Phenylalanine monooxygenase and the sulfur oxygenation of S-carboxymethyl-l-cysteine in mice

1.?The extent of sulfoxidation of the drug, S-carboxymethyl-l-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-l-cysteine has been investigated.

2.?Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased C_max, AUC_{(0–180?min)} and AUC_{(0–∞?min)} values (15?- to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-l-cysteine (13?- to 22-fold lower).

3.?Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-l-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.     

Sapropterin dihydrochloride, 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin,in the treatment of phenylketonuria

Sapropterin dihydrochloride, 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is being introduced in the US for treatment of phenylketonuria (PKU). This compound has been in use in Europe to treat mild forms of PKU. Tetrahydrobiopterin is the cofactor in the hydroxylation reaction of the three aromatic amino acids phenylalanine, tyrosine and tryptophan. It is also involved in other reactions, which are not the focus of this review. The cofactor BH4 is synthesized in many tissues in the body. The pathway of BH4 biosynthesis is complex, and begins with guanosine triphosphate (GTP). The first reaction that commits GTP to form pterins is GTP cyclohydrolase. Several reactions follow resulting in the active cofactor BH4. During the hydroxylation reaction BH4 is oxidized to quinonoid-BH2, which is recycled by dihydropteridine reductase, resulting in the active cofactor. It was discovered that some patients with PKU had a decline in blood phenylalanine after oral intake of BH4. This response to BH4 is not the result of change in the synthesis or regeneration of the cofactor, but rather an effect on the mutant enzyme phenylalanine hydroxylase either by accommodating the higher K_m of the mutant enzyme or by acting as a chaperone for the mutant enzyme. This response has become of intense interest in the treatment of PKU.     

Inhibitory effects of sevoflurane on pacemaking activity of sinoatrial node cells in guinea-pig heart

BACKGROUND AND PURPOSE

The volatile anaesthetic sevoflurane affects heart rate in clinical settings. The present study investigated the effect of sevoflurane on sinoatrial (SA) node automaticity and its underlying ionic mechanisms.

EXPERIMENTAL APPROACH

Spontaneous action potentials and four ionic currents fundamental for pacemaking, namely, the hyperpolarization-activated cation current (I_f), T-type and L-type Ca²⁺ currents (I_Ca,T and I_Ca,L, respectively), and slowly activating delayed rectifier K⁺ current (I_Ks), were recorded in isolated guinea-pig SA node cells using perforated and conventional whole-cell patch-clamp techniques. Heart rate in guinea-pigs was recorded ex vivo in Langendorff mode and in vivo during sevoflurane inhalation.

KEY RESULTS

In isolated SA node cells, sevoflurane (0.12–0.71 mM) reduced the firing rate of spontaneous action potentials and its electrical basis, diastolic depolarization rate, in a qualitatively similar concentration-dependent manner. Sevoflurane (0.44 mM) reduced spontaneous firing rate by approximately 25% and decreased I_f, I_Ca,T, I_Ca,L and I_Ks by 14.4, 31.3, 30.3 and 37.1%, respectively, without significantly affecting voltage dependence of current activation. The negative chronotropic effect of sevoflurane was partly reproduced by a computer simulation of SA node cell electrophysiology. Sevoflurane reduced heart rate in Langendorff-perfused hearts, but not in vivo during sevoflurane inhalation in guinea-pigs.

CONCLUSIONS AND IMPLICATIONS

Sevoflurane at clinically relevant concentrations slowed diastolic depolarization and thereby reduced pacemaking activity in SA node cells, at least partly due to its inhibitory effect on I_f, I_Ca,T and I_Ca,L. These findings provide an important electrophysiological basis of alterations in heart rate during sevoflurane anaesthesia in clinical settings.     

Cadmium inhibits acid secretion in stimulated frog gastric mucosa

Cadmium, a toxic environmental pollutant, affects the function of different organs such as lungs, liver and kidney. Less is known about its toxic effects on the gastric mucosa. The aim of this study was to investigate the mechanisms by which cadmium impacts on the physiology of gastric mucosa. To this end, intact amphibian mucosae were mounted in Ussing chambers and the rate of acid secretion, short circuit current (I_sc), transepithelial potential (V_t) and resistance (R_t) were recorded in the continuous presence of cadmium. Addition of cadmium (20 µM to 1 mM) on the serosal but not luminal side of the mucosae resulted in inhibition of acid secretion and increase in NPPB-sensitive, chloride-dependent short circuit current. Remarkably, cadmium exerted its effects only on histamine-stimulated tissues. Experiments with TPEN, a cell-permeant chelator for heavy metals, showed that cadmium acts from the intracellular side of the acid secreting cells. Furthermore, cadmium-induced inhibition of acid secretion and increase in I_sc cannot be explained by an action on: 1) H₂ histamine receptor, 2) Ca²⁺ signalling 3) adenylyl cyclase or 4) carbonic anhydrase. Conversely, cadmium was ineffective in the presence of the H⁺/K⁺-ATPase blocker omeprazole suggesting that the two compounds likely act on the same target. Our findings suggest that cadmium affects the functionality of histamine-stimulated gastric mucosa by inhibiting the H⁺/K⁺-ATPase from the intracellular side. These data shed new light on the toxic effect of this dangerous environmental pollutant and may result in new avenues for therapeutic intervention in acute and chronic intoxication.     

Toxicodynamic effects of ciclosporin are reflected by metabolite profiles in the urine of healthy individuals after a single dose

AIMS

The immunosuppressant ciclosporin is an efficient prophylaxis against transplant organ rejection but its clinical use is limited by its nephrotoxicity. Our previous systematic studies in the rat indicated urine metabolite pattern changes to be sensitive indicators of the negative effects of ciclosporin on the kidney. To translate these results, we conducted an open label, placebo-controlled, crossover study assessing the time-dependent toxicodynamic effects of a single oral ciclosporin dose (5 mg kg⁻¹) on the kidney in 13 healthy individuals.

METHODS

In plasma and urine samples, ciclosporin and 15-F_2t-isoprostane concentrations were assessed using HPLC-MS and metabolite profiles using ¹H-NMR spectroscopy.

RESULTS

The maximum ciclosporin concentrations were 1489 ± 425 ng ml⁻¹ (blood) and 2629 ± 1308 ng ml⁻¹ (urine). The increase in urinary 15-F_2t-isoprostane observed 4 h after administration of ciclosporin indicated an increase in oxidative stress. 15-F_2t-isoprostane concentrations were on average 2.9-fold higher after ciclosporin than after placebo (59.8 ± 31.2 vs. 20.9 ± 19.9 pg mg⁻¹ creatinine, P < 0.02). While there were no conclusive changes in plasma 15-F_2t-isoprostane concentrations or metabolite patterns, non-targeted metabolome analysis using principal components analysis and partial least square fit analysis revealed significant changes in urine metabolites typically associated with negative effects on proximal tubule cells. The major metabolites that differed between the 4 h urine samples after ciclosporin and placebo were citrate, hippurate, lactate, TMAO, creatinine and phenylalanine.

CONCLUSION

Changes in urine metabolite patterns as a molecular marker are sufficiently sensitive for the detection of the negative effects of ciclosporin on the kidney after a single oral dose.     

The kinetics of cohesive powder de-agglomeration from three inhaler devices

Purpose

The purpose of the current investigation is to understand the kinetics of de-agglomeration (k_d) of micronised salbutamol sulphate (SS) and lactohale 300 (LH300) under varying air flow rates (30-180 l min⁻¹) from three dry powder inhaler devices (DPIs), Rotahaler^® (RH), Monodose Inhaler^® (MI) and Handihaler^® (HH).

Results

Cumulative fine particle mass vs. time profiles were obtained from the powder concentration, emitted mass and volume percent <5.4 μm, embedded in the particle size distributions of the aerosol at specific times. The rate of de-agglomeration (k_d), estimated from non-linear least squares modelling, increased with increasing air flow rates. The k_dvs. air flow rate profiles of SS and LH300 were significantly different at high air flow rates. The k_d was highest from RH and lowest from MI. Differences in k_d between the devices were related to device mode of operation while the differences between the materials were due to the powder bed structure.

Conclusion

This approach provided a methodology to measure the rate constant for cohesive powder de-agglomeration following aerosolisation from commercial devices and an initial understanding of the influence of device, air flow rate and material on these rate constants.     

Structural and mechanistic implications of incorporating naturally occurring aberrant mutations of human dihydropteridine reductase into a rat model

Phenylketonuria (PKU) is a debilitating hereditary disorder related to an individual's inability to convert phenylalanine to its usual tyrosine product. The genetic errors occur in three regions: in the cooperative enzymes phenylalanine hydroxylase (PAH) and dihydropteridine reductase (DHPR), and in the biosynthetic pathway from GTP to the hydroxylation cofactor, tetrahydrobiopterin (BH₄). Many instances of naturally occurring defects in DHPR metabolism have been identified, and in most cases the error has been equated with an altered enzyme gene sequence. Using computer graphics, this report analyses the altered structural characteristics of eight of the enzymes encoded by mutant gene sequence and provides logical explanations for their diminished enzyme activities. In one instance, that of a threonine insertion, a mutant construct of the rat analog has been expressed in Escherichia coli and the DHPR isolated and characterised, confirming the marked changes this insert can create.     

Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) is mediated mainly by cytochrome P₄₅₀ monooxygenases (CYP) CYP1A1, 1A2 and 1B1. Several PAH are known to induce these CYP via aryl hydrocarbon receptor (AhR) signaling. Recently, it was shown that the PAH benzo[a]pyrene (BaP) can induce CYP3A4 as well. The induction was suggested to be mediated by the pregnane X receptor (PXR) rather than AhR. Metabolism by CYP3A4 is only known for dihydrodiol metabolites of PAH but not for their parent compounds.     

BGC20-1531, a novel,potent and selective prostanoid EP4 receptor antagonist: a putative new treatment for migraine headache

Background and purpose:

Prostanoid EP₄ receptor antagonists may have therapeutic utility in the treatment of migraine since EP₄ receptors have been shown to be involved in prostaglandin (PG)E₂-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP₄ receptor antagonist.

Experimental approach:

BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP₄ receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo.

Key results:

BGC20-1531 exhibited high affinity at recombinant human EP₄ receptors expressed in cell lines (pK_B 7.6) and native EP₄ receptors in human cerebral and meningeal artery (pK_B 7.6–7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE₂-induced vasodilatation of human middle cerebral (pK_B 7.8) and meningeal (pK_B 7.6) arteries in vitro, but had no effect on responses induced by PGE₂ on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1–10 mg·kg⁻¹ i.v.) caused a dose-dependent antagonism of the PGE₂-induced increase in canine carotid blood flow in vivo.

Conclusions and implications:

BGC20-1531 is a potent and selective antagonist at EP₄ receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache.     

Co-operative signalling through DP(1) and DP(2) prostanoid receptors is required to enhance leukotriene C(4) synthesis induced by prostaglandin D(2) in eosinophils

BACKGROUND AND PURPOSE

Prostaglandin (PG) D₂ has emerged as a key mediator of allergic inflammatory pathologies and, particularly, PGD₂ induces leukotriene (LT) C₄ secretion from eosinophils. Here, we have characterized how PGD₂ signals to induce LTC₄ synthesis in eosinophils.

EXPERIMENTAL APPROACH

Antagonists and agonists of DP₁ and DP₂ prostanoid receptors were used in a model of PGD₂-induced eosinophilic inflammation in vivo and with PGD₂-stimulated human eosinophils in vitro, to identify PGD₂ receptor(s) mediating LTC₄ secretion. The signalling pathways involved were also investigated.

KEY RESULTS

In vivo and in vitro assays with receptor antagonists showed that PGD₂-triggered cysteinyl-LT (cysLT) secretion depends on the activation of both DP₁ and DP₂ receptors. DP₁ and DP₂ receptor agonists elicited cysLTs production only after simultaneous activation of both receptors. In eosinophils, LTC₄ synthesis, but not LTC₄ transport/export, was activated by PGD₂ receptor stimulation, and lipid bodies (lipid droplets) were the intracellular compartments of DP₁/DP₂ receptor-driven LTC₄ synthesis. Although not sufficient to trigger LTC₄ synthesis by itself, DP₁ receptor activation, signalling through protein kinase A, did activate the biogenesis of eosinophil lipid bodies, a process crucial for PGD₂-induced LTC₄ synthesis. Similarly, concurrent DP₂ receptor activation used Pertussis toxin-sensitive and calcium-dependent signalling pathways to achieve effective PGD₂-induced LTC₄ synthesis.

CONCLUSIONS AND IMPLICATIONS

Based on pivotal roles of cysLTs in allergic inflammatory pathogenesis and the collaborative interaction between PGD₂ receptors described here, our data suggest that both DP₁ and DP₂ receptor antagonists might be attractive candidates for anti-allergic therapies.

LINKED ARTICLE

This article is commented on by Mackay and Stewart, pp. 1671–1673 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01236.x     

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

BACKGROUND AND PURPOSE

A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF₁) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan.

EXPERIMENTAL APPROACH

A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF₁ antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF₁ receptor binding of novel compounds can be predicted on the basis of in vitro assays.

KEY RESULTS

The non-competitive antagonist behaviour appeared to be correlated to the CRF₁ receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations.

CONCLUSIONS AND IMPLICATIONS

This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF₁ receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF₁ receptor antagonists in an efficient manner in a drug discovery setting.     

Different effects of gadolinium on IKR,IKS and IK1 in guinea-pig isolated ventricular myocytes

1. Using the whole cell configuration of the patch clamp technique, we studied the potential blocking effects of gadolinium (1 μM to 1 mM) on potassium currents: I_KR, I_KS and I_K1. The study was performed on guinea-pig isolated ventricular myocytes.
2. The background current, I_K1 was insensitive to Gd³⁺. Thus, we found that no obvious screening of surface charges was visible with concentrations of Gd³⁺ up to 100 μM.
3. By use of three different protocols: tail currents fitting, analysis of envelope of tails and electrophysiological dissection, we found that I_KR was the only component of I_K that was sensitive to Gd³⁺. The sensitivity was apparently different depending on the protocol used.
4. Comparison of the results obtained with the different protocols revealed that the rapid component of I_KR is more sensitive to Gd³⁺ than the slow one.
5. Of the different protocols used to distinguish between I_KR and I_KS, the electrophysiological dissection seems to be the more accurate.

     

Effect of green tea on pharmacokinetics of 5-fluorouracil in rats and pharmacodynamics in human cell lines in vitro

Tea drinking is widely practiced in the world and has recently increased among cancer patients. However, the effects of concurrent consumption of tea on the bioavailability and the net therapeutic potential of co-administered chemical drugs are not clear. In this study, the effects of green tea on the pharmacokinetics of 5-fluorouracil (5-FU) in rats and the pharmacodynamics in human cell lines in vitro were studied. The pharmacokinetic experiment indicated that there was an approximately 151% increase in the maximum plasma concentration (C_max) and an approximately 425% increase in the area under the plasma concentration curve (AUC) of 5-FU in the green tea-treated group compared with the control group. Green tea consumption increased the plasma concentration of 5-FU. In addition, the pharmacodynamics experiment showed that at the moderate dose level (equivalent to <6 cups daily in human), neither fresh green tea extract nor (−)-epigallocatechin-3-gallate (EGCG) showed significant additive effects on the cytotoxicity of 5-FU in human cell lines. The results showed that it is crucial to perform therapeutic drug monitoring (TDM) when the cancer patients have a habit of drinking green tea.     

Nitric oxide donor trans-[RuCl([15]aneN4)NO]2+ as a possible therapeutic approach for Chagas' disease

Background and purpose:

Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas'' disease. However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activated macrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities against T. cruzi, of the NO donor, trans-[RuCl([15]aneN₄)NO]²⁺.

Experimental approach:

Trans-[RuCl([15]aneN₄)NO]²⁺was incubated with a partially drug-resistant T. cruzi Y strain and the anti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treated during the acute phase of Chagas'' disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiac parasitism, myocarditis and the curative rate.

Key results:

Trans-[RuCl([15]aneN₄)NO]²⁺ was 10- and 100-fold more active than Bz against amastigotes and trypomastigotes respectively. Further, trans-[RuCl([15]aneN₄)NO]²⁺ (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms) in vitro. Trans-[RuCl([15]aneN₄)NO]²⁺ induced permanent suppression of parasitaemia and 100% survival in a murine model of acute Chagas'' disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of the animals treated with the NO donor (3.33 µmol·kg⁻¹·day⁻¹) and Bz (385 µmol·kg⁻¹·day⁻¹), respectively, but when given together, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and a normalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses.

Conclusions and implications:

These findings indicate that trans-[RuCl([15]aneN₄)NO]²⁺is a promising lead compound for the treatment of human Chagas'' disease.This article is commented on by Machado et al., pp. 258–259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00662.x and to view a related paper in this issue by Silva et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00524.x     

Treatment with the Kv7 potassium channel activator flupirtine is beneficial in two independent mouse models of pulmonary hypertension

Background and purpose:

Voltage-gated potassium (K_v) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K_v7 channels has been recently proposed. We investigated the effect of the K_v7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT⁺ mice).

Experimental approach:

Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg·kg⁻¹·day⁻¹). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K_v channel blocking drugs, including the K_v7 channel blocker linopirdine, were measured.

Key results:

In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT⁺ mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT⁺ mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K_v channel blockers did not.

Conclusions and implications:

Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT⁺ mice, apparently via K_v7 channel activation. These results provide the first direct evidence that drugs activating K_v7 channels may be of benefit in the treatment of PAH with different aetiologies.     

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE

KCNQ-encoded voltage-gated potassium channels (K_v7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

EXPERIMENTAL APPROACH

Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K_v7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels.

KEY RESULTS

KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K_v7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K_v7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K⁺ currents. In contrast, the K_v7.1-specific blocker chromanol 293B did not affect vascular tone. Two K_v7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K_v7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991.

CONCLUSIONS AND IMPLICATIONS

This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo.

LINKED ARTICLE

This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x     

In vitro and in vivo oxidative metabolism and glucuronidation of anastrozole

AIMS

Little information is available regarding the metabolic routes of anastrozole and the specific enzymes involved. We characterized anastrozole oxidative and conjugation metabolism in vitro and in vivo.

METHODS

A sensitive LC-MS/MS method was developed to measure anastrozole and its metabolites in vitro and in vivo. Anastrozole metabolism was characterized using human liver microsomes (HLMs), expressed cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs).

RESULTS

Hydroxyanastrozole and anastrozole glucuronide were identified as the main oxidative and conjugated metabolites of anastrozole in vitro, respectively. Formation of hydroxyanastrozole from anastrozole was markedly inhibited by CYP3A selective chemical inhibitors (by >90%) and significantly correlated with CYP3A activity in a panel of HLMs (r = 0.96, P = 0.0005) and mainly catalyzed by expressed CYP3A4 and CYP3A5. The K_m values obtained from HLMs were also close to those from CYP3A4 and CYP3A5. Formation of anastrozole glucuronide in a bank of HLMs was correlated strongly with imipramine N-glucuronide, a marker of UGT1A4 (r = 0.72, P < 0.0001), while expressed UGT1A4 catalyzed its formation at the highest rate. Hydroxyanastrozole (mainly as a glucuronide) and anastrozole were quantified in plasma of breast cancer patients taking anastrozole (1 mg day⁻¹); anastrozole glucuronide was less apparent.

CONCLUSION

Anastrozole is oxidized to hydroxyanastrozole mainly by CYP3A4 (and to some extent by CYP3A5 and CYP2C8). Once formed, this metabolite undergoes glucuronidation. Variable activity of CYP3A4 (and probably UGT1A4), possibly due to genetic polymorphisms and drug interactions, may alter anastrozole disposition and its effects in vivo.     

Self-augmentation of the lengthening of repolarization is related to the shape of the cardiac action potential: implications for reverse rate dependency

Background and purpose:

The aims of the present work were to study the mechanism of the reverse rate dependency of different interventions prolonging cardiac action potential duration (APD).

Experimental approach:

The reverse rate-dependent lengthening effect of APD-prolonging interventions and the possible involvement of I_Kr (rapid component of the delayed rectifier potassium current) and I_K1 (inward rectifier potassium current) were studied by using the standard microelectrode and the whole-cell patch-clamp techniques in dog multicellular ventricular preparations and in myocytes isolated from undiseased human and dog hearts.

Key results:

All applied drugs – dofetilide (1 µmol·L⁻¹), BaCl₂ (10 µmol·L⁻¹), BAY-K-8644 (1 µmol·L⁻¹), veratrine (1 µg·mL⁻¹) – lengthened APD in a reverse rate-dependent manner regardless of their mode of action, suggesting that reverse rate dependency may not represent a specific mechanism of APD prolongation. The E-4031-sensitive current (I_Kr) and the Ba²⁺-sensitive current (I_K1) were recorded during repolarizing voltage ramps having various steepness and also during action potential waveforms with progressively prolonged APD. Gradually delaying repolarization results in smaller magnitude of I_Kr and I_K1 currents at an isochronal phase of the pulses. This represents a positive feedback mechanism, which appears to contribute to the reverse rate-dependent prolongation of action potentials.

Conclusions and implications:

Action potential configuration may influence the reverse rate-dependent APD prolongation due to the intrinsic properties of I_Kr and I_K1 currents. Drugs lengthening repolarization by decreasing repolarizing outward, or increasing depolarizing inward, currents are expected to cause reverse rate-dependent APD lengthening with high probability, regardless of which current they modify.     

The S-oxidation of S-carboxymethyl-L-cysteine in hepatic cytosolic fractions from BTBR and phenylketonuria enu1 and enu2 mice

1. Mice that were heterozygous dominant for the enu1 and enu2 mutation in phenylalanine monooxygenase/phenylalanine hydroxylase (PAH) resulted in hepatic PAH assays for S-carboxymethyl-L-cysteine (SCMC) that had significantly increased calculated K_m (wild type (wt)/enu1, 1.84–2.12 fold increase and wt/enu2 a 2.75 fold increase in PAH assays). The heterozygous dominant phenotypes showed a significantly reduced catalytic turnover of SCMC (wt/enu1, 6.11 fold decrease and wt/enu2 an 11.25 fold decrease in calculated V_max). Finally, these phenotypes also had a significantly reduced clearance, C_LE (wt/enu1, 13.02 fold and wt/enu2, a 30.80–30.94 fold decrease) The homozygous recessive phenotype (enu1/enu1) was also found to have significantly increased calculated K_m (2.16 fold increase), a significantly reduced calculated V_max (11.35–12.33 fold decrease) and C_LE (24.75–25.00 fold decrease). The enu2/enu2, homozygous recessive phenotype had no detectable PAH activity using SCMC as substrate.

2. The identity of the enzyme responsible for the C-oxidation of L-phenylalanine (L-Phe) and the S-oxidation of SCMC in wt/wt (BTBR) mice was identified using monoclonal antibody and selective chemical inhibitors and was found to be PAH.

3. This in vitro mouse hepatic cytosolic fraction metabolism investigation provides further evidence to support the hypothesis that an individual possessing one variant allele for PAH will result in a poor metaboliser phenotype that is unable to produce significant amounts of S-oxide metabolites of SCMC.

     

Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid concentrations in rhesus monkeys

Brain penetration of drugs which are subject to P-glycoprotein (Pgp)-mediated efflux is attenuated, as manifested by the fact that the cerebrospinal fluid concentration (C_CSF), a good surrogate of the unbound brain concentration (C_ub), is lower than the unbound plasma concentration (C_up) for Pgp substrates. In rodents, the attenuation magnitude of brain penetration by Pgp-mediated efflux has been estimated by correlating the ratio of CSF to plasma exposures (C_CSF/C_p) with the unbound fraction in plasma (f_u) upon the incorporation of the in vivo or in vitro Pgp-mediated efflux ratios (ERs). In the present work, we investigated the impact of Pgp-mediated efflux on C_CSF in monkeys. Following intravenous administration to cisterna magna ported rhesus monkeys, the CSF and plasma concentrations were determined for 25 compounds from three discovery programs. We also evaluated their f_u in rhesus plasma and ER in human and African green monkey MDR-transfected LLC-PK1 cells. These compounds varied significantly in the f_u (0.025-0.73), and 24 out of 25 are considered Pgp substrates based on their appreciable directional transport (ER > 2). The C_CSF/C_p was significantly lower than the corresponding f_u (≥3-fold) for 16 compounds regardless of a significant correlation (R² = 0.59, p = 4 × 10⁻⁵) when the C_CSF/C_p was plotted against the f_u. When the f_u was normalized to the ER (f_u/ER) the correlation was improved (R² = 0.75, p = 8 × 10⁻⁸). More importantly, only one compound showed the C_CSF/C_p that exceeded 3-fold of the normalized f_u. The results suggest that the impact of Pgp-mediated efflux in monkeys, similar to the case in rodents, is reasonably reflected by the gradient between the free concentrations in plasma and in CSF. Therefore, f_u and Pgp ER may serve as useful measurements in estimating in vivo C_CSF/C_p ratios in monkeys, and potentially in humans.