Udom Chantharaksri

UGT1A6 genotype-related pharmacokinetics of deferiprone (L1) in healthy volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT UGT1A6 has been proposed as the predominant isoform responsible for the glucuronidation of deferiprone. UGT1A6*2 allele has been associated with the altered enzyme activity. WHAT THIS STUDY ADDS There is no statistically significant effect of UGT1A6 genotype on the single-dose pharmacokinetics of deferiprone in healthy volunteers. Gender influences serum pharmacokinetics of deferiprone. Body iron stores reflected by serum ferritin levels may have an influence on the extent of extravascular deferiprone distribution. AIMS To examine the effects of UGT1A6 polymorphisms on the pharmacokinetics of deferiprone in healthy volunteers. METHODS Twenty-two healthy volunteers were enrolled and grouped according to UGT1A6 genotype. After an overnight fast, the subjects received a single oral dose of 25 mg kg(-1) deferiprone. Blood samples were collected at 0, 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min after dosing. Urine output was collected at 0, 0-2, 2-4, 4-8, 8-12 and 12-24 h. Deferiprone (L1) and deferiprone-glucuronide (L1G) concentrations in serum and urine were determined using a validated high-performance liquid chromatography method. UGT1A6 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS No statistically significant differences in any pharmacokinetic parameters of either deferiprone or deferiprone-glucuronide among the genotype groups were noted. Likewise, there were no statistically significant differences in 24-h urinary deferiprone and deferiprone-glucuronide excretion among the genotype groups. Significant differences between men and women were found in AUC(0-infinity), V(d)/F, and CL/F of deferiprone. Gender differences in 24-h urinary deferiprone and its metabolite excretion, however, failed to reach statistical significance. The V(d)/F of deferiprone was found to correlate significantly with serum ferritin (r(s) = 0.665; P = 0.001). CONCLUSION The studied single nucleotide polymorphisms in UGT1A6 do not appear to exert statistically significant effects on the single-dose pharmacokinetics of deferiprone. Gender appears to influence the serum pharmacokinetics of deferiprone, but not urinary excretion of deferiprone and its metabolite. Body iron stores may have an influence on the extent of extravascular deferiprone distribution.

Hemin: A Possible Cause of Oxidative Stress in Blood Circulation of β-Thalassemia/Hemoglobin E Disease

A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of g -thalassemia/hemoglobin E disease ( g -thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 w M in serum of g -thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of g -thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of g -thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum hemin, r =0.778 (p <0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p =0.259) and 0.519 (p =0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of g -thal/Hb E.

The effects of vitamin E on platelet activity in β‐thalassaemia patients

Summary.  A double‐blind, crossover, placebo‐controlled study of the effect of vitamin E on platelet functions was performed on nine splenectomized and 16 non‐splenectomized β‐thalassaemia/haemoglobin E (β‐thalassaemia/HbE) patients. The patients were supplemented with a daily dose of vitamin E (525 IU) for 3 months. The functions of platelets were assessed by adenosine diphosphate (ADP)‐induced platelet aggregation and adenosine triphosphate release. Plasma α‐tocopherol, plasma thiobarbituric reactive substances (TBARs) and serum ferritin levels represented patients’ antioxidant status, lipid peroxidation status and iron status respectively. Before experimentation, all patients had low plasma α‐tocopherol levels. The splenectomized patients showed severe iron overload iron, had higher plasma TBAR levels and their platelets were more reactive to ADP than those of non‐splenectomized patients. Three months of daily vitamin E supplementation resulted in a significant increase in plasma α‐tocopherol levels and reduction in plasma TBAR levels in all patients. Serum ferritin levels of the patients were not altered; however, vitamin E reduced the platelet reactivity of the splenectomized patients towards normal levels. The influence of vitamin E on platelet reactivity may result in delaying hypoxaemia and pulmonary occlusion that commonly occurs in splenectomized β‐thalassaemia/HbE patients.

Effects of Combined UDP-Glucuronosyltransferase (UGT) 1A1*28 and 1A6*2 on Paracetamol Pharmacokinetics in β-Thalassemia/HbE

In addition to pathophysiological changes, genetic variations can alter drug pharmacokinetics in patients with thalassemia. Numerous drugs are metabolized by UDP-glucuronosyltransferases (UGT) including paracetamol (PCM), a widely used analgesic. Co-occurrence of the UGT1A1 polymorphism (UGT1A1*28) and the UGT1A6 polymorphism (UGT1A6*2) may affect PCM glucuronidation. To elucidate the effect of these combined polymorphisms on the PCM metabolism in thalassemic patients, 15 β-thalassemia/hemoglobin E subjects with three different UGT1A genotypes received a single oral dose of 1,000 mg PCM. Drug disposition was determined by HPLC. Patients who have UGT1A6*2 without UGT1A1*28 showed a significant, lower area under concentration-time curve (AUC₀→∞) of PCM, PCM-glucuronide and PCM-sulfate than those of the patients with wild-type UGT1A1 and UGT1A6 (p < 0.05). In addition, a high elimination rate constant and clearance of PCM and its metabolites were also found in these patients (p < 0.05). Ourstudy suggests that a subtherapeutic level of PCM may occur in patients who have UGT1A6*2 without UGT1A1*28.

Atorvastatin affects TLR4 clustering via lipid raft modulation

Statins, HMG-CoA reductase inhibitors, are used widely in the treatment of hypercholesterolemia. Apart from lowering lipid levels, statins have been shown to have anti-inflammatory effects. Previously we showed that atorvastatin inhibits NF-kappaB activation, dose and time dependently, in LPS-TLR4 signaling pathway. In this study, we investigated the anti-inflammatory mechanism of atorvastatin via Toll-like receptor 4 (TLR4) in murine pro-B cell lines transfected with TLR4. Co-treatment of LPS-stimulated cells with both atorvastatin and mevalonate rescued NF-kappaB activation and TLR4 blockade demonstrated that atorvastatin does not exert its inhibitory effect via TLR4 receptor-ligand binding mechanism. Further investigation into the anti-inflammatory mechanism has shown that atorvastatin causes an impairment of TLR4 recruitment into the lipid raft thereby affecting anti-inflammatory responses. In contrast, mevalonate repaired lipid raft function leading to TLR4 clustering in the lipid raft. Together, these data suggest that atorvastatin exerts its anti-inflammatory effect via lipid raft modification. This novel finding offers another insight into the pleiotropic effects of atorvastatin and may be applicable to other pattern recognition receptors that utilize membrane lipid raft as a platform for signal transduction.

Pharmacokinetics of deferiprone in patients with β-thalassaemia: impact of splenectomy and iron status.

Background and ObjectiveIron-rich transfusions and/or a compensatory increase in iron absorption ultimately result in iron loading in patients with β-thalassaemia. Hence, without iron chelation, iron accumulates relentlessly. Deferiprone has been shown to be capable of reducing the iron burden in patients with b-thalassaemia. However, there is wide interpatient variation in deferiprone-induced urinary iron excretion (UIE). We hypothesized that splenectomy and iron status might influence the pharmacokinetic profiles of deferiprone in patients with β-thalassaemia/haemoglobin E, and the present study was aimed at examining this hypothesis.Study Participants and MethodsThirty-one patients with β-thalassaemia/haemoglobin E (20 splenecto-mized and 11 non-splenectomized patients) were enrolled in the study. After an overnight fast, the subjects received a single oral dose of deferiprone 25 mg/kg of body weight. Blood samples were collected pre-dosing and at 15, 30, 45, 60, 90, 120, 180, 240, 300, 360 and 480 minutes after dosing. Urine output was pooled and collected at 0–2, 2–4, 4–8, 8–12 and 12–24 hour intervals. Serum and urine concentrations of deferiprone and its metabolite deferiprone glucuronide were determined using a validated high-performance liquid chromatography method. Serum deferiprone-chelated iron and UIE were determined using a validated colourimetric method.ResultsNo significant difference in the pharmacokinetic parameters of non-conjugated deferiprone was observed between splenectomized and non-splenectomized patients. However, the maximum serum concentration (Cmax) and the area under the serum concentration-time curve (AUC) from time zero to infinity (AUC∞) values of deferiprone glucuronide were significantly lower (both p < 0.05) in splenectomized patients (median 53.2µmol/L and 12 634 µmol · min/L, respectively) than in non-splenectomized patients (median 70.5 µmol/L and 20 601 mmol · min/L, respectively). The Cmax and the AUC from time zero to the time of the last measurable concentration (AUClast) values of serum deferiprone-chelated iron, as well as UIE, were significantly higher (p < 0.001) in splenectomized patients (median values 7.1 µmol/L, 1645 mmol · min/L and 77.1 mmol, respectively) than in non-splenectomized patients (median values 3.1 µmol/L, 545 mmol · min/L and 12.5 µmol, respectively). Urinary excretion of non-conjugated deferiprone and deferiprone glucuronide did not differ between the two groups. Further analyses using multiple linear regressions indicated that the iron profiles (non-transferrin-bound iron and ferritin) were significant predictors of the pharmacokinetic parameters of non-conjugated deferiprone, deferiprone-chelated iron and UIE. In addition, splenectomy status was identified as the strongest predictor of the AUClast of deferiprone-chelated iron and UIE.ConclusionBoth iron and splenectomy status have significant effects on the pharmacokinetics and iron chelation efficacy of deferiprone. A greater degree of iron overload in splenectomized patients results in alterations in pharmacokinetic parameters (the Cmax and AUC) of deferiprone glucuronide and deferiprone-chelated iron, as well as a significant increase in UIE.

Rat uterine contractility and the activities of uterine adenyl cyclase and phosphodiesterase during the estrus cycle

Abstract A study has been made of the activities of rat uterus adenyl cyclase and phosphodiesterase, and the inhibitory effect of theophylline on uterine contractions at various stages of the estrus cycle. The activities of adenyl cyclase and phosphodiesterase at proestrus were found to be 2.52 ± 0.28 pmoles/min/mg of protein and 1.90 ± 0.30 nmoles/min/mg of protein, respectively. Activities of both the enzymes increased from proestrus to peak values at metestrus (early metestrus for adenyl cyclase and late metestrus for phosphodiesterase) and then fell until the following proestrus. Theophylline inhibition of oxytocin-induced maximum uterine contractions was found to be greatest at early metestrus. Similarly, the oxytocin concentration producing 40 per cent maximum contraction in the presence and absence of the theophylline Oxytocin (E 40 )+ theophylline Oxytocin (E 40 ) was also highest at early metestrus. These findings indicate that, at early metestrus, cellular turnover of cyclic AMP may be high and that the exaggerated inhibition of oxytocin- induced maximum contraction and the high value of Oxytocin (E 40 )+ theophylline Oxytocin (E 40 ) could be due to extensive accumulation of cyclic AMP produced from theophylline inhibition of phosphodiesterase.

Pharmaco/ferrokinetic-related pro-oxidant activity of deferiprone in β-thalassemia

The potential of free radical formation in serum of β-thalassemia/Hb E patients receiving a single oral dose of 25 mg/kg body weight of deferiprone, a bidentate orally active iron chelator, was evaluated using EPR/spin trapping technique. In the presence of ascorbic acid and tert-butylhydroperoxide, EPR signals of ascorbyl radical (aH=0.18 mT) and DMPO-carbon centred adduct (aH=2.37 mT, aN=1.65 mT) were detected. Shortly after deferiprone administration, EPR signal intensities decreased concomitant with an increase in serum levels of deferiprone. Unfortunately, enhanced EPR signal intensities were observed at 300 min after dosing in patients with serum molar ratio of deferiprone to iron less than 3, suggesting the formation of incomplete iron-deferiprone complexes and consequently free radical formation. To avoid adverse effects of deferiprone, a dosage regimen should be designed according to iron status of the patients and aimed at maintaining an adequate ratio of serum chelator-to-iron concentration.

The reduction of cholesteryl linoleate in lipoproteins: an index of clinical severity in β-thalassemia/Hb E

Abstract Background: Oxidative modification of lipoproteins has been reported in β-thalassemia and has been suggested to relate to atherogenesis-risk. This study focused on the change in cholesteryl esters in plasma lipoproteins under oxidative stress resulting from iron overload in β-thalassemia/hemoglobin E (β-thal/Hb E) patients. Methods: Markers of oxidative damage and cholesteryl esters (CEs) were measured in plasma and lipo-proteins from 30 β-thal/Hb E patients and compared to those from 10 healthy volunteers. CEs in plasma, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were separated and identified using HPLC. Results: β-Thal/Hb E patients presented iron overload, a precipitous decrease in α-tocopherol and increased lipid peroxidation (thiobarbituric acid-reactive substances; TBARs) in both plasma and lipoproteins. Cholesteryl linoleate, the most abundant CE in lipoproteins, showed a reduction of 70% in LDL, while other CEs showed a lower reduction (50%). An inverse relationship between the cholesteryl linoleate/cholesteryl oleate ratio (CL/CO) and the degree of clinical severity suggested that the CL/CO ratio is an index of damaged lipoproteins and could be used as a pathologic marker of underlying iron overload. Good correlation of non-transferrin-bound iron (NTBI) and TBARs (r=0.8, p<0.01) in LDL strongly supported the contention that iron overload is responsible for initiating the lipid peroxidation in β-thal/Hb E. Conclusions: This study suggests that cholesteryl linoleate is the primary target of oxidative modification induced by NTBI in β-thal/Hb E patients and that reduction in cholesteryl linoleate in lipoproteins could be used as a severity index for β-thal/Hb E.

Paraoxonase and platelet-activating factor acetylhydrolase activities in lipoproteins of β-thalassemia/hemoglobin E patients

Abstract Background: Iron-induced oxidative stress may be implicated in the alteration of the lipoprotein-associated antioxidant enzymes paraoxonase 1 (PON1) and platelet-activating factor acetylhydrolase (PAF-AH), leading to atherosclerosis-related vascular complication in patients with β-thalassemia hemoglobin E (β-thal/Hb E). Methods: Plasma and lipoprotein enzyme activities of PON1 and PAF-AH were studied in 13 mild to moderate and 15 severe cases of β-thal/Hb E in comparison with 15 normal subjects. Results: PON1 activity was significantly reduced in association with oxidative stress in the patients. There were significant correlations between high-density lipoprotein (HDL)-PON1 activity and oxidative stress markers, including plasma levels of α-tocopherol (r=0.694 p<0.001) and the ratio of cholesteryl linoleate to cholesteryl oleate (CL/CO, r=0.662, p<0.001) in HDL. On the other hand, PAF-AH activity was markedly increased in patients by approximately two-fold and three- to four-fold in plasma and lipoproteins, respectively. Significant correlations of low-density lipoprotein (LDL) and HDL-PAF-AH activity with plasma iron, α-tocopherol and the CL/CO ratio were also demonstrated. Conclusions: We suggest that impairment of PON1 activity may be directly caused by oxidative damage, while increased PAF-AH activity possibly results from oxidative stress-induced inflammatory response in β-thal/Hb E patients. Clin Chem Lab Med 2007;45:884–9.