Pharmacokinetic aspects and in vitro–in vivo correlation potential for lipid-based formulations

Lipid-based formulations have been an attractive choice among novel drug delivery systems for enhancing the solubility and bioavailability of poorly soluble drugs due to their ability to keep the drug in solubilized state in the gastrointestinal tract. These formulations offer multiple advantages such as reduction in food effect and inter-individual variability, ease of preparation, and the possibility of manufacturing using common excipients available in the market. Despite these advantages, very few products are available in the present market, perhaps due to limited knowledge in the in vitro tests (for prediction of in vivo fate) and lack of understanding of the mechanisms behind pharmacokinetic and biopharmaceutical aspects of lipid formulations after oral administration. The current review aims to provide a detailed understanding of the in vivo processing steps involved after oral administration of lipid formulations, their pharmacokinetic aspects and in vitro in vivo correlation (IVIVC) perspectives. Various pharmacokinetic and biopharmaceutical aspects such as formulation dispersion and lipid digestion, bioavailability enhancement mechanisms, impact of excipients on efflux transporters, and lymphatic transport are discussed with examples. In addition, various IVIVC approaches towards predicting in vivo data from in vitro dispersion/precipitation, in vitro lipolysis and ex vivo permeation studies are also discussed in detail with help of case studies.KEY WORDS: Pharmacokinetics, Lipolysis, IVIVC, Efflux transporters, Lymphatic delivery, Food effectAbbreviations: ADME, absorption/distribution/metabolism/elimination; AUC, area under the curve; BCS, biopharmaceutics classification system; BDDCS, biopharmaceutics drug disposition classification system; CACO, human epithelial colorectal adenocarcinoma cells; C_max, maximum plasma concentration; CMC, critical micellar concentration; CYP, cytochrome; DDS, drug delivery systems; FaSSGF, fasted-state simulated gastric fluid; FaSSIF, fasted-state simulated intestinal fluid; FeSSIF, fed-state simulated intestinal fluid; GIT, gastrointestinal tract; IVIVC, in vitro in vivo correlation; LCT, long chain triglyceride; LFCS, lipid formulation classification system; log P, n-octanol/water partition coefficient; MCT, medium chain triglyceride; MDCK, Madin–Darby canine kidney cells; NCE, new chemical entity; P-app, apparent permeability; P-gp, permeability glycoprotein; SCT, short chain triglyceride; SEDDS, self-emulsifying drug delivery system; SIF, simulated intestinal fluid; SMEDDS, self-microemulsifying drug delivery system; SNEDDS, self-nanoemulsifying drug delivery system; Vit E, vitamin E     

The serological response in dogs inoculated with canine distemper virus vaccine at the acupuncture point governing vessel-14: A randomized controlled trial

The improvement of immunity to vaccination has historically focused on manipulation of antigen presentation rather than the host. Immune modulation by stimulating specific acupuncture points along the Meridian System has been practiced in Traditional Chinese Medicine. The purpose of this study was to quantitatively determine whether acupoint vaccination, in which vaccine is administered at an acupuncture point in dogs, has the potential to enhance the immune response.A randomized controlled trial was conducted to compare the effectiveness of acupoint vaccination versus a conventional method, based on humoral immune response in dogs given Canine Distemper Vaccine (CDV). One hundred client-owned dogs were admitted to the study with following characteristics: (1) passed a routine physical exam, (2) aged between 1 and 10?years old, (3) had no history of chronic disease, and (4) were not on immunomodulating medications. Dogs were randomly assigned to either the Acupuncture group inoculated at the acupoint Governing Vessel (GV)-14, or to the Control group inoculated conventionally at a non-acupuncture site. Mean changes from Day0 to Day14 of the response to CDV vaccination, measured by serum neutralization (SN) titers with log-transformation for reducing outlier effects, were compared between groups. No significant difference was found between groups in age, weight, or sex (all p?>0.2). Both groups had significant increases of CDV SN titer post-vaccination (p?<?0.001). The mean increase in Acupuncture group (0.72; SD?=?0.79) was significantly greater than that of the Control group (0.36; SD?=?0.67); p?=?0.019. Inference on percentage of change in raw SN titer data further revealed that the effects in the Acupuncture group was significantly greater than the Control group (242% vs. 83%; p?=?0.02).This study demonstrated that Acupoint vaccination at GV-14 resulted in a significantly elevated humoral immune response to CDV vaccine compared to Controls, which suggests the potential of acupoint vaccination to enhance the immune response.     

In vitro anti-hepatitis B and SARS virus activities of a titanium-substituted-heteropolytungstate

A structural determined heteropolytungstate, [K₄(H₂O)₈Cl][K₄(H₂O)₄PTi₂W₁₀O₄₀]·NH₂OH 1, has been synthesized and evaluated for in vitro antiviral activities against hepatitis B (HBV) and SARS virus. The identity and high purity of compound 1 were confirmed by elemental analysis, NMR, IR analysis and single-crystal X-ray diffraction. The compound 1, evaluated in HepG 2.2.15 cells expressing permanently HBV, significantly reduced the levels of HBV antigens and HBV DNA in a dose-dependent and time-dependent manner. EC₅₀ values were determined to be 54 μM for HBeAg, 61 μM for HBsAg and 2.66 μM for supernatant HBV DNA, as compared to 1671, 1570, 169 μM, respectively, for the commercially-available hepatitis B drug adefovir dipivoxil (ADV). Intracellular cccDNA, pgRNA and HBcAg were also found to be decreased by compound 1 in a concentration-dependent manner. Cytotoxicity results showed that compound 1 has low toxicity in HepG 2 cells with CC₅₀ value of 515.20 μM. The results indicate that compound 1 can efficiently inhibit HBV replication in HepG 2.2.15 cells line in vitro. Additionally, compound 1 also shows high anti-SARS activity at an EC₅₀ of 7.08 μM and toxicity with a CC₅₀ of 118.6 μM against MDCK cells.     

Effect of amiodarone on dispersion of ventricular repolarization in a canine congestive heart failure model

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Glucuronidation as a mechanism of intrinsic drug resistance in colon cancer cells: contribution of drug transport proteins

We have recently shown that drug conjugation catalysed by UDP-glucuronosyltransferases (UGTs) functions as an intrinsic mechanism of resistance to the topoisomerase I inhibitors 7-ethyl-10-hydroxycamptothecin and NU/ICRF 505 in human colon cancer cells and now report on the role of drug transport in this mechanism. The ability of transport proteins to recognise NU/ICRF 505 as a substrate was evaluated in model systems either transfected with breast cancer-resistance protein 1 (Bcrp1), multidrug-resistance protein 2 (Mrp2) or Mrp3, or overexpressing MRP1 or P-170 glycoprotein. Results from chemosensitivity assays suggested that NU/ICRF 505 was not a substrate for any of the above proteins. In drug accumulation studies in human colon cancer cell lines NU/ICRF 505 was taken up avidly and retained in cells lacking UGTs (HCT116), whereas, following equally rapid uptake, it was cleared rapidly from cells displaying UGT activity (HT29) as glucuronide metabolites. HT29 cells were shown to express MRP1 and 3, but not P-170 glycoprotein, MRP2 or breast cancer-resistance protein. The major glucuronide of NU/ICRF 505 inhibited ATP-dependent transport of estradiol 17-beta-glucuronide in Sf9 insect cell membrane vesicles containing MRP1 or MRP3, while co-incubation of HT29 cells with the MRP antagonist, MK571, significantly restored intracellular concentrations of NU/ICRF 505. These data lead us to conclude that the presence of a glucuronide transporter is essential for glucuronidation to represent a major de novo resistance mechanism and that UGTs will contribute more as a primary resistance mechanism when the parent drug (e.g. NU/ICRF 505) is not itself recognised by transport proteins.     

An in vitro and in silico study on the flavonoid-mediated modulation of the transport of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through Caco-2 monolayers

The present study describes the effect of different flavonoids on the absorption of the pro-carcinogen PhIP through Caco-2 monolayers and the development of an in silico model describing this process taking into account passive diffusion and active transport of PhIP. Various flavonoids stimulated the apical to basolateral PhIP transport. Using the in silico model for flavone, kaempferol and chrysoeriol, the apparent Ki value for inhibition of the active transport to the apical side was estimated to be below 53 muM and for morin, robinetin and taxifolin between 164 and 268 microM. For myricetin, luteolin, naringenin and quercetin, the apparent Ki values were determined more accurately and amounted to 37.3, 12.2, 11.7 and 5.6 microM respectively. Additional experiments revealed that the apical to basolateral PhIP transport was also increased in the presence of a typical BCRP or MRP inhibitor with apparent Ki values in the same range as those of the flavonoids. This observation together with the fact that flavonoids are known to be inhibitors of MRPs and BCRP, corroborates that inhibition of these apical membrane transporters is involved in the flavonoid-mediated increased apical to basolateral PhIP transport. Based on the apparent Ki values obtained, it is concluded that the flavonols, at the levels present in the regular Western diet, are capable of stimulating the transport of PhIP through Caco-2 monolayers from the apical to the basolateral compartment. This points to flavonoid-mediated stimulation of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients.     

曲美他嗪对犬急性心肌梗死后延迟再灌注中缺血区心肌细胞凋亡的影响

目的探讨曲美他嗪(trimetazidine)在犬急性心肌梗死后延迟再灌注中对梗死周边缺血区心肌细胞的保护作用及其机制。方法24只健康成年犬随机平均分为3组:假手术组、单纯延迟再灌注组(late reperfusion group,LR)、延迟再灌注预加曲美他嗪组(late reperfusion+trimetazidinegroup,LR+TMZ)。假手术组和LR组灌服生理盐水,LR+TMZ组灌服曲美他嗪(2mg·kg-1.d-1)共14d。所有犬于d14灌药2h后开胸,假手术组不结扎冠状动脉;LR和LR+TMZ在高位结扎左冠状动脉前降支10h,再灌注10h,制成AMI延迟再灌注模型。采用TUNEL法测心肌细胞凋亡指数(AI),免疫组化法测Bcl-2、Bax、细胞色素C和AIF蛋白表达。结果与LR组相比,LR+TMZ组心肌细胞AI明显降低,Bax、细胞色素C和AIF蛋白的表达明显减少(P<0.01),而Bcl-2蛋白的表达明显增高(P<0.01);分别与假手术组比较,差异均有显著性(P<0.01)。结论曲美他嗪在急性心肌梗死后延迟再灌注中可减少梗死周边缺血区心肌细胞的凋亡,其机制可能与增加Bcl-2蛋白的表达,减少Bax、细胞色素C、AIF蛋白的表达有关。     

活血通脉片对麻醉犬心肌缺血程度及心肌梗塞范围的影响

目的：观察活血通脉片（ＨＸＴＭＴ）对实验犬心肌缺血及民肌梗塞的影响。方法：采用心外膜电图标测心肌缺血范围及程度,定量组织学（Ｎ－ＢＴ染色法）测定心肌梗塞范围。结果：实验结果表明,活血通脉片消化道给药具有明显改善犬急性心肌缺血程度（∑－ＳＴ）（Ｐ〈０．５￣Ｐ〈０．０１）及心肌缺血范围（Ｎ－ＳＴ）（Ｐ〈０．００１）,明显减少经Ｎ－ＢＴ染色所显示的心肌梗塞区（Ｐ〈０．０１）结论：活血通脉片为用于心肌缺血     

左肺移植的实验研究

目的为临床肺移植积累经验。方法分析总结29例犬左肺移植的资料。其中15例的供体肺用Euro-Collings液在4℃下保存6h,14例用UW液保存22～25h,作同种异体移植,并阻断受体的右肺动脉,以测定移植左肺的功能。结果所有的移植左肺均能胜任气体交换,循环稳定。结击本左肺移植方法可靠,可供临床应用时参考。     

Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of alpha,beta-unsaturated carbonyl compounds

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of alpha,beta-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. Alpha,beta-unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition.