Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Expression of cytochrome P450 enzymes and metabolism of timolol in human ocular tissue

Glaucoma is a group of progressive optic neuropathies causing irreversible blindness if not diagnosed and treated in the early state of progression. Disease is often, but not always, associated with increased intraocular pressure (IOP), which is also the most important risk factor for glaucoma. Ophthlamic timolol preparations have been used for decades to lower increased intraocular pressure (IOP). Timolol is locally well tolerated but may cause e.g. cardiovascular and pulmonary adverse effects due to systemic absorption. It has been reported that approximately 80% of a topically administered eye drop is systemically absorbed. However, only limited information is available on timolol metabolism in the liver or especially in the human eye. The aim of this work was to investigate metabolism of timolol in human liver and human ocular tissues. The expression of drug metabolizing cytochrome P450 (CYP) enzymes in the human ciliary epithelial cells was studied. The metabolism of timolol and the interaction potential of timolol with other commercially available medicines were investigated in vitro using different liver preparations. The absorption of timolol to the aqueous humor from two commercially available products: 0.1% eye gel and 0.5% eye drops and the presence of timolol metabolites in the aqueous humor were investigated in a clinical trial. Timolol was confirmed to be metabolized mainly by CYP2D6 as previously suggested. Potent CYP2D6 inhibitors especially fluoxetine, paroxetine and quinidine inhibited the metabolism of timolol. The inhibition may be of clinical significance in patients using ophthalmic timolol products. CYP1A1 and CYP1B1 mRNAs were expressed in the human ciliary epithelial cells. CYP1B1 was also expressed at protein level and the expression was strongly induced by a known potent CYP1B1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The CYP1B1 induction is suggested to be mediated by aryl hydrocarbon receptor (AHR). Low levels of CYP2D6 mRNA splice variants were expressed in the human ciliary epithelial cells and very low levels of timolol metabolites were detected in the human aqueous humor. It seems that negligible amount of CYP2D6 protein is expressed in the human ocular tissues. Timolol 0.1% eye gel leads to aqueous humor concentration high enough to achieve therapeutic effect. Inter-individual variation in concentrations is low and intraocular as well as systemic safety can be increased when using this product with lower timolol concentration instead of timolol 0.5% eye drops.

Literature Review: Investigating Drug Transport at the Blood-Brain Barrier and the Effects of P-glycoprotein on the Brain Distribution of Drugs.

The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.

Drug-drug interaction studies of cardiovascular drugs involving P-glycoprotein, an efflux transporter, on the pharmacokinetics of edoxaban, an oral factor Xa inhibitor.

BACKGROUND: Edoxaban, an oral direct factor Xa inhibitor, is in development for thromboprophylaxis, including prevention of stroke and systemic embolism in patients with atrial fibrillation (AF). P-glycoprotein (P-gp), an efflux transporter, modulates absorption and excretion of xenobiotics. Edoxaban is a P-gp substrate, and several cardiovascular (CV) drugs have the potential to inhibit P-gp and increase drug exposure. OBJECTIVE: To assess the potential pharmacokinetic interactions of edoxaban and 6 cardiovascular drugs used in the management of AF and known P-gp substrates/inhibitors. METHODS: Drug-drug interaction studies with edoxaban and CV drugs with known P-gp substrate/inhibitor potential were conducted in healthy subjects. In 4 crossover, 2-period, 2-treatment studies, subjects received edoxaban 60 mg alone and coadministered with quinidine 300 mg (n = 42), verapamil 240 mg (n = 34), atorvastatin 80 mg (n = 32), or dronedarone 400 mg (n = 34). Additionally, edoxaban 60 mg alone and coadministered with amiodarone 400 mg (n = 30) or digoxin 0.25 mg (n = 48) was evaluated in a single-sequence study and 2-cohort study, respectively. RESULTS: Edoxaban exposure measured as area under the curve increased for concomitant administration of edoxaban with quinidine (76.7 %), verapamil (52.7 %), amiodarone (39.8 %), and dronedarone (84.5 %), and exposure measured as 24-h concentrations for quinidine (11.8 %), verapamil (29.1 %), and dronedarone (157.6 %) also increased. Administration of edoxaban with amiodarone decreased the 24-h concentration for edoxaban by 25.7 %. Concomitant administration with digoxin or atorvastatin had minimal effects on edoxaban exposure. CONCLUSION: Coadministration of the P-gp inhibitors quinidine, verapamil, and dronedarone increased edoxaban exposure. Modest/minimal effects were observed for amiodarone, atorvastatin, and digoxin.

Role of P-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of Glycyrrhiza glabra.

Purpose. Glabridin is a major active constituent of Glycyrrhiza glabra which is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases. Recently, we have found that glabridin is a substrate of P-glycoprotein (PgP/MDR1). This study aimed to investigate the role of PgP in glabridin penetration across the blood–brain barrier (BBB) using several in vitro and in vivo models.
Materials and Methods. Cultured primary rat brain microvascular endothelial cells (RBMVECs) were used in the uptake, efflux and transcellular transport studies. A rat bilateral in situ brain perfusion model was used to investigate the brain distribution of glabridin. The brain and tissue distribution of glabridin in rats with or without coadministered verapamil or quinidine were examined with correction for the tissue residual blood. In addition, the brain distribution of glabridin in mdr1a(-/-) mice was compared with the wild-type mice. Glabridin in various biological matrices was determined by a validated liquid chromatography mass spectrometric method.
Results. The uptake and efflux of glabridin in cultured RBMVECs were ATP-dependent and significantly altered in the presence of a PgP or multi-drug resistance protein (Mrp1/2) inhibitor (e.g. verapamil or MK-571). A polarized transport of glabridin was found in RBMVEC monolayers with
facilitated efflux from the abluminal (BL) to luminal (AP) side. Addition of a PgP or Mrp1/2 inhibitor in both luminal and abluminal sides attenuated the polarized transport across RBMVECs. In a bilateral in situ brain perfusion model, the uptake of glabridin into the cerebrum increased from 0.42 T 0.09% at 1 min to 9.27 T 1.69% (ml/100 g tissue) at 30 min and was significantly greater than that for sucrose. Coperfusion of a PgP or Mrp1/2 inhibitor significantly increased the brain distribution of glabridin by 33.6j142.9%. The rat brain levels of glabridin were only about 27% of plasma levels when corrected by tissue residual blood and it was increased to up to 44% when verapamil or quinidine was coadministered. The area under the brain concentration-time curve (AUC) of glabridin in mdr1a(-/-) mice was 6.0-fold higher than the wild-type mice.
Conclusions. These findings indicate that PgP limits the brain penetration of glabridin through the BBB and PgP may cause drug resistance to glabridin (licorice) therapy for CNS diseases and potential drugglabridin interactions. However, further studies are needed to explore the role of other drug transporters (e.g. Mrp1-4) in restricting the brain penetration of glabridin.

Transport of cryptotanshinone, a major active triterpenoid in Salvia miltiorrhiza Bunge widely used in the treatment of stroke and Alzheimers disease, across the blood-brain

Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer's disease. Our recent study indicates that CTS is a substrate for Pglycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides attenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 ± 0.1% at 1 min to 11.13 ± 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1-163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further studies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.

Induction of propranolol metabolism by Ginkgo biloba extract EGb 761 in rats

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. When used in the elderly, Ginkgo has a high potential for interactions with cardiovascular drugs. This study aimed to investigate the effects of the standard Ginkgo biloba extract (EGB 761) treatment on the pharmacokinetics of propranolol and its metabolism to form Ndesisopropylpropranolol (NDP) in rats. We also examined the activity and expression of cytochrome P450 (CYP) 1A and other CYPs in rats treated with EGb 761 at 10 and 100 mg/kg/day for 10 days. A single oral dose of propranolol (10 mg/kg) was administered on day 11 and the concentrations of both propranolol and NDP were determined using validated liquid chromatography-mass spectrometry (LC-MS) methods. The levels of mRNA and protein of various CYPs were determined by RT-PCR and Western blotting analysis, respectively. Pretreatment of EGb 761 at 100 mg/kg, but not 10 mg/kg, for 10 days significantly reduced the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (C max) of propranolol, whereas those values of NDP were significantly increased. CYP1A1, 1A2, 2B1/2, and 3A1 activities and gene expression in the rat liver were significantly increased in a dose-dependent manner by pretreatment with EGb 761. The ex-vivo formation of NDP in liver microsomes from rats pretreated with EGb 761 was markedly enhanced. The formation of NDP from propranolol in liver microsomes was significantly inhibited by α- naphthoflavone (ANF, a selective CYP1A2 inhibitor), but not by quinidine (a CYP2D inhibitor). These results indicated that EGb 761 pretreatment decreased the plasma concentrations of propranolol by accelerated conversion of parental drug to NDP due to induction of CYP1A2. EGb 761 pretreatment also significantly induced CYP2B1/2 and CYP3A1, suggesting potential interactions with substrate drugs for these two enzymes. Further study is needed to explore the potential for gingko-drug interactions and the clinical impact.

Dearomatizzazione organocatalitica regio- ed enantioselettiva di sali di piridinio attivati

In this work, we present the first regio- and enantioselective organocatalytic nucleophilic dearomatization of activated N-alkyl pyridinium salts. In particular, N-benzyl pyridinium bromides bearing electron-withdrawing substituents at the C3 position of the pyridine ring were chosen as substrates. These compounds were easily obtained through an alkylation reaction between benzyl bromides and the corresponding 3-substituted pyridines. Then, a wide range of nucleophiles and organocatalysts was tested, providing the best results when indole, a thiourea derived from quinidine and 1-benzyl-3-nitropyridinum bromide were employed as the nucleophile, the catalyst and the pyridinium salt, respectively. Subsequently, the reaction conditions were optimised evaluating different bases, solvents, N-benzylic protecting groups, molar concentrations and temperatures. With the optimized condition in hand, the scope of the reaction with different substituted indoles was explored, affording the corresponding 1,4-dihydropyridines in good yields, regio- and enantio-selectivities. In addition, several experiments were carried out in order to understand the mechanism of the reaction, showing an unusual pathway involving a covalently bound intermediate formed by addition of the catalyst to the pyridine unit.

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2-5 Hz and consist of 4-12 mV large, 40-150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K+]o of 6.25 mM and decreased [Ca2+]o of 1.5 mM and [Mg2+]o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K+]o and can be prevented by the K+ channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABAA) receptor response. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl-D-aspartate (NMDA), or GABA(A) receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA(A) receptor desensitization increases its duration and amplitude. The apparent reversal potential of -27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.

On the molecular mechanism of chloroquine's antimalarial action.

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.

Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels.

Voltage-gated Na+ channels are the molecular targets of local anesthetics, class I antiarrhythmic drugs, and some anticonvulsants. These chemically diverse drugs inhibit Na+ channels with complex voltage- and frequency-dependent properties that reflect preferential drug binding to open and inactivated channel states. The site-directed mutations F1764A and Y1771A in transmembrane segment IVS6 of type IIA Na+ channel alpha subunits dramatically reduce the affinity of inactivated channels for the local anesthetic etidocaine. In this study, we show that these mutations also greatly reduce the sensitivity of Na+ channels to state-dependent block by the class Ib antiarrhythmic drug lidocaine and the anticonvulsant phenytoin and, to a lesser extent, reduce the sensitivity to block by the class Ia and Ic antiarrhythmic drugs quinidine and flecainide. For lidocaine and phenytoin, which bind preferentially to inactivated Na+ channels, the mutation F1764A reduced the affinity for binding to the inactivated state 24.5-fold and 8.3-fold, respectively, while Y1771A had smaller effects. For quinidine and flecainide, which bind preferentially to the open Na+ channels, the mutations F1764A and Y1771A reduced the affinity for binding to the open state 2- to 3-fold. Thus, F1764 and Y1771 are common molecular determinants of state-dependent binding of diverse drugs including lidocaine, phenytoin, flecainide, and quinidine, suggesting that these drugs interact with a common receptor site. However, the different magnitude of the effects of these mutations on binding of the individual drugs indicates that they interact in an overlapping, but nonidentical, manner with a common receptor site. These results further define the contributions of F1764 and Y1771 to a complex drug receptor site in the pore of Na+ channels.

Functional expression of mouse Mdr1 in an outer membrane permeability mutant of Escherichia coli.

Functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in Escherichia coli is providing an appropriate system for structure/function studies and might provide an invaluable tool to screen potential P-gp substrates and inhibitors. The major problem encountered in such studies, however, is the impermeability of the outer membrane of Gram-negative bacteria, which protects microorganisms against the cytotoxic effects of many lipophilic cancer drugs and blocks accessibility of P-gp reversal agents. In the present study we have constructed, by mutagenesis, a "leaky" (containing a permeable outer membrane) strain of E. coli, which is significantly more susceptible to the toxic effect of known P-gp substrates and cytotoxic agents. Expression of mouse Mdr1 in the mutant confers cross-resistance to daunomycin, quinidine, chloroquine, rhodamine 6G, and puromycin. Most importantly, reserpine and doxorubicin completely abolish Mdr1-mediated rhodamine resistance. The results provide strong support for previous observations, suggesting that Mdr1 can be expressed functionally in E. coli and indicate that the leaky mutant will be useful for further structure/function studies of the heterologously expressed eukaryotic drug efflux protein.

Enantioselective alkylation of β-keto esters promoted by dimeric Cinchona-derived ammonium salts as recoverable organocatalysts

Dimeric anthracenyldimethyl-derived Cinchona ammonium salts are used as chiral organocatalysts in 5 mol% for the phase-transfer enantioselective alkylation reaction of 2-alkoxycarbonyl-1-indanones with activated bromides. The corresponding adducts bearing a new all-carbon quaternary center are obtained usually in high yield and with moderate and opposite enantioselectivity (up to 55%) when using ammonium salts derived from quinidine and its pseudoenantiomer quinine as organocatalysts. These catalysts can be almost quantitatively recovered by precipitation in ether and reused.

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.