Active Metabolite Oxypurinol Research Articles

Pharmacokinetic/Pharmacodynamic Modelling of Allopurinol, its Active Metabolite Oxypurinol, and Biomarkers Hypoxanthine, Xanthine and Uric Acid in Hypoxic-Ischemic Encephalopathy Neonates.

BackgroundAllopurinol, an xanthine oxidase (XO) inhibitor, is a promising intervention that may provide neuroprotection for neonates with hypoxic-ischemic encephalopathy (HIE). Currently, a double-blind, placebo-controlled study (ALBINO, NCT03162653) is investigating the neuroprotective effect of allopurinol in HIE neonates.ObjectiveThe aim of the current study was to establish the pharmacokinetics (PK) of allopurinol and oxypurinol, and the pharmacodynamics (PD) of both compounds on hypoxanthine, xanthine, and uric acid in HIE neonates. The dosage used and the effect of allopurinol in this population, either or not undergoing therapeutic hypothermia (TH), were evaluated.MethodsForty-six neonates from the ALBINO study and two historical clinical studies were included. All doses were administered on the first day of life. In the ALBINO study (n = 20), neonates received a first dose of allopurinol 20 mg/kg, and, in the case of TH (n = 13), a second dose of allopurinol 10 mg/kg. In the historical cohorts (n = 26), neonates (all without TH) received two doses of allopurinol 20 mg/kg in total. Allopurinol and oxypurinol population PK, and their effects on inhibiting conversions of hypoxanthine and xanthine to uric acid, were assessed using nonlinear mixed-effects modelling.ResultsAllopurinol and oxypurinol PK were described by two sequential one-compartment models with an autoinhibition effect on allopurinol metabolism by oxypurinol. For allopurinol, clearance (CL) was 0.83 L/h (95% confidence interval [CI] 0.62–1.09) and volume of distribution (Vd) was 2.43 L (95% CI 2.25–2.63). For metabolite oxypurinol, CL and Vd relative to a formation fraction (fm) were 0.26 L/h (95% CI 0.23–0.3) and 11 L (95% CI 9.9–12.2), respectively. No difference in allopurinol and oxypurinol CL was found between TH and non-TH patients. The effect of allopurinol and oxypurinol on XO inhibition was described by a turnover model of hypoxanthine with sequential metabolites xanthine and uric acid. The combined allopurinol and oxypurinol concentration at the half-maximal XO inhibition was 0.36 mg/L (95% CI 0.31–0.42).ConclusionThe PK and PD of allopurinol, oxypurinol, hypoxanthine, xanthine, and uric acid in neonates with HIE were described. The dosing regimen applied in the ALBINO trial leads to the targeted XO inhibition in neonates treated with or without TH.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-021-01068-0.

Detection of allopurinol and oxypurinol in canine urine by HPLC/MS-MS: Focus on veterinary clinical pharmacokinetics

Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite’s RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 μm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 μg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 μg/mL, and oxypurinol at 10−200 μg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment.

HPTLC-Densitometric Determination of Allopurinol and its Metabolite Oxypurinol in Human Plasma and Allopurinol in Tablet Dosage Form

A simple, accurate and precise high-performance thin layer chromatography (HPTLC) method has been developed for the simultaneous determination of allopurinol (AP), a xanthine oxidase inhibitor and its active metabolite oxypurinol (OP) in human plasma and AP in tablet dosage form. The proposed method was developed and validated on aluminium plates pre-coated with silica gel 60G F254 as the stationary phase using methanol: chloroform: ammonia (2.0:7.9: 0.1, v/v/v) as the developing solvent system. Densitometric measurements were made at 206 nm and the retention factors (Rf) obtained were 0.38 ± 0.01 and 0.65 ± 0.01 for AP and OP respectively. System suitability testing parameters were evaluated to determine the performance of the chromatographic method. The regression plots were linear (r2 > 0.9993) in the concentration range of 100-700 ng/band for both the analytes. The limit of detection and limit of quantitation of the method were 19.56 and 59.29 ng/band for AP and 19.01 and 57.59 ng/band for OP respectively for plasma samples. For spiked plasma samples, protein precipitation with formic acid in acetonitrile afforded mean recovery of 84.67 % and 86.21 % for AP and OP, respectively. The method was validated for linearity, specificity, accuracy and precision, repeatability, stability and robustness.

Determination of Allopurinol and Oxypurinol in Dogs Plasma by High- Performance Liquid Chromatography with an Ultraviolet Detector: Application for Pharmacokinetic Studies

High performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed and validated for quantification of allopurinol and its active metabolite oxypurinol; in dog plasma with naturally acquired zoonotic visceral leishmaniasis. Allopurinol is a potent inhibitor of the xanthine oxidase enzyme; an enzyme that catalyzes the conversion of hypoxanthine to xanthine and from xanthine to uric acid. In veterinary medicine allopurinol is indicated in the treatment of canine visceral leishmaniasis. Allopurinol is utilized to inhibit the synthesis of Leishmania RNA. The conditions defined for development of the chromatographic analysis of dog plasma samples by utilizing the mobile phase of the HPLC-UV consist of a mixture of 0.1% water, 88% formic acid and 0.25% acetonitrile. Allopurinol and oxypurinol were separated on a LiChroCART® 125-4 LiChrospher® 100 RP-8 (5 μm) column utilizing a flow rate of 0.7 mL min-1 and detector operation was at a wavelength of 254 nm. Acyclovir was utilized as an internal standard. The validation of the HPLC-UV method was determined by limits of detection and quantification, linearity, reproducibility and repeatability. The method had a lower quantification limit of 0.1 μg.mL-1 for both allopurinol and oxypurinol. The analysis was linear at concentrations of 0.1-20.0 μg.mL-1 for both allopurinol and its oxypurinol. The precision value intracurrent and intercurrent for all concentrations presented had a coefficient variation lower than 15%. The confidence limits of HPLC-UV for analysis of allopurinol and oxypurinol in dog plasma indicates that the method is applicable to the multiple dose pharmacokinetic study of allopurinol in dogs. It is efficient, accurate and sensitive. This study of Pharmacokinetic research of allopurinol and oxypurinol in dogs with Stage I and II visceral leishmaniasis resulted in similar research outcomes that correlated with the healthy dog investigation.

Therapeutic drug monitoring in rheumatic diseases: utile or futile?

Rapid and effective suppression of inflammation is a primary goal in the treatment of rheumatic diseases. However, the therapeutic effect of most medications may be slow to manifest, in the order of weeks or months in the case of DMARDs. Monitoring of drug concentrations allows the possibility of appropriate dose adjustment or changes in medication to achieve more rapid or better outcomes. We review the evidence for drug concentration monitoring. Despite the theoretical utility for monitoring of MTX polyglutamate concentrations in red blood cells in patients with RA, studies have not shown a clear association between concentrations and either efficacy or toxicity and routine measurement is not yet recommended. Small studies associating disease control with concentrations of anti-TNF therapies and anti-drug antibodies suggest that routine monitoring may be useful in the future. However, the data are not yet sufficient for this recommendation. With the use of allopurinol in gout, there is a putative therapeutic range for the active metabolite oxypurinol; however, adjusting the allopurinol dose to achieve a target urate concentration is likely to be most effective, and measuring oxypurinol may be best suited to assessing drug adherence. Although measuring thiopurine metabolite concentrations with AZA therapy has been shown to be useful in IBD, studies in rheumatic diseases have so far failed to confirm a useful association between concentrations and disease control or drug toxicity. Whole blood concentrations of HCQ have been associated with disease control in SLE and future studies may be able to determine a therapeutic range.

Determination of allopurinol and oxypurinol in human plasma and urine by liquid chromatography-tandem mass spectrometry

Allopurinol is used widely for the treatment of gout, but its pharmacokinetics is complex and some patients show hypersensitivity, necessitating careful monitoring and improved detection methods. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed to determine the concentrations of allopurinol and its active metabolite oxypurinol in human plasma and urine using 2,6-dichloropurine as the internal standard (IS). Analytes and the IS were extracted from 0.5ml aliquots of plasma or urine using ethyl acetate and separated on an Agilent Eclipse Plus C18 column using methanol and ammonium formate-formic acid buffer containing 5mM ammonium formate and 0.1% formic acid (95:5, v/v) as the mobile phase (A) for allopurinol or methanol plus 5mM ammonium formate aqueous solution (95:5, v/v) as the mobile phase (B) for oxypurinol. Allopurinol was detected in positive ion mode and the analysis time was about 7min. The calibration curve was linear from 0.05 to 5μg/mL allopurinol in plasma and 0.5-30μg/mL in urine. The lower limit of quantification (LLOQ) was 0.05μg/mL in plasma and 0.5μg/mL in urine. The intra- and inter-day precision and relative errors of quality control (QC) samples were ≤11.1% for plasma and ≤ 8.7% for urine. Oxypurinol was detected in negative mode with an analysis time of about 4min. The calibration curve was linear from 0.05 to 5μg/mL in plasma (LLOQ, 0.05μg/mL) and from 1 to 50μg/mL in urine (LLOQ, 1μg/mL). The intra- and inter-day precision and relative errors were ≤7.0% for plasma and ≤9.6% for urine. This method was then successfully applied to investigate the pharmacokinetics of allopurinol and oxypurinol in humans.

Chronic Hyperuricemia, Uric Acid Deposit and Cardiovascular Risk

Hyperuricemia is commonly associated with traditional risk factors such as dysglicemia, dyslipidemia, central obesity and abnormal blood pressure, i.e. the metabolic syndrome. Concordantly, recent studies have revived the controversy over the role of circulating uric acid, hyperuricemia, and gout as an independent prognostic factor for cardiovascular morbidity and mortality. In this regard, different studies also evaluated the possible role of xanthine inhibitors in inducing blood pressure reduction, increment in flow-mediated dilation, and improved cardiovascular prognosis in various patient settings. The vast majority of these studies have been conducted with either allopurinol or its active metabolite oxypurinol, i.e. two purine-like non-selective inhibitors of xanthine oxidase. More recently, the role of uric acid as a risk factor for cardiovascular disease and the possible protective role exerted by reduction of hyperuricemia to normal level have been evaluated by the use of febuxostat, a selective, non purine-like xanthine oxidase inhibitor. In this review, we will report current evidence on hyperuricemia in cardiovascular disease. The value of uric acid as a biomarker and as a potential therapeutic target for tailored old and novel “cardiometabolic” treatments will be also discussed.

Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia

Cachexia is a common co-morbidity in cancer occurring in up to 80% of patients depending on the type of cancer. Uric acid (UA), the end-product of the purine metabolism, is elevated in cachexia due to tissue wasting and upregulated xanthine oxidase (XO) activity. High serum UA levels indicate increased XO-dependent production of oxygen free radicals (reactive oxygen species; ROS) and correlate with metabolic illness and poor survival. We hypothesized that XO-inhibition might reduce inflammatory signals accounting for tissue wasting and improve survival in experimental cancer cachexia. Animals were inoculated intraperitoneally with AH-130 hepatoma cells and treated with two XO-inhibitors: allopurinol [Allo, low (LD) and high dose (HD) 4 and 40 mg/kg/d] and its more effective active metabolite oxypurinol (Oxy, 4 and 40 mg/kg/d) or placebo for 15 days. Weight loss and tissue wasting of both fat and lean tissue (assessed by NMR-scanning) was reduced by both LD and HD Allo and LD-Oxy, but not by HD-Oxy. A robust induction of XO-activity for generation of reactive oxygen species was seen in the placebo group (assessed by electron paramagnetic spectroscopy), which was reduced by XO-inhibition. Increased ROS induced cytokine signaling, proteolytic activity and tissue degradation were all attenuated by XO inhibition. Survival was significantly and dose dependently improved. Food intake and spontaneous locomotor activity were higher, indicating a higher quality of life. Inhibition of XO can reduce tissue wasting and improve survival in cancer cachexia and clearly clinical studies are needed.

Maternal Allopurinol During Fetal Hypoxia Lowers Cord Blood Levels of the Brain Injury Marker S-100B

Fetal hypoxia is an important determinant of neonatal encephalopathy caused by birth asphyxia, in which hypoxia-induced free radical formation plays an important role. Maternal treatment with allopurinol, will cross the placenta during fetal hypoxia (primary outcome) and reduce S-100B and free radical formation (secondary outcome). In a randomized, double-blind feasibility study, 53 pregnant women in labor (54 fetuses) with a gestational age of >36 weeks and fetal hypoxia, as indicated by abnormal/nonreassuring fetal heart rate tracing or fetal scalp pH of <7.20, received 500 mg of allopurinol or placebo intravenously. Severity of fetal hypoxia, brain damage and free radical formation were assessed by arterial cord blood lactate, S-100B and non-protein-bound-iron concentrations, respectively. At birth, maternal and cord blood concentrations of allopurinol and its active metabolite oxypurinol were determined. Allopurinol and oxypurinol concentrations were within the therapeutic range in the mother (allopurinol > 2 mg/L and/or oxypurinol > 4 mg/L) but not always in arterial cord blood. We therefore created 3 groups: a placebo (n = 27), therapeutic allopurinol (n = 15), and subtherapeutic allopurinol group (n = 12). Cord lactate concentration did not differ, but S-100B was significantly lower in the therapeutic allopurinol group compared with the placebo and subtherapeutic allopurinol groups (P < .01). Fewer therapeutic allopurinol cord samples had measurable non-protein-bound iron concentrations compared with placebo (P < .01). Maternal allopurinol/oxypurinol crosses the placenta during fetal hypoxia. In fetuses/newborns with therapeutic allopurinol/oxypurinol concentrations in cord blood, lower plasma levels of the brain injury marker protein S-100B were detected. A larger allopurinol trial in compromised fetuses at term seems warranted. The allopurinol dosage must be adjusted to achieve therapeutic fetal allopurinol/oxypurinol concentrations.

Placental transfer and pharmacokinetics of allopurinol in late pregnant sows and their fetuses

At present no standard pharmacological intervention strategy is available to reduce these adverse effects of birth asphyxia. In the present study we aimed to evaluate placental transfer of allopurinol, an inhibitor of XOR. For this purpose, fetal catheterization of the jugular vein was conducted in five late pregnant sows (one fetus per sow). At 24-48 h after surgery, sows received allopurinol (15 mg/kg body weight; i.v.) and pharmacokinetics of allopurinol and its active metabolite oxypurinol were measured in both late pregnant sows and fetuses. Maternal and fetal blood samples were collected during and after allopurinol administration. Maternal C(max) values averaged 41.90 microg/mL (allopurinol) and 3.68 microg/mL (oxypurinol). Allopurinol crossed the placental barrier as shown by the average fetal C(max) values of 5.05 microg/mL at 1.47 h after allopurinol administration to the sow. In only one fetus low plasma oxypurinol concentrations were found. Incubations of subcellular hepatic fractions of sows and 24-h-old piglets confirmed that allopurinol could be metabolized into oxypurinol. In conclusion, we demonstrated that allopurinol can cross the placental barrier, a prerequisite for further studies evaluating the use of allopurinol as a neuroprotective agent to reduce the adverse effects following birth asphyxia in neonatal piglets.

Studies on Bi-Layer Osmotic Pump Tablets of Water-Insoluble Allopurinol with Large Dose: In Vitro and In Vivo

Controlled release bi-layer osmotic pump tablets (BOPT) of water—insoluble allopurinol with large dose (150 mg/BOPT) were successfully prepared merely with sodium chloride as osmotic promoting agent and polyethylene oxide (PEO) as suspending agent. Formulations of the two kinds of agents were investigated in order to discuss their effects on the release behavior of BOPT, and then the optimal formulation was evaluated. The pharmacokinetics studies of allopurinol and its active metabolite oxypurinol in two-preparation and two-period crossover design relative to the equivalent dose of commercially common allopurinol tablets were evaluated in six Beagle dogs. And the pharmacokinetics results showed that allopurinol BOPT were able to provide a slow release of allopurinol, and oxypurinol were bioequivalent between allopurinol BOPT and common allopurinol tablets. A good in vitro–in vivo correlation of allopurinol was also proved. In conclusion, water-insoluble drugs with large dose can be designed to BOPT for efficacy and safety use.

Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol.

The prototypical xanthine oxidase (XO) inhibitor allopurinol, has been the cornerstone of the clinical management of gout and conditions associated with hyperuricemia for several decades. More recent data indicate that XO also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, inflammatory diseases, and chronic heart failure. Allopurinol and its active metabolite oxypurinol showed considerable promise in the treatment of these conditions both in experimental animals and in small-scale human clinical trials. Although some of the beneficial effects of these compounds may be unrelated to the inhibition of the XO, the encouraging findings rekindled significant interest in the development of additional, novel series of XO inhibitors for various therapeutic indications. Here we present a critical overview of the effects of XO inhibitors in various pathophysiological conditions and also review the various emerging therapeutic strategies offered by this approach.

Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol

A simple and sensitive capillary zone electrophoresis method with UV absorbance detection is described for the quantitation of allopurinol and its metabolite oxypurinol in aqueous solution. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was systematically investigated; these parameters included the nature and concentration of the separation buffer, pH and applied voltage. A buffer consisting of 15 mM 2-[ N-cyclohexylamino]ethanesulfonic acid (CHES) adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of these compounds. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing these compounds in serum and allopurinol in commercial pharmaceutical preparations.

Determination of allopurinol and its active metabolite oxypurinol by capillary electrophoresis with end-column amperometric detection

A simple method was proposed for the separation of allopurinol (AP) and its active metabolite oxypurinol (OP) by capillary electrophoresis with end-column amperometric detection. A running buffer composed of 15 mM Na 2HPO 4/NaH 2PO 4 at a pH 9.55, electrokinetic injection 7 s at 5 kV, separation voltage at 15 kV and detection potential at 1.20 V were investigated to be the optimal condition for the separation. The method exhibited low detection limit ( S/ N=3) as 1×10 −8 mol/l for AP and OP, wide linearity range of 2×10 −7 to 1×10 −4 mol/l, 1×10 −7 to 1×10 −4 and high efficiency of 1.2×10 5 and 1.8×10 5 N/m for AP and OP, respectively. The potential application examined for the method was the determination of the spiked urine sample, which was proved to be sensitive and efficient.

The Pharmacokinetics of Injectable Allopurinol in Newborns With the Hypoplastic Left Heart Syndrome

The purpose of this investigation was to determine the pharmacokinetic disposition of intravenous allopurinol and its metabolite oxypurinol in neonates with the hypoplastic left heart syndrome (HLHS) and to evaluate the subsequent degree of xanthine oxidase inhibition using serum uric acid as a marker. Pharmacokinetic data were evaluated in 12 stable preoperative neonates with HLHS after a single intravenous allopurinol administration of 5 mg/kg or 10 mg/kg. Pharmacokinetic parameters were determined for elimination half-life, clearance, volume of distribution, and mean residence time. Xanthine oxidase inhibition, measured by serum uric acid reduction, was also measured. Pharmacokinetic parameters revealed no statistically significant differences between a 5-mg/kg and 10-mg/kg dose of intravenous allopurinol on elimination half-life, clearance, volume of distribution, and mean residence time. Mean serum uric acid levels were significantly reduced from baseline by 39.99 and 42.94%, respectively, in the 5- and 10-mg/kg treatment groups. The enzyme xanthine oxidase plays a key biochemical role in the generation of toxic oxygen-derived free radicals during ischemia-reperfusion conditions. Allopurinol and its active metabolite oxypurinol inhibit xanthine oxidase, and significantly reduce the conversion of hypoxanthine to xanthine and xanthine to uric acid. Cell injury may be caused by toxic oxygen free radicals produced by ischemia-reperfusion injury such as could occur during the repair of HLHS under hypothermic total circulatory arrest. We hypothesize that allopurinol may provide protection from cellular injury in this clinical context.

Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.

The effects of the xanthine oxidase inhibitors, allopurinol and oxypurinol on the pattern of purine release from hypoxic rat cerebral cortex

Release of purines from the normoxic and hypoxic/ischemic rat cerebral cortex was studied with the cortical cup technique. Both allopurinol (100 mg/kg, i.v.) and its active metabolite oxypurinol (20 mg/kg, i.v.) reduced the release of uric acid to 10% of predrug levels, indicating an effective block of xanthine oxidase activity. Xanthine and hypoxanthine, substrates for xanthine oxidase, increased in the cortical perfusates following drug administration. Allopurinol treatment resulted in a decreased release of adenosine during the first postdrug hypoxic challenge, whereas oxypurinol, and allopurinol during the second postdrug challenge, increased the hypoxia-evoked level of adenosine and inosine in the perfusate. The purine sparing effects of allopurinol and oxypurinol, in addition to the prevention of xanthine oxidase-mediated free radical generation, may account for their protective action in reperfusion injury paradigms.