Drug Metabolism Letters - Volume 1, Issue 3, 2007

Inflammation is a hallmark of various diseases like infections, autoimmune syndromes, cardiovascular and neurodegenerative disorders, and cancer. Thereby, Th1-type cytokine interferon-γ is a central pro-inflammatory mediator. Antioxidant compounds counteract the activation of T-cells and macrophages. This anti-inflammatory activity could be important for the development of immunotolerance and for treatment of above-mentioned disorders.

Three neurotransmitters, namely adrenaline, serotonin and tryptamine inhibit the in vitro activity of several cytochrome P450 (CYP) isozymes (CYP1A2, CYP2C9, CYP2D6 and CYP3A). In order to test whether this effect is related to inhibition of the CYP-coupled NADPH reductase activity, we assayed the potential inhibitory effect of these neurotransmitters and their main metabolites on the NADPH reductase activity. Of the five compounds analyzed: tryptamine, tryptophol, serotonin, 5-hydroxytryptamine and adrenaline, only adrenaline significantly decreased NADPH reductase activity at the fixed concentration of 500 μM. However, the effect became negligible when adrenaline concentration was decreased to 100 μM: whereas a high inhibitory effect was observed in CYP2D6, CYP2C9 and CYP3A4 enzyme activities, the NADPH reductase activity remains unchanged. This study indicates that the effect of these endogenous neurotransmitters on CYP enzymes is not related to changes in the reductase activity. In the light of these findings further studies on the inhibitory effect of these neurotransmitters on CYP enzymes can be designed ruling out the modulation of the coupled NADPH reductase activity as a confounding factor.

Certain anti-cancer prodrugs are subject to cytochrome P450 (CYP)-mediated metabolism and become more active. Because CYP activity may be regulated by phosphorylation via adenylyl cyclase/protein kinase A, selective adenylyl cyclase subtype activators may be utilized in future chemotherapy to regulate CYP activity as a switch in a tumor tissue-specific manner.

Profiling of rat plasma using a highly sensitive LC-ARC-MS technique showed that [3H] mefenamic acid was metabolized to several products, including a sulfate conjugate and a hydroxylated analogue as major metabolites. This technique of detecting low levels of radioactivity in plasma was superior to previously used methods, such as β-RAM detectors.

The transport of hydroxyurea, a ribonucleoside reductase inhibitor, over biological membranes is slow and it has therefore been suggested that the substance could interact with an active efflux transporter. The transport of [11C]hydroxyurea into the rat brain was therefore studied after administration of the multidrug resistance protein inhibitor probenecid (50 and 150 mg/kg), the P-glycoprotein inhibitor cyclosporin A (25 mg/kg), hydroxyurea (50, 150 and 450 mg/kg) and mannitol (25%). None of the intervention drugs affected the brain uptake of [11C]hydroxyurea. The brain-toplasma concentration ratios (Kp), with or without intervention drug, were in the range 0.12-0.25 after 60 min of [11C]hydroxyurea infusion. [11C]Verapamil, a P-glycoprotein substrate with low brain penetration, was used to study the ability of hydroxyurea to inhibit P-glycoprotein. Administration of hydroxyurea (150 and 450 mg/kg) did not increase brain concentrations of [11C]verapamil. It is therefore unlikely that hydroxyurea is a substrate for or an inhibitor of Pglycoprotein or a substrate for a probenecid sensitive transport system. The low brain concentrations may instead be the result of slow uptake due to the hydrophilic nature of hydroxyurea.

Antagonism of the melanocortin 4 receptor (MC4R) has been proposed as a therapeutic intervention for the prevention of lean body mass waste, as in cachexia. Pharmacokinetic profiles of substituted 1,2-diarylethane MC4R antagonists were determined in rats after a single intravenous (IV) administration at 1 mg/kg. Brain and plasma concentrations of these compounds were determined at 1 and 4 hours after an oral dose at 10 mg/kg, since oral administration is the intended clinical dosing route and the pharmacological target is the central nervous system. The brain to plasma concentration ratios (0.10 - 50) after oral dosing correlated well with Vdss (2.21 to 81.4 L/kg; R2=0.810) determined after IV administration. A good correlation was also observed between the brain AUC0-4hr (119 - 18400 nM*hr) and Vdss (R2=0.981). Thus, further screening and ranking of substituted 1,2-diarylethanes for their brain uptakes could be carried out more efficiently via the simple and indirect Vdss screen after intravenous administration in rats.

There is an increasing application of protein/peptide drugs in the treatment of various diseases such as cancer and autoimmune diseases in clinical settings. However, data is scant on the potential for modulation of cytochrome P450s (CYPs) by these protein/peptide drugs. In this study, we examined the effects of recombinant human thymosin-α1 (rh- Tα1) on hepatic cytochrome P450 (CYP) enzyme activity in rats in vitro and in vivo. For the in vitro experiments, rh-Tα1 was incubated with the probe drugs and the liver microsomes from rats, while rh-Tα1 was administered to rats subcutaneously at 150, 300, or 600 μg/kg daily for two weeks in in vivo studies. The activities of six rat hepatic CYP enzymes, namely CYP1A2, CYP2C6, CYP2C11, CYP2D2, CYP2E1, and CYP3A1/2, were determined by a cocktail of probe drugs including phenacetin (O-deethylation), tolbutamide (4-hydrolylation), omeprazole (5-hydroxylation), dextromethorphan (O-demethylation), chlorzoxazone (6-hydroxylation), and nifedipine (N-dehydrogenation), respectively. Co-incubation of rh-Tα1 at the concentration of 20 and 50 μmol/l with the liver microsomes significantly inhibited CYP2E1 activity, whereas there was no significant effect on the activities of CYP1A2, CYP2C6, CYP2C11, CYP2D2, and CYP3A1/2. As to in vivo studies, treatment of rh-Tα1 at either dosage did not significantly alter the liver weight. However, an ex vivo study demonstrated that the activity of rat hepatic CYP2E1 was significantly increased by pretreatment of rh-Tα1 at the three doses for two weeks, and the activities of CYP1A2, CYP2D2, and CYP3A1/2 were also significantly increased in rats pretreated with rh-Tα1 at 600 μg/kg. These data indicate that rh-Tα1 can modulate the activities of major rat CYP isoforms, and further studies are needed to investigate its effect on human CYP activities and the potential for causing drug interactions.

Tanshinone IIB (TSB) is a major constituent of Salvia miltiorrhiza, which is widely used in treatment of cardiovascular and central nervous system (CNS) diseases such as coronary heart disease and stroke. This study aimed to investigate the role of various drug transporters in the brain penetration of TSB using several in vitro and in vivo mouse and rat models. The uptake and efflux of TSB in rat primary microvascular endothelial cells (RBMVECs) were ATPdependent and significantly altered in the presence of a P-glycoprotein (P-gp) or multidrug resistance associated protein (Mrp1/2) inhibitor. A polarized transport of TSB was found in RBMVEC monolayers with facilitated efflux from the abluminal to luminal side. Addition of a P-gp inhibitor (e.g. verapamil) in both abluminal and luminal sides attenuated the polarized transport. In an in situ rat brain perfusion model, TSB crossed the blood-brain barrier (BBB) and bloodcerebrospinal fluid barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a P-gp or Mrp1/2 inhibitor. The brain levels of TSB were only about 30% of that in the plasma and it could be increased to up to 72% of plasma levels when verapamil, quinidine, or probenecid was co-administered in rats. The entry of TSB to CNS increased by 67-97% in rats subjected to middle cerebral artery occlusion or treatment with the neurotoxin, quinolinic acid, compared to normal rats. Furthermore, The brain levels of TSB in mdr1a(-/-) and mrp1(-/-) mice were 28- to 2.6-fold higher than those in the wild-type mice. TSB has limited brain penetration through the BBB due to the contribution of P-gp and to a lesser extent of Mrp1 in rodents. Further studies are needed to confirm whether these corresponding transporters in humans are involved in limiting the penetration of TSB across the BBB and the clinical relevance.

Hammerhead ribozymes (HRz), catalytic RNA molecules capable of inducing the site-specific cleavage of a phosphodiester bond within an RNA molecule, are typically introduced into target cells by specific constructs (such as viral vectors) able to drive their expression from defined expression cassettes (promoter). This strategy implies the presence of promoter-derived sequences bound to the hammerhead ribozyme structure, a fact which can unpredictably affect HRz cleavage efficiency and eventually the biological effect. We explored the effects of promoter-derived sequences on the cleavage kinetics of an HRz targeted against a relevant cell cycle regulator, i.e. cyclin E1, implicated in the pathogenesis of several human diseases including in-stent restenosis and hepatocellular carcinoma. Sequences derived form the most commonly used promoters (CMV, T7, Pol I and Pol III promoters) were added to the minimal HRz structure and their effects on the cleavage kinetic constants kcat and Km evaluated in vitro under single turn-over conditions, using a mathematical model we recently developed. The different promoter derived sequences variably affected HRz cleavage efficiency (kcat/Km) with those derived from the pol III and from a truncated form of T7 promoter (T7-S), impairing maximally and minimally kcat/Km, respectively. Additionally, the extra sequences tend to increase Km and to reduce kcat. The extent of this effect depends both on the secondary RNA structure and on the length of the added sequences. In conclusion, these data, together with further work in cultured cells, can lead to the selection of optimal expression cassettes thus contributing to improve HRz efficacy, bringing these molecules closer to practical applications.

Metabolite profiling of 100- and 1,000-fold diluted urine and plasma samples from a conventional radiolabeled human ADME study is described using a highly sensitive LC-AMS technique. The concentration of radioactivity and the metabolic profiles in urine and plasma determined using this technique were similar to those employing standard off-line (i.e. LSC) or in-line (i.e. β-RAM or LC-ARC dynamic-flow) radioactivity monitoring techniques. The results indicate that at a simulated ca. 100 nCi clinical dose, plasma and urine concentrations of 14C, as well as their metabolic profiles, may be determined routinely by LC-AMS. This approach opens the possibility of using LC-AMS for both the highthroughput quantitation of biological samples and the generation of high-resolution chromatographic profiles of complex mixtures at a lower cost than current AMS analyses that require the conversion of sample carbon to graphite, a laborious and time consuming process.

Site-specific mono-PEGylations were performed in different conformational regions of Thymosin alpha 1 (Tα1) by introducing one cysteine residue into the chosen site and coupling with thiol-specific mPEG-MAL reagent. Results demonstrated that PEGylated sites and regions influenced the conformations and pharmacokinetic profiles of the peptide greatly with following order: α-helix, β-turn, random coil and terminals, but little on the immunoactivity.