Development of an in vivo rat screen model to predict pharmacokinetic interactions of CYP3A4 substrates

With the advent of polytherapy, drug interactions have become a common clinical problem. Although in vitro data are routinely used for the prediction of drug interactions, in vitro systems are not dynamic and sometimes fail to predict drug interactions. We sought to use the rat as an in vivo screening model to predict pharmacokinetic interactions with ketoconazole. The pharmacokinetic studies were conducted following an oral dose of CYP3A substrates and an optimized oral regimen of ketoconazole. In vitro reaction phenotyping was conducted using individual human and rat cDNA-expressed CYP enzymes and human or rat liver microsomes in the presence of ketoconazole. The in vitro experiments indicated that the test compounds were largely metabolized by CYP3A in both human and rat. The compounds could be rank-ordered with respect to the increase in C_max and area under the curve (AUC) values relative to midazolam in the presence of ketoconazole. The degree of pharmacokinetic interaction with ketoconazole was dependent, in part, upon their in vitro metabolism in the presence of rat CYP3A1/3A2 and in rat and human microsomes, co-incubated with ketoconazole, and on their fraction metabolized (f_m) in the rat relative to other disposition pathways. Based on the rank-order of interaction, the compounds could be prioritized for further preclinical development.     

Polymorphic behaviour of Compritol®888 ATO as bulk lipid and as SLN and NLC

Compritol®888 ATO (glycerol behenate) is widely used as a pharmaceutical excipient in the field of solid dosage forms due to its lubricating properties. It is an amphiphilic material with a high melting point (～70°C) and, therefore, it can also be used to prepare aqueous colloidal dispersions. The aim of this paper is to study the suitability of Compritol®888 ATO for the production of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for the entrapment of a lipophilic model drug. This study assesses the crystalline structure of the bulk lipid, as well as the changes that occur in its crystal lattice with the addition of ‘impurities’, such as oil (α-tocopherol) and drug (ketoconazole), using DSC and X-ray diffraction analysis before and after thermal stress. Aqueous SLN and NLC dispersions were produced using an appropriate surfactant/co-surfactant system and their physicochemical stability was assessed by PCS, LD, DSC and by WAXS. It was found that the crystalline lattice of Compritol®888 ATO is composed of very small amounts of the unstable α polymorphic form characteristic of triacylglycerols, which disappears after thermal stress of bulk lipid. Mixing oils and drug molecules which are soluble in this lipid decreased its lattice organization and, thus, was revealed to be suitable for production of lipid nanoparticles containing ketoconazole. However, particle growth could not be avoided during shelf life.     

The Combination of GIS and Biphasic to Better Predict In Vivo Dissolution of BCS Class IIb Drugs,Ketoconazole and Raloxifene

The formulation developments and the in vivo assessment of Biopharmaceutical Classification System (BCS) class II drugs are challenging due to their low solubility and high permeability in the human gastrointestinal (GI) tract. Since the GI environment influences the drug dissolution of BCS class II drugs, the human GI characteristics should be incorporated into the in vitro dissolution system to predict bioperformance of BCS class II drugs. An absorptive compartment may be important in dissolution apparatus for BCS class II drugs, especially for bases (BCS IIb) because of high permeability, precipitation, and supersaturation. Thus, the in vitro dissolution system with an absorptive compartment may help predicting the in vivo phenomena of BCS class II drugs better than compendial dissolution apparatuses. In this study, an absorptive compartment (a biphasic device) was introduced to a gastrointestinal simulator. This addition was evaluated if this in vitro system could improve the prediction of in vivo dissolution for BCS class IIb drugs, ketoconazole and raloxifene, and subsequent absorption. The gastrointestinal simulator is a practical in vivo predictive tool and exhibited an improved in vivo prediction utilizing the biphasic format and thus a better tool for evaluating the bioperformance of BCS class IIb drugs than compendial apparatuses.     

HPLC法同时测定复方酮康唑凝胶中酮康唑、硝酸咪康唑的含量

目的：建立同时测定复方酮康唑凝胶中酮康唑、硝酸咪康唑含量的方法。方法：采用HPLC法,色谱柱为HypersilBDSC18（416mm×200mm,5μm）;以0.5%醋酸铵溶液-0.2%三乙胺的甲醇溶液（20∶80）为流动相;流速为1.0ml·min-1;检测波长为230nm,柱温：30℃,进样量20μl。结果：酮康唑在5.1～510.0mg·L-1浓度范围内有良好的线性关系,r=0.9999。平均回收率为100.3%（RSD=0.38%,n=6）。硝酸咪康唑在5.0～500.0mg·L-1浓度范围内有良好的线性关系,r=0.9999,平均回收率=99.9%（RSD=0.79%,n=6）。结论：该方法能简便、快速、准确、灵敏地同时测定酮康唑、硝酸咪康唑的含量,可以用于复方酮康唑凝胶的质量控制。     

HPLC法同时测定离体透皮实验接收液中萘替芬和酮康唑浓度

目的:建立HPLC法同时测定离体透皮实验接收液中萘替芬和酮康唑浓度。方法:氯雷他定作内标,透皮液经乙醚提取后,采用Hypersil C18柱(5μm,5.0 mm×200 mm),以乙腈-甲醇-水-三乙胺(35:40:25:0.8,用冰醋酸调至pH 7.0)为流动相,流速1.2 mL·min-1;在紫外265 nm处检测。结果:萘替芬和酮康唑线性范围分别为0.08040-2.010μg·mL-1(r=0.9936)和0.2016-5.040μg·mL-1(r=0.9999),最低检出浓度分别为0.0201μg·mL-1和0.0403μg·mL-1,不同浓度的平均回收率(n=15)分别为102.6％和100.1％。结论:本法灵敏度、准确度及回收率均能达到离体透皮实验接收液中药物浓度的测定要求,也可用于药代动力学研究。     

高效液相色谱法测定酮康唑乳膏中酮康唑含量

目的建立测定酮康唑乳膏中酮康唑含量的方法。方法采用反相高效液相色谱法,固定相为十八烷基硅烷键舍硅胶柱（150mm×4．6mm,5um）,流动相为0．5％醋酸铵溶液-0．2％二异丙铵的甲醇溶液（23：77）,流速为1．0mL／min,检测波长为240nm。结果酮康唑质量浓度在0．1—2．0mg／mL范围内与峰面积呈良好的线性关系（r=0．9999）,最低检测限为0．2ug／mL。平均加样回收率为100．39％,RSD为0．58％（n=9）。结论该方法专属性好,准确、灵敏,可用于酮康唑乳膏中酮康唑的含量测定。     

Effect of ketoconazole on the pharmacokinetics of udenafil in healthy Korean subjects

AIMS

Udenafil is a phosphodiesterase 5 inhibitor used for the treatment of erectile dysfunction. It is metabolized to DA-8164, a major metabolite, by CYP3A4. This study was performed to investigate the effect of ketoconazole, a known CYP3A4 inhibitor, on the pharmacokinetics of udenafil.

METHODS

An open-label, two-period, fixed-sequence crossover study was performed in 12 healthy male volunteers. They received a single 100-mg oral dose of udenafil. Following a 5-day interval, 400 mg of ketoconazole was administered once a day for three consecutive days. On day 3 of ketoconazole treatment, a second 100 mg of udenafil was dosed concomitantly. Blood samples were collected at time points up to 48 h without ketoconazole treatment and up to 72 h with ketoconazole co-administration. The plasma concentration of udenafil was determined using liquid chromatography–tandem mass spectrometry.

RESULTS

Following ketoconazole co-administration, the mean C_max and AUC_last of udenafil (95% confidence interval) increased 1.9-fold (1.60, 2.27) and 3.2-fold (2.82, 3.63), respectively. The median time to reach the C_max was delayed in the co-administrated treatment, while the mean terminal elimination half-life (t_1/2) remained relatively unchanged regardless of ketoconazole co-administration. The metabolic AUC ratio (AUC_last of DA-8164/AUC_last of udenafil) was 1.71 when udenafil was administered alone, and the value decreased to 0.19 when udenafil was dosed in the presence of ketoconazole. Regarding safety assessments, no clinically significant difference or serious adverse event was observed.

CONCLUSIONS

The systemic exposure of udenafil increased significantly when it was administered with ketoconazole. Dose adjustment may be required when these drugs are used together.     

In vitro antifungal susceptibility testing of filamentous fungi with Sensititre Yeast One

Sensititre is a colorimetric microdilution method for in vitro antifungal susceptibility testing based on the M27-A document (National Committee for Clinical Laboratory Standards) for yeasts. Difference between both methods is the presence of Alamar-blue and RPMI 1640 (glucose 2%) as culture medium. Antifungal susceptibility to amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine, 100 opportunistic filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp.) obtained from pathological samples was determined by the Sensititre method. Induction to conidium and sporangiospore formation at 35 degrees C was used to get inoculum and plates were covered by 1 ml of saline and suspensions were made by gently probing by a sterile loop. Optical densities of the conidial suspensions were adjusted to 80-82% transmittance for Aspergillus spp. and 68-70% for the rest of strains tested. Final inoculum concentration size was 0.4 x 10(4)-5 x 10(4) CFU ml(-1). Readings were made at 72 h of incubation at 35 degrees C; amphotericin B and itraconazole was active against Aspergillus fumigatus with CMI90 1 and 0.5 microg ml(-1), respectively, opposite to Scedosporium prolificans and Scedosporium apiospermum. As it was expected, a CMI90 of 256 microg ml(-1) for fluconazole and CMI90 for flucytosine amounting to 64 g ml(-1) were obtained. Sensititre Yeast One is a useful method and an alternative to reference methods to determine antifungal susceptibility of filamentous fungi for clinical laboratory routine. Correlation with microdilution results is studied. New triazole derivatives should be included as soon as their clinical use will be feasible.     

有限采样法评价肝脏CYP3A代谢活性的实验研究

目的:以细胞色素P450(CYP)3A探针药物咪哒唑仑(MDZ)的系统清除率(Cls)为指标,评价有限采样法(LSS)预测肝脏CYP3A抑制状态下代谢活性的可行性。方法:采用系列剂量的CYP3A选择性抑制剂酮康唑预处理大鼠,静脉注射MDZ后在若干时间点收集血浆样品并检测MDZ浓度,经逐步回归分析和Jack-knife验证建立LSS模型。对经相同处理的另一随机群体进行验证分析,评价该LSS模型方程的准确性和重现性。结果:由两点(60、90min)或三点(30、60、90min或30、60、120min)血浆药物浓度建立的LSS预测模型所得到的Cls估计值(Clest)与实际计算值(Clobs)之间具有良好的相关性,误差小,特别是两点LSS模型则更为简便。结论:本研究结果表明,以MDZCls为指标,采用LSS评价大鼠肝脏CYP3A抑制状态下的代谢活性是一种准确而简便的方法,为今后推广到临床评价肝脏代谢功能从而制定和调整治疗药物的给药方案提供了理论依据和实验室证据。     

Evaluation of a 15-day screening assay using intact male rats for identifying steroid biosynthesis inhibitors and thyroid modulators.

An in vivo screening assay using intact adult male rats has been evaluated for its ability to detect four endocrine-active compounds (EACs) via oral (gavage) administration. The test compounds included the aromatase inhibitor fadrozole (FAD), the testosterone biosynthesis inhibitor ketoconazole (KETO), and the thyroid modulators phenobarbital (PB) and propylthiouracil (PTU). Three of the test compounds (KETO, PB, and PTU) have been previously evaluated in the 15-day intact male assay with compound administration via intraperitoneal injection (ip). For the current studies, male rats were dosed for 15 days via oral gavage and euthanized on the morning of test day 15. The endpoints evaluated included final body and organ weights (liver, thyroid gland, testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit [ASG]), serum hormone concentrations (testosterone [T], estradiol [E2], dihydrotestosterone [DHT], luteinizing hormone [LH,] follicle stimulating hormone [FSH], prolactin [PRL], T(3), T(4), thyroid stimulating hormone [TSH]), and histopathology of the testis, epididymis, and thyroid gland; positive results for each endpoint are described below. In addition, an evaluation of immune system endpoints (humoral immune function, spleen and thymus weights, and spleen cell number) was conducted on a subset of animals dosed with either KETO or PB. FAD and KETO decreased the weights for the androgen-dependent tissues and caused similar patterns of hormonal alterations (decreased serum T and DHT; increased serum FSH and/or LH). In addition, KETO caused spermatid retention. For FAD and KETO, effects on thyroid parameters were not indicative of thyroid toxicity. PB and PTU caused thyroid effects consistent with thyroid modulators (increased thyroid weight, decreased serum T(3) and T(4), increased serum TSH, thyroid follicular cell hypertrophy/hyperplasia, and colloid depletion). In addition, PB increased relative liver weight and altered reproductive hormone concentrations (decreased serum DHT, PRL, LH; increased serum E2). Orally administered KETO and PB did not alter the primary humoral immune response to sheep red blood cells (SRBC), although spleen weights were increased at the highest doses for both compounds. In the current study, all four test substances were identified as endocrine-active. The effects that were observed in the current study via oral (gavage) compound administration were similar to the responses that were observed by the ip route in previous studies for KETO, PB, and PTU. Overall, the sensitivity (i.e., the dose required to elicit similar magnitude responses) between the ip and oral routes of administration were similar for the three EACs that were examined by both routes of administration. This article, in addition to the > 20 compounds that have already been examined using the 15-day intact male assay, supports this assay as a viable screening assay for detecting EACs, and also illustrates that the ability to identify EACs using the intact male assay will be equivalent regardless of the route of compound administration.