Analysis of In Vivo Nuclear Factor-B Activation during Liver Inflammation in Mice: Prevention by Catalase Delivery

ABSTRACT Nuclear factor-B (NF-B) is a transcription factor that plays crucial roles in inflammation, immunity, cell proliferation, and apoptosis. Until now, there have been few studies of NF-B activation in whole animals because of experimental difficulties. Here, we show that mice receiving a simple injection of plasmid vectors can be used to examine NF-B activation in the liver. Two plasmid vectors, pNF-B-Luc (firefly luciferase gene) and pRL-SV40 (Renilla reniformis luciferase gene), were injected into the tail vein of mice by the hydrodynamics-based procedure, an established method of gene transfer to mouse liver. Then, the ratio of the firefly and R. reniformis luciferase activities (F/R) was used as an indicator of the NF-B activity in the liver. Injection of thioacetamide or lipopolysaccharide plus D-galactosamine increased the F/R ratio in the liver, and this was significantly (P Ͻ 0.001) inhibited by an intravenous injection of catalase derivatives targeting liver nonparenchymal cells. Imaging the firefly luciferase expression in live mice clearly demonstrated that the catalase derivatives efficiently prevented the NF-B-mediated expression of the firefly luciferase gene. Plasma transaminases and the survival rate of mice supported the findings obtained by the luminescence-based analyses. Thus, this method, which requires no genetic recombination techniques, is highly sensitive to the activation of NF-B and allows us to continuously examine the activation in live animals. In conclusion, this novel, simple, and sensitive method can be used not only for analyzing the NF-B activation in the organ under different inflammatory conditions but also for screening drug candidates for the prevention of liver inflammation

Minireview Current Approaches for Absorption, Distribution, Metabolism, and Excretion Characterization of Antibody-Drug Conjugates: An Industry White Paper

ABSTRACT An antibody-drug conjugate (ADC) is a unique therapeutic modality composed of a highly potent drug molecule conjugated to a monoclonal antibody. As the number of ADCs in various stages of nonclinical and clinical development has been increasing, pharmaceutical companies have been exploring diverse approaches to understanding the disposition of ADCs. To identify the key absorption, distribution, metabolism, and excretion (ADME) issues worth examining when developing an ADC and to find optimal scientifically based approaches to evaluate ADC ADME, the International Consortium for Innovation and Quality in Pharmaceutical Development launched an ADC ADME working group in early 2014. This white paper contains observations from the working group and provides an initial framework on issues and approaches to consider when evaluating the ADME of ADCs

Assessment of the value of high-performance thin-layer chromatography for the detection and characterisation of drugs and metabolites in biological fluids

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Reversed-phase extraction coupled with capillary gas chromatography Development of a fully automated technique for bioanalysis

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Pharmacokinetics and metabolism of lacidipine

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DMD Fast Forward

. Due to broad substrate specificity of CYP enzymes, it is possible for more than one enzyme to be involved in the metabolism of a single compound. It is also possible for one CYP enzyme to catalyze two or more metabolic pathways for the same drug. In vitro methods have been established to determine which CYP isoform(s) is (are) involved in the metabolism of a specific drug. This process, also referred to as CYP reaction phenotyping, integrates data obtained from native human liver microsomes, intact cell models, recombinant CYPs, and inhibition studies with CYP selective chemical inhibitors and specific antibodies (Parkinson, 1996; Rodrigues, 1999 This was shown by Guengerich and colleagues (1987; 1988) who demonstrated the CYP catalyzed oxidative ester cleavage of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester. The product, 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester, was formed in the presence of substrate, enzymatically active microsomes and an NADPH generating system. The formation of these products was observed with five different CYP enzymes, however the extent of formation varied with the enzyme (Guengerich, 1987; Guengerich et al., 1988). CYP Chemical Inhibitor Incubations with Pooled Human Liver Microsomes. Of the CYP inhibitors studied at 100 µ M 13 are known to mediate dexamethasone induction, with the GR requiring nanomolar levels and PXR requiring micromolar levels for activation. In addition to the liver, the expression of hCE-1 and hCE-2 has been observed in small intestine, colon, testis, kidney, spleen, and heart classes of carboxylesterases, respectively. As well, more general esterase classification systems have been proposed, based on substrate and inhibitor specificity or interactions with organophosphorous insecticides The role of esterases in the metabolism of compounds is becoming increasingly important as, during the discovery of new drugs, candidate molecules are being synthesized which may contain an ester function. However, due to the non-selective substrate and inhibition specificity of esterases, phenotyping of specific esterases involved in the metabolism of a given compound is difficult. Esterases have been reported to exhibit clinically relevant polymorphisms which can lead to variable clearance of drugs. Cholinesterase, or butyrylcholinesterase, is abundant in human plasma and serum and hydrolyzes the muscle relaxant succinylcholine to succinylmonocholine and choline (Lockridge, 1990; Daly, 1993). Succinylcholine was introduced for use in 1951 having a quick onset of action resulting in complete paralysis, and a rapid recovery free of toxic side-effects. In some patients however, paralysis lasted for hours, instead of minutes, which often required life-saving intervention. For detailed discussion of the biochemistry and pharmacogenomics of cholinesterase, the authors refer the reader to Lockridge (1990); Darvesh et al. (2003); and Kalow (2004). Another esterase identified as having genetic variants is paraoxonase-1 (PON1). PON1 is a serum enzyme that catalyzes the hydrolysis of organophosphate esters, carbamates and aromatic carboxylic acid esters. For detailed discussion of the pharmacogenomics and catalytic efficiency of PON1, refer to Costa et al. (2003). Since it has been shown that CYP enzymes, in addition to esterases, catalyze the cleavage of ester-containing compounds, it becomes important to understand the involvement of these different enzyme families in the metabolism of a drug candidate. The determination of which enzyme family is involved may have an impact on the Using FD to measure human liver microsomal esterase activity, alphanaphthoflavone, clotrimazole ketoconazole, miconazole, nicardipine, and verapamil significantly inhibited the formation of the fluorescein product. The apparent IC 50 values of esterase activity for alpha-naphthoflavone and ketoconazole were 18 µ M and 6.5 µ M, respectively Much attention is directed towards the CYP metabolism of drugs and as a result, many other biotransformation enzymes, such as esterases, often get overlooked. Esterases however, are responsible for the metabolism of a number of endogenous and exogenous compounds. They can be induced, inhibited, and are subject to genetic polymorphisms, which can have clinical implications for the development of a drug. The results from the current study suggest that CYP chemical inhibitors should not be used to assess the role of CYP enzymes in the biotransformation of esters. Their potential to inhibit human liver microsomal esterase activity may result in an overestimation of the contribution of CYP enzymes in the metabolism of esters leading to a misinterpretation of potential drug-drug interactions. In contrast, CYP mAbs may be a useful tool to determine the contribution of CYP enzymes on the metabolism of esters as they were shown to have no effect on human liver microsomal esterase activity. Additional experiments to assess the contribution of oxidative enzymes in the metabolism of esters may include incubations in the presence and absence of β -nicotinamide adenine dinucleotide 2'-phosphate reduced (β-NADPH). DMD #9704 17 ACKNOWLEDGMENT

Copyright © 2007 by Institute ofPharmacology Polish Academy ofSciences

Citrus juices can interact with a number of medications. As little as 250 ml of grapefruit or orange juice can change the metabolism of drugs. Most drugs affected by citrus juices are known to be metabolized by CYP3A4. Recently, some studies have shown an in vitro inhibition of CYP2D6 metabolism by grapefruit juice. CYP2D6 enzyme is an important factor in the metabolism of 20–25 % of clinically important drugs, such as antidepressants, neuroleptics, antiemetics, cardiologic drugs and opioids. The aim of our study was to investigate the effects of orange juice on human cytochrome CYP2D6 enzyme activity. The study involved twenty unrelated healthy volunteers. Ten of them received 250 ml (one glass) of orange juice daily for 5 days. Control group consisted of ten persons who received 250 ml (one glass) of tap water. The CYP2D6 phenotype was analyzed before and after 5 days of juice/water ingestion, using sparteine as a model drug. Sparteine and its metabolites were determined in urine by the method of Eichelbaum. Metabolic ratio (MR) was calculated as the ratio of amount of parent drug to the total amount of metabolites. Mean MR, measured after 5 days of drinking the juice, increased by only 7 % in group receiving orange juice, and by 10 % in control group receiving tap water. Our results suggest that orange juice has no significant influence on metabolism and excretion of CYP2D6-dependent drugs. So it is not necessary to advise patients against drinking orange juice at the same time when they take those drugs. Key words: orange juice, CYP2D6, drug metabolism Pharmacological Reports, 2007, 59, suppl. 1, 173–176 173Abbreviations: ADR – adverse drug reaction, AUC – area under curve, C max – maximal concentratio

Inhibition of intestinal OATP2B1 by the calcium receptor antagonist ronacaleret results in a significant drug-drug interaction by causing a two-fold decrease in exposure of rosuvastatin

Abbreviations: AUC, area under the curve; BCRP, human breast cancer resistance protein; Cmax, maximal plasma concentration; DDI, drug-drug interaction; MRP, multidrug resistance protein; NTCP, sodium-taurocholate cotransporting polypeptide; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; P-gp, P-glycoprotein; Tmax, time to maximal plasma concentrations; t 1/2 , apparent half-life DMD #72397 4 ABSTRACT Rosuvastatin is a widely prescribed anti-hyperlipidemic which undergoes limited metabolism, but is an in vitro substrate of multiple transporters (OATP1B1, OATP1B3, OATP1A2, OATP2B1, NTCP, BCRP, MRP2, MRP4, OAT3). It is therefore frequently used as probe substrate in clinical drug-drug interaction (DDI) studies to investigate transporter inhibition. While each of these transporters is believed to play a role in rosuvastatin disposition, multiple pharmacogenetic studies confirm that OATP1B1 and BCRP play an important role in vivo. Ronacaleret, a drug development candidate for treatment of osteoporosis (now terminated), was shown to inhibit OATP1B1 in vitro (IC 50 11 µM), while it did not inhibit BCRP. Since a DDI risk through inhibition of OATP1B1 could not be discharged, a clinical DDI study wa