A possible covalent xanthine oxidase inhibitor TS10: Inhibition mechanism, metabolites identification and PDPK assessment.

Xanthine oxidase (XO) inhibitors are widely used in the control of serum uric acid levels in the clinical management of gout. Our continuous efforts in searching novel amide-based XO inhibitors culminated in the identification of N-(4-((3-cyanobenzyl)oxy)-3-(1H-tetrazol-1-yl)phenyl)isonicotinamide (TS10), which exhibited comparable in vitro inhibition to that of topiroxostat (TS10, IC50 = 0.031 µM; topiroxostat, IC50 = 0.020 µM). According to the molecular modeling, we speculated that, as well as topiroxostat, TS10 would be biotransformed by XO to yield TS10-2-OH. In this work, TS10-2-OH was successfully identified in XO targeted metabolism study, demonstrated that TS10 underwent a covalent binding with XO via a TS10-O-Mo intermediate after anchoring in the XO molybdenum cofactor pocket. Furthermore, TS10-2-OH is a weak active metabolite, and its potency was explained by the molecular docking. In metabolites identification, TS10 could be oxidized by CYP2C9, CYP3A4 and CYP3A5 to generate two mono-hydroxylated metabolites (not TS10-2-OH); and could occur degradation in plasma to mainly generate a hydrolytic metabolite (TS10-hydrolysate). In pharmacokinetic assessment, the low oral system exposure was observed (Cmax = 14.73 ± 2.66 ng/mL and AUClast = 9.17 ± 1.42 h⋅ng/mL), which could be explained by the poor oral absorption property found in excretion studies. Nonetheless, in pharmacodynamic evaluation, TS10 exhibited significant uric acid-lowering effect after oral administration in a dose-dependent manner. Briefly, in addition to allopurinol and topiroxostat, TS10 is possibly another explicitly mechanism-based XO inhibitor with powerful covalent inhibition.

Assessment of anti-nociceptive effect of allopurinol in a neuropathic pain model.

BACKGROUND: This study aimed to investigate the antinociceptive effect of allopurinol, a xanthine oxidase inhibitor, in the chronic constriction injury (CCI) to sciatic nerve rat model of neuropathic pain. METHODS: Allopurinol administration (30, 60, 90 mg/kg, i.p.) was started at the time of nerve injury, and given for 14 continuous days. Behavioural tests (von Frey filaments, acetone drop, hot plate) were conducted on days 0, 3, 7, 10 and 14. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on the spinal cord of CCI animals on day 14. The contribution of adenosine (A) receptors was tested using the methylxanthine theophylline, a non-selective A receptor antagonist and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1 receptor antagonist, administered 30 min before allopurinol on day 10. RESULTS: CCI of the sciatic nerve resulted in a persistent mechanical allodynia, cold allodynia, and heat hyperalgesia, together with increased iNOS, bax/bcl2, iba-1 and TNF-α expression in the lumbar spinal cord of animals. The highest-dose group of allopurinol (90 mg/kg) attenuated pain-like behaviors compared with the normal saline treated group, and this was accompanied by normalization of iNOS, bax/bcl2, caspase 3, iba-1 and TNF-α gene expression changes. DPCPX and theophylline reversed the thermal anti-hyperalgesic effect of allopurinol. In contrast, the mechanical anti-allodynic effect was only prevented by theophylline. CONCLUSION: Allopurinol through interacting with different aspects of neuropathic pain, via anti-oxidant effects, protection against neuroinflammation, and activating adenosine receptors, could be useful in the treatment of patients with neuropathic pain.

Urinary cystatin C and NGAL as early biomarkers for assessment of renal ischemia-reperfusion injury: a serum marker to replace creatinine?

PURPOSE: To assess application of cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) as biomarkers for renal ischemic injury. We also evaluated the use of allopurinol as a renoprotective agent. A second goal was to assess cystatin C as a biomarker in patients undergoing partial nephrectomy. METHODS: Using 58 Sprague-Dawley rats, we evaluated urinary cystatin C (n=26) and NGAL (n=32) as a biomarker for renal ischemia injury. Half of the rats were pretreated with allopurinol; the other cohort served as a control. The right renal hilum was ligated in all rats, thereby creating a solitary kidney model. After a 30-minute stabilization period, the left hilum was clamped for time periods of 15, 30, and 60 minutes. Urinary levels of cystatin C and NGAL were then measured at the following time points: Preclamp (after the 30-minute stabilization period) and postclamp (30, 45, and 60 minute periods after unclamping). For our clinical subjects, serum cystatin C levels (n=17) were obtained preoperatively, at the induction of anesthesia before robot-assisted partial nephrectomy, immediately postoperatively, and on postoperative days 1 and 2. Three of these patients had their tumors excised off clamp and served as controls. We then estimated glomerular filtration rate by using the Creatinine-Cystatin C Equation. RESULTS: Urinary levels of cystatin C and NGAL increased after renal clamping. The 30-minute period of ischemia demonstrated the greatest increase of these biomarkers. Allopurinol did appear to serve a renoprotective function in those animals undergoing 30-minute clamp times. In our clinical patients, the serum cystatin C levels did increase at each postoperative time point, but remained nonelevated in the control group. CONCLUSIONS: Cystatin C and NGAL both appear to be useful biomarkers of renal injury. Studies with larger numbers are needed, however. Also, allopurinol does exhibit renoprotective effects against ischemic injury.

Assessment of the anti-protozoal activity of crude Carica papaya seed extract against Trypanosoma cruzi.

In order to determine the in vivo activity against the protozoan Trypanosoma cruzi, two doses (50 and 75 mg/kg) of a chloroform extract of Carica papaya seeds were evaluated compared with a control group of allopurinol. The activity of a mixture of the three main compounds (oleic, palmitic and stearic acids in a proportion of 45.9% of oleic acid, 24.1% of palmitic and 8.52% of stearic acid previously identified in the crude extract of C. papaya was evaluated at doses of 100, 200 and 300 mg/kg. Both doses of the extracts were orally administered for 28 days. A significant reduction (p < 0.05) in the number of blood trypomastigotes was observed in animals treated with the evaluated doses of the C. papaya extract in comparison with the positive control group (allopurinol 8.5 mg/kg). Parasitemia in animals treated with the fatty acids mixture was also significantly reduced (p < 0.05), compared to negative control animals. These results demonstrate that the fatty acids identified in the seed extracts of C. papaya (from ripe fruit) are able to reduce the number of parasites from both parasite stages, blood trypomastigote and amastigote (intracellular stage).

Assessment of the role of multidrug resistance-associated proteins in MPTP neurotoxicity in mice.

GOALS: The available scientific data indicate that the pathomechanism of Parkinson's disease (PD) involves the accumulation of endogenous and exogenous toxic substances. The disruption of the proper functioning of certain transporters in the blood-brain barrier and in the blood-cerebrospinal fluid barrier in PD would accompany to that accumulation. Although there is an emerging role of the dysfunction of multidrug resistance-associated proteins (MRPs), members of ATP-b nding cassette (ABC) transporter superfamily, in neurodegenerative disorders, there is only a few available data as regards PD. So the aim of our study was the assessment of the role of certain MRPs (1 ,2, 4 and 5) in neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine METHODS: Following the intraperitoneal administration of silymarin (with MRP1, 2, 4 and 5 inhibitory effects), naringenin (with MRP1, 2 and 4 stimulatory effects), sulfinpyrazone (with MRP1, 4 and 5 inhibitory and MRP2 stimulatory effects) and allopurinol (with MRP4 stimulatory effect in doses of 100 mg/kg, 100 mg/kg, 100 mg/kg and 60 mg/kg, respectively, for one week before and after the administration of MPTP in C57B/6 mice in acute dosing regimen the striatal concentrations of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid has been measured using high-performance liquid chromatography. RESULTS: Although the results of these experiments showed that neither of these substances exerted significant influence on MPTP-induced striatal depletion of dopamine and its metabolites, naringenin exerted a slight prevention of dopamine decrease, while allopurinol considerably enhanced the MPTP-induced lethality in mice. The explanation of these findings would be that the stimulation of MRP1- and MRP2-mediated transport of glutathione conjugates of toxic substances may have slight beneficial effects, while stimulation of MRP4-mediated efflux of brain urate, which has an important antioxidant potency, may worsen the effects of oxidative stress.

A randomized study comparing IGL-1 to the University of Wisconsin preservation solution in liver transplantation.

BACKGROUND: Institut Georges Lopez-1 (IGL-1) is a new preservation solution with lower potassium and lower viscosity than University Wisconsin solution (UW). These characteristics which improve liver preservation lead us to evaluate clinical effects of IGL-1 in a randomized controlled study with UW. MATERIAL/METHODS: From June 2007 to July 2009, after exclusion of partial graft, combined transplantation and fulminant hepatic failure, 140 deceased donor allografts were randomly assigned to IGL-1 (n=48) or UW (n=92) solution. Variables concerning donors and recipients were collected including liver tests (total serum bilirubin, prothrombin time and transaminases) were analyzed until postoperative day 30. Incidences of hepatic artery thrombosis (HAT), primary non function (PNF) and biliary non anastomotic strictures (NAS) were analyzed. The comparative analysis of costs was realized. RESULTS: Donor and recipients characteristics were similar in both groups. Volume of preservation solution utilized for harvesting was identical. Duration of cold ischemia (472±142 vs. 477±122 min), surgery (427±97 vs. 437±94 min) and proportion of extended criteria donor was similar. Postoperative kinetic and level liver tests were similar. Rate of PNF (2% vs. 4%), early retransplantation (6% vs. 7%), incidence of biliary NAS (2% vs. 3%) and HAT (6% vs. 4%) were similar. Mean intensive care unit (ICU) stay was similar (5.6 vs. 6.1 days). However costs related to preservation solution for one liver procurement were 992.0 for IGL-1 vs. 1609.0 Euros for UW. CONCLUSIONS: Results of this randomized study shows that the efficacy and safety of IGL-1 are comparable to those of the reference UW with a lower cost.

Comparative analysis of clinical efficacy and cost between University of Wisconsin solution and histidine-tryptophan-ketoglutarate.

OBJECTIVE: To compare University of Wisconsin solution (Viaspan), the universal standard for organ preservation, with histidine-tryptophan-ketoglutarate solution. An analysis of each solution, in reference to clinical trials with specific organs, is presented and assessed to find the efficacy of each in a clinical environment. Also to view each solution from an economical standpoint, and in the end develop an overall understanding of the key similarities and differences between each solution in order to assess appropriate use of each in a clinical setting. DATA SOURCES: A literature search was conducted by using PubMed, MEDLINE, BIOSIS, Embase, and other online data bases to find the most recent studies of University of Wisconsin and histidine-tryptophan-ketoglutarate solutions. Search terms included University of Wisconsin solution, histidine-tryptophan-ketoglutarate, preservation solution, cost analysis, biliary complication, and other related subjects. STUDY SELECTION: Previous research was selected from the literature search to provide basic information on the 2 solutions and also to provide clinical examples of each solution and the efficacy of each with specific organs. DATA SYNTHESIS: Information and published articles on the 2 solutions were gathered for descriptive and comparative purposes. CONCLUSIONS: The 2 solutions appear equally effective in organ preservation. Each solution has its own organ-specific qualities, and each has different complications. The studies reviewed here indicate that the differences are minor and thus suggest that the 2 solutions are equally acceptable for clinical use. Of the 2 solutions, histidine-tryptophan-ketoglutarate costs less than University of Wisconsin solution.

A modified CZ-1 preserving solution for organ transplantation: comparative study with UW preserving solution.

BACKGROUND: The University of Wisconsin colloid based preserving solution (UW solution) is the most efficient preserving solution for multiorgan transplantation. Unfortunately, unavailability of delayed organ preserving solutions hindered further progression of cardinal organ transplantation in China. In this study, we validated an organ preserving Changzheng Organ Preserving Solution (CZ-1 solution) and compared it with UW solution. METHODS: A series of studies were conducted on how and how long CZ-1 solution could preserve the kidneys, livers, hearts, lungs and pancreas of New Zealand rabbits and SD rats. Morphology of transplanted organs was studied by visible microscopy and electron microscopy; biochemical and physiological functions and the survival rate of the organs during prolonged cold storage were studied. RESULTS: There was no significant difference between CZ-1 and UW solutions in preserving the kidneys, livers, hearts or lungs of rabbits; kidneys, livers, intestinal mucosa or pancreases of SD rats or five deceased donors' testicles. In some aspects, such as preserving rabbits' hearts, rats' intestinal mucosa and pancreases, the effect of CZ-1 solution was superior to UW solution. CZ-1 could safely preserve kidneys for 72 hours, livers for 24 hours, hearts for 18 hours and lungs for 8 hours for SD rats. Twelve kidneys preserved in cold CZ-1 solution for 22 - 31 hours were transplanted successfully and the mean renal function recovery time was (3.83 +/- 1.68) days. CONCLUSIONS: CZ-1 solution is as effective as UW solution for organ preservation. The development of CZ-1 solution not only reduces costs and improves preservation of organs, but also promotes future development of organ transplantation in China.

In vivo assessment of free radical activity during shock wave lithotripsy using a microdialysis system: the renoprotective action of allopurinol.

PURPOSE: Shock wave lithotripsy is believed to cause renal damage directly through cellular injury from high energy shock waves and indirectly through vascular injury and resultant ischemia, which gives rise to oxygen free radical compounds. The transient and volatile nature of free radicals and derived products makes their detection difficult. Moreover, certain medications may provide a protective effect against shock wave lithotripsy induced renal parenchymal injury. We introduced an innovative microdialysis system for in vivo sampling of interstitial fluids that can be analyzed for free radical mediated lipid peroxidation products after shock wave lithotripsy treatment in the swine model. In addition, this system was used to test the antioxidant or renoprotective action of allopurinol. MATERIALS AND METHODS: Ten juvenile swine were assigned to a nonmedicated control group that underwent shock wave lithotripsy or to a group that was premedicated with allopurinol before shock wave lithotripsy. Each group of animals underwent shock wave lithotripsy to the lower pole of the right kidney and received a total of 10,000 shock waves. Dialysate fluid was collected at 1,000-shock wave increments via probes surgically implanted into the lower pole of the right and left kidneys before lithotripsy. Samples were immediately preserved in liquid nitrogen and subsequently analyzed for the presence and concentration of conjugated diene levels, a measure of lipid peroxidation. Five additional juvenile swine were assigned to a sham treated group that did not undergo shock wave lithotripsy. Dialysate fluid was collected from the lower pole of the right and left kidneys to establish baseline or pre-lithotripsy levels of conjugated dienes. RESULTS: After shock wave lithotripsy conjugated diene levels increased almost 100-fold over that in the right kidneys of the nonmedicated control group. The difference was statistically significant compared to levels in the contralateral untreated kidneys (p <0.01). Right kidneys in the group premedicated with allopurinol did not demonstrate an increase in conjugated diene levels during shock wave lithotripsy. CONCLUSIONS: The results of this study confirm shock wave lithotripsy induced free radical activity as well the antioxidant and protective nature of allopurinol. The newly described microdialysis system enables real-time sampling of interstitial fluids during shock wave lithotripsy. It represents a unique method for assessing free radical formation and evaluating the protective effects of additional antioxidant medications.

Intraluminal casein model of necrotizing enterocolitis for assessment of mucosal destruction, bacterial translocation, and the effects of allopurinol and N-acetylcysteine.

An intraluminal casein model (ICM) of necrotizing enterocolitis (NEC) is able to produce small-bowel changes reminiscent of human NEC in neonatal animals. We studied bacterial translocation (BT) in NEC induced by using the ICM in neonatal piglets. We also studied whether allopurinol (AL) and N-acetylcysteine (NAC) have an effect on BT and mucosal changes in the ICM of NEC. Twenty-eight neonatal piglets were randomized into four groups. NEC was induced in 21 by injecting casein-d-gluconate into a loop of terminal ileum: group Cas (n = 7) had no premedication, in group Cas/AL (n = 7) intravenous (i.v.) Al (100 mg/kg), and in group Cas/NAC (n = 7) i.v. NAC (200 mg/kg) was given. Group Sham (n = 7) had the ileum injected with 0.9% saline with no premedication. Immediately after the injection a mesenteric lymph node (MLN) adjacent to the loop was harvested for quantitative aerobic bacterial culture; 4 h after the injection another MLN and samples of spleen, liver, kidney, and lung were harvested and cultured. Comparison of the incidence of samples with positive bacterial cultures and the number of colony-forming units (CFU) in samples was made between groups. The severity of NEC in the ileum was graded from 0 to 3 according to macroscopic and histologic findings. NEC changes in the bowel were most severe in Cas piglets, less severe in Cas/NAC piglets ( P < 0.5), and sham piglets had the least severe changes ( P < 0.05). piglets with NEC changes in the ileum had a higher incidence of BT into the MLN than piglets without NEC changes ( P < 0.05), but the difference in CFU was not significant ( P > 0.05). In Cas and Cas/NAC piglets a high incidence of BT into the MLN was noted as early at -5 min after casein injection. The incidence of BT into the MLN was significantly higher in Cas and Cas/NAC piglets than in Sham piglets ( P < 0.05), the difference in CFU being not significant ( P > 0.05). BT in Cas/Al piglets was not significantly different from that of Cas piglets ( P > 0.05), but less than in Cas/NAC piglets ( P < 0.05). Four hours after casein injection into the ileum there was significant BT into the MLN. Premedication with NAC was associated with less severe NEC changes, but neither NAC nor AL significantly affected BT.

Flow cytometric assessment of allopurinol susceptibility in Leishmania infantum promastigote.

BACKGROUND: Leishmaniasis is a major tropical and subtropical parasitic disease. Sodium stibogluconate, N-methyl -D-glucamine antimoniate, amphotericin B, pentamidine, and ketoconazole are drugs used to treat this disease. Some of these drugs cause severe adverse side effects and treatment failures are common. Allopurinol, a purine analog, has been used to treat leishmaniasis, alone or combined with the previously mentioned drugs. Low cost, ease of administration (oral), and lack of toxicity make allopurinol a particularly appealing candidate. METHODS: The effect of allopurinol on Leishmania infantum (MCAN/ES/89/IPZ229/1/89, zymodeme MON1) wild-type promastigotes (wt-p229), and an altered form of these promastigotes (allo-p229) resulting from long term in vitro exposure to allopurinol, was determined by [(3)H]-thymidine incorporation assays and by diverse flow cytometric approaches. RESULTS: Allopurinol arrested the proliferative capacity of wt-p229 promastigotes, reduced the proportion of viable cells, and decreased their total protein content. In contrast, allo-p229 promastigote proliferation was only slightly decelerated and the proportion of viable cells and the protein content were not affected by the allopurinol treatment. CONCLUSIONS: The flow cytometry approach allowed us to demonstrate differences in allopurinol susceptibility of the two promastigote forms, expanding the spectrum of flow cytometry applications in studies of parasite resistance.

Assessment of hepatic viability during cold ischemic preservation.

A reliable and easy method for assessing the viability of a cold ischemia-preserved donor liver prior to transplantation into the recepient is needed. Based on an earlier study, we hypothesized that liver free fatty acid (FFA) leakage into the preservation fluid may be a useful, atraumatic indicator of irreversible ischemic injury. The aim of the present study was to determine the time course and magnitude of liver FFA release into the preservation solution and its correlation with the duration of cold ischemic preservation compatible with survival after transplantation. Rat livers (n = 48) were flushed and preserved with University of Wisconsin (UW) solution at 4 degrees C for 0, 12, 24, and 48 h. Thereafter, half of the livers were analyzed for preservation fluid FFA (gas-liquid chromatography) and protein. The other half were perfused with Krebs-Henseleit (KH) solution at 37 degrees C for 1 h. Bile secretion and liver enzyme release (SGOT, SGPT, and LDH) were measured in addition to perfusate FFA and protein. Total FFA in the preservation fluid was 24 micrograms/g wet tissue after 12 h; it increased sharply 2.6-fold after 24 h and 3.7-fold after 48 h of preservation. Bile production was normal after 12 h of preservation but fell by 20% and 54% after 24 h and 48 h, respectively. LDH release rose from a value of 20 U/l at 0 time to 120 U/l and 260 U/l after 24 h and 48 h of preservation. These results suggest that liver viability declines sharply between 12 and 24 h of cold ischemic preservation, which corresponds with a sharp decrease in the 1-week survival from 100% to 33% after 12 h and 24 h, respectively, of cold ischemic preservation. We conclude that measuring FFA and LDH in the preservation solution of donor livers may be a useful means of assessing the quality of the cold-preserved liver before insertion into the recipient. We also speculate that a "threshold" FFA level in the UW preservation fluid indicating irreversible damage may be in the order of 35 micrograms total FFA/g liver. Studies on the clinical applicability of our findings are currently under way.

Functional and morphological assessment of rat aorta stored in University of Wisconsin and Eurocollins solutions.

University of Wisconsin (UW) and Eurocollins (EC) solutions are widely used for preservation of organs before transplantation. However, effect of storage solutions on vascular interface for transplant success is not known. In this study, we have used rat aorta as a model and assessed the effects of cold storage in UW and EC solutions on smooth muscle and endothelial function and the morphology. Smooth muscle and endothelial functions of the rat aorta were assessed using in vitro isometric tension measurement. Morphologic studies were done with scanning and transmission electron microscopy. No significant difference in contractile response to either norepinephrine (NE) or potassium chloride was observed between control aorta and aorta stored in UW solution for 1 hr or 24 hr. In contrast, sensitivity, but not the reactivity to NE and KCl, was increased in aorta stored in EC solution for 1 hr. If the tissues were stored in EC solution for 24 hr, both sensitivity and reactivity to NE and KCl were significantly reduced. Relaxatory response to acetylcholine, in endothelium-intact vessels were reduced in aortas stored in EC solution, but not in UW solution. The magnitude of relaxations observed in tissues stored in the EC solution for 24 hr was less than in tissues stored for 1 hr. Sodium nitroprusside elicited similar relaxatory response in endothelium-denuded control tissue and in tissues stored in UW and EC solution. Electron microscopy data revealed marked swelling of the cell, loss of mitochondria and other intracellular organelles, and striking calcium deposits after preservation of the vessels in EC for 1 or 24 hr. In aorta stored in UW solution for 24 hr, endothelial and smooth muscle cells were intact, with moderate-size vacuoles in the cytoplasm. These results suggest that the UW solution is more suitable than EC solution for short-term preoperative storage of blood vessels.