pharmacokinetic and the dissolution models employed. Considering the role of subjectivity in identifying pharmacokinetic and dissolution models, these findings suggest a need to develop objective criteria to identify models a priori to IVIVC analysis.     

3-methylindole-induced toxicity to human bronchial epithelial cell lines.

Transfected BEAS-2B cells that express different cytochrome P450 enzymes were used to assess whether human bronchial epithelial cell lines are target cells for 3-methylindole (3MI)-induced damage. Four different transfected BEAS-2B lines overexpressing P450s 2A6, 3A4, 2F1, and 2E1 (B-CMV2A6, B-CMV3A4, B-CMV2F1, and B-CMV2E1), respectively, were compared. The B-CMV2F1 and B-CMV3A4 cells were the most susceptible to 3MI-mediated cytotoxicity, measured by leakage of lactate dehydrogenase into the medium after a 48-h incubation. The toxicity was ameliorated by pretreatment with 1-aminobenzotriazole (ABT). Depletion of glutathione with diethylmaleate decreased the onset and increased the extent of cell death with 3MI. Thus, 3MI is cytotoxic to immortalized bronchial epithelial cells overexpressing 2F1 without concomitant depletion of GSH, but depletion of GSH modestly enhances the cytotoxicity of 3MI to human lung cells. Additional studies clearly demonstrated that a low concentration of 3MI (10 micro M) induced apoptosis in BEAS-2B cells that was measured by DNA fragmentation, and apoptosis was inhibited by the presence of ABT. The B-CMV2F1 cells overexpressing 2F1 demonstrated increased apoptosis (measured by Annexin-V binding) at 24 h with 100 micro M 3MI. Therefore, CYP2F1 in human bronchial epithelial lung cells may bioactivate 3MI to 3-methyleneindolenine, which induces programmed cell death at relatively low concentrations. Human lung cells may be susceptible to this prototypical pneumotoxicant.     

Phenytoin I: in vitro-in vivo correlation for 100-mg phenytoin sodium capsules

Dissolution profiles for 11 brands of phenytoin sodium capsules were carried out by the basket and paddle methods (USP) and the spin-filter method. The results from the dissolution studies have been correlated with observed differences in in vivo parameters (Cmax and tmax). The dissolution by the basket method at 50 rpm in water gave a correlation greater than 0.9. The results suggest the existence of two types of phenytoin sodium products on the market.     

In vitro-in vivo correlation for intrinsic clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo clearance for compounds metabolized by monoamine oxidase

Oxidative deamination of the GABA(A) partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CL(int)) values were determined and compared to the corresponding in vivo oral clearance (CL(PO)) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A. The intrinsic clearance, CL(int), of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CL(int), values were calculated as 0.008 and 0.002 ml/mg/min for CP-409,092 and sumatriptan, respectively). A similar correlation was observed from the in vivo metabolic data where the unbound oral clearance, CL(u)(PO), values in humans were calculated as 724 and 178 ml/min/kg for CP-409,092 and sumatriptan, respectively. The present work demonstrates that it is possible to predict in vivo metabolic clearance from in vitro metabolic data for drugs metabolized by the enzyme monoamine oxidase.     

In vitro-in vivo correlation for intrinsic clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo clearance for compounds metabolized by monoamine oxidase

1. Oxidative deamination of the GABA_A partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds.

2. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A.

3. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CL_int) values were determined and compared to the corresponding in vivo oral clearance (CL_PO) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A.

4. The intrinsic clearance, CL_int, of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CL_int, values were calculated as 0.008 and 0.002?ml/mg/min for CP-409,092 and sumatriptan, respectively). A similar correlation was observed from the in vivo metabolic data where the unbound oral clearance, CL(u)_PO, values in humans were calculated as 724 and 178?ml/min/kg for CP-409,092 and sumatriptan, respectively.

5. The present work demonstrates that it is possible to predict in vivo metabolic clearance from in vitro metabolic data for drugs metabolized by the enzyme monoamine oxidase.

     

Benomyl-induced effects of ORMDL3 overexpression via oxidative stress in human bronchial epithelial cells

The respiratory system is a major site of exposure route during pesticide use. Although pesticide exposure is associated with chronic respiratory diseases including asthma, the underlying pathophysiological mechanism remains to be elucidated. In this study, we investigated the in vitro effects of benomyl-induced ORMDL3 overexpression on the toxicological mechanism using the human bronchial epithelial cell line 16HBE14o-. Benomyl increased reactive oxygen species and Ca²⁺ levels, and asthma-related ADAM33 and ORMDL3 expression in 16HBE14o− cells. Considering the change in Ca²⁺ level and protein expression, we focused on ORMDL3 to elucidate the mechanism of benomyl-induced asthma. Antioxidant treatment showed that benomyl-induced ORMDL3 and endoplasmic reticulum stress could be triggered by oxidative stress. Furthermore, ORMDL3 knockdown alleviated the effects of benomyl on intracellular Ca²⁺, and the expression of metalloproteinases, and proinflammatory cytokines involved in the pathogenesis of asthma. In conclusion, our results suggest that benomyl-induced ORMDL3 overexpression via oxidative stress might be a mechanism involved in asthma. Moreover, antioxidants and alleviating mechanisms that reduce ORMDL3 levels could serve as promising therapeutic targets for pesticide-induced asthma.