Systematic analysis and identification of unexpected interactions from the neuroprotein drug interactome in hydrocephalus pharmacological intervention.

Hydrocephalus is a neurological condition caused by an abnormal accumulation of cerebrospinal fluid; pharmacological intervention of the disease has been found to elicit a variety of adverse drug reactions (ADRs) in central nervous system (CNS) by unexpectedly targeting certain functional neuroproteins. Here, a systematic neuroprotein drug interactome (SNDI) is created for 11 hydrocephalus drugs/metabolites plus 20 control drugs across 518 druggable pockets on the surface of 472 CNS neuroproteins via a large-scale molecular docking approach. Heuristic clustering analysis of the SNDI profile divides the 31 investigated drug ligands into a distinct panel and a background panel; the former consists of two hydrocephalus drugs (Furosemide and Triamterene) and their respective metabolites (Furosemide glucuronide and Hydroxytriamterene) that are inferred to have generally high affinity towards the whole array of neuroprotein pockets. A total of 13 neuroproteins are enriched in gene ontology semantic mining as putative unexpected targets of the distinct panel, and their intermolecular interactions with hydrocephalus drugs/metabolites are investigated in detail using dynamics simulation and energetics analysis. We also perform kinase assay and viability test to substantiate the interactome analysis. It is found that the Furosemide and Triamterene have significant cytotoxic effects on normal human astrocytes, in which the Triamterene can inhibit the neurokinase ROCK2, a representative of putative unexpected targets, with a high activity, which is comparable with the sophisticated ROCK2 inhibitor Fasudil.

In vitro cytotoxicity and DNA/HSA interaction study of triamterene using molecular modelling and multi-spectroscopic methods.

The anticancer activity of triamterene on HCT116 and CT26 colon cancer cells lines was investigated. Furthermore, the mechanism of interaction between triamterene and calf thymus DNA (ct-DNA) and also human serum albumin (HSA) was conducted using spectroscopic and molecular docking techniques. In vitro cytotoxicity of triamterene against HCT116 and CT26 cells showed promising anticancer effects with IC50 values of 31.30 and 24.45 µM, respectively. Competitive studies of the triamterene with NR (neutral red) and MB (methylene blue) as intercalator probes showed that triamterene can be replaced by these probes. The viscosity data also confirmed that triamterene binds to calf-thymus DNA through intercalation binding mode. Binding properties of triamterene with HSA in the presence of warfarin and ibuprofen showed that triamterene competes with warfarin for the site I of human serum albumin (HSA). In addition, the binding modes of triamterene with DNA and HSA were verified by molecular docking technique. Abbreviations ct-DNA calf thymus DNA CV cyclic voltammetry DNA deoxyribonucleic acid DPV differential pulse voltammetry FBS fetal bovine serum HSA human serum albumin NR neutral red MB methylene blue MTT 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide Communicated by Ramaswamy H. Sarma.

Quantitative investigation of the role of breast cancer resistance protein (Bcrp/Abcg2) in limiting brain and testis penetration of xenobiotic compounds.

The role of breast cancer resistance protein (BCRP/ABCG2) in limiting the brain and testis penetration of xenobiotic compounds in the blood-brain and -testis barriers was investigated using Bcrp(-/-) mice. Tissue/plasma concentration ratios in the brain (K(p,brain)) and testis (K(p,testis)) obtained under steady-state conditions were significantly increased in Bcrp(-/-) mice for PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), N-hydroxyl PhIP, MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), dantrolene, and prazosin. In addition, the K(p,brain) of triamterene and the K(p,testis) of 4'-hydroxyl PhIP were also significantly increased in Bcrp(-/-) mice. The effect of functional impairment of Bcrp on the brain uptake of PhIP, dantrolene, and daidzein in Bcrp(-/-) mice determined using in situ brain perfusion was weaker than that observed on the K(p) values. In vitro transcellular transport experiments using cell lines expressing mouse Bcrp or P-glycoprotein (Mdr1a/Abcb1a) showed that, among the tested Bcrp substrates, PhIP, MeIQx, prazosin, and triamterene are common substrates of Bcrp and P-glycoprotein. The K(p) values of common substrates exhibited a smaller increase both in the brain and testis of Bcrp(-/-) mice than expected from the in vitro Bcrp activities. The Bcrp-specific substrates were weak acids, whereas basic or neutral BCRP substrates were also P-glycoprotein substrates. These results suggest that BCRP limits the tissue penetration of xenobiotic compounds in the blood-brain and -testis barriers, but its in vivo importance is also modulated by P-glycoprotein activity.

Rate-limiting biotransformation of triamterene is mediated by CYP1A2.

OBJECTIVE: Triamterene (TA), a potassium-sparing diuretic, is extensively metabolized by hydroxylation in 4'-position and subsequent conjugation by cytosolic sulfotransferases. To identify the cytochrome P450 enzyme(s) catalyzing hydroxylation of triamterene (the rate-limiting step in the formation of the sulfate ester (STA)), in vitro incubation studies were performed with human liver microsomes. METHODS: Initial rates of TA hydroxylation (0 - 300 microM) were determined during a ten-minute-incubation period with liver microsomes of two donors. The role of individual CYP enzymes was determined by pre-incubation with selective inhibitors/alternative substrates. Vice versa, the effect of TA (0 - 500 microM) on 3-demethylation of caffeine (0 - 1,000 microM) was assessed. Metabolite concentrations were estimated by reversed-phase HPLC methods. RESULTS: TA Km values without inhibitors were 60 and 142 microM, Vmax was 177 and 220 pmol/min/mg protein, respectively. Mean inhibitor induced changes of 4'-hydroxy-TA formation were as follows: Furafylline 25 microM (CYP1A2), complete inhibition (-100%); omeprazole 250 microM (CYP1A2 inhibitor/CYP2C 19 substrate), -30%; coumarin 25 microM (CYP2A6), -11%; quinidine 25 microM (CYP2D6), -9%; ketoconazole 25 microM (CYP3A), -18%; and erythromycin 250 microM (CYP3A), -8%. In the reverse inhibition studies, TA competitively inhibited caffeine 3-demethylation with Ki values of 65 and 111 microM, respectively. CONCLUSION: 4'-hydroxylation of TA in humans appears to be mediated exclusively by CYP1A2. Inhibition or induction of CYP1A2 will change the time course of both TA and its active phase-II metabolite. The net pharmacodynamic effect of such changes is difficult to predict and needs to be evaluated in clinical studies.

Physical stability of solid dispersions containing triamterene or temazepam in polyethylene glycols.

The effect of storage on the physical stability of solid dispersions of triamterene or temazepam in polyethylene glycols was studied using differential scanning calorimetry (DSC), particle-size analysis and dissolution methods. The enthalpies of fusion of the carriers, without included drug and previously fused and crystallized, increased on storage. Analysis of similarly treated solid dispersions, containing either 10% temazepam or 10% triamterene, showed that each drug influenced the morphology of the polyethylene glycol (PEG). The enthalpies and melting points of the solidus components of the dispersions' carriers were initially reduced after preparation, but on storage these increased. The particle sizes of the drugs dispersed in the PEGs increased on storage. The changes in dissolution after storage of triamterene or temazepam dispersions were smaller for dispersions in PEG 1500 than for dispersions in PEGs of higher molecular weight (PEG 2000, PEG 4000 or PEG 6000) in which the reduction in dissolution was particularly marked during the first month of storage. The rank order of changes in dissolution were PEG 1500 < < PEG 2000 < PEG 4000 approximately PEG 6000.

BIBW22 BS, potent multidrug resistance-reversing agent, binds directly to P-glycoprotein and accumulates in drug-resistant cells.

The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.

[Animal experimental and human pharmacologic studies with phase-II metabolites of triamterene]. / Tierexperimentelle und humanpharmakologische Untersuchungen mit dem Phase-II-Metaboliten von Triamteren.

Animal Experiments and Human Studies with the Phase-II Metabolite of Triamterene The described animal experiments as well as the pharmacological investigations in man show the diuretic efficacy of the phase-II metabolite of triamterene (CAS 396-01-0), OH-TA-ester. As demonstrated in the present investigation, a phase-II metabolite can not be considered only as an ineffective conjugation product, formed to accomplish the excretion of a foreign compound, but also it must be considered as an important factor in the therapy, if its pharmacologic efficacy has been established, moreover if as a result of disease-related alterations in the pharmacokinetics the phase-II metabolite cumulates.

Effect of H2 antagonists on the differential secretion of triamterene and its sulfate conjugate metabolite by the isolated perfused rat kidney.

The isolated perfused rat kidney model was used to examine the effect of the histamine H2 antagonists cimetidine, ranitidine, and famotidine and the organic anion inhibitor probenecid on the differential renal handling of triamterene and its active metabolite p-hydroxytriamterene sulfate. The kidneys were perfused with a Krebs-Henseleit buffer containing albumin, glucose, and amino acids to pH 7.4, and drug concentrations were measured by HPLC. At an initial triamterene concentration of 0.5 mg/liter, the unbound renal clearance to glomerular filtration rate (GFR) ratio was 11.0 +/- 2.5 (mean +/- SD): 1, indicating substantial tubular secretion of the drug. Cimetidine and ranitidine reduced the tubular secretion by about 80% (p less than 0.01), famotidine by between 35 and 60% (p = 0.05), whereas probenecid had no inhibitory effect. For p-hydroxytriamterene sulfate, its unbound renal clearance to GFR ratio was 41 +/- 25:1; this was not affected by cimetidine, ranitidine, or famotidine, whereas probenecid significantly (p less than 0.01) reduced the rate of tubular secretion by 80%. These data indicate that the renal tubular secretion of triamterene is mediated by the organic cation system, whereas for p-hydroxytriamterene sulfate its tubular secretion is via the organic anion system. Famotidine is a weaker inhibitor of the organic cation system compared with cimetidine and ranitidine. These results have implications for drug-drug interaction studies involving renal elimination pathways.

Deconjugation of p-hydroxytriamterene sulphate in the rat.

1. The extent of deconjugation of p-hydroxytriamterene sulphate was studied in rats, by h.p.l.c., after i.v., i.p., and oral administration. 2. After i.v. administration, deconjugation accounted for 29-54% of the recovered dose as free p-hydroxytriamterene in urine and faeces. Following i.p. administration, 70-88% was deconjugated and 72-96% was deconjugated after oral administration. Most of the p-hydroxytriamterene was recovered in faeces.

Dyazide-induced reversible acute renal failure associated with intracellular crystal deposition.

Acute interstitial nephritis due to Dyazide therapy, ie, a combination of hydrochlorothiazide (25 mg) and triamterene (50 mg), has been recently reported in the literature. This had been characterized by nonoliguric renal failure after a long latent period (weeks) following exposure to the drug. Pathologic data have indicated a drug-induced hypersensitivity reaction. We report here one case of oliguric acute renal failure after a massive Dyazide intoxication. Based on the results of the renal biopsy and clinical course, we propose that the oliguria was secondary to a direct toxic effect on the tubules, and intrarenal obstruction was secondary to triamterene crystals and crystal-laden cells. In addition, pathologic findings also suggested a moderate hypersensitivity reaction. After hemodialysis and short-term steroid therapy, the patient achieved complete recovery of renal function within 12 days. Recent knowledge of triamterene-induced nephrolithiasis helps to explain the pathogenesis of acute renal failure in this patient, and is briefly reviewed here.

Correlation between mechanistic biotransformation and biochemical toxicology of some antihypertensive drugs.

Mechanistic biochemistry (consideration of metabolism in the context of knowledge of contemporary biochemistry) was applied to propanolol (1), hydrochlorothiazide (2), hydralazine (3), and triamterene (4), representative of the main types of anti-hypertensive drugs in common use. Three routes of metabolism, that is, acetylation, generation of free radicals (leading to peroxidation of lipids), and osazone formation were considered in relation to the structures of these drugs. The possibility that acetylation can lead to hepatic toxicity, lipid peroxidation to membrane lesion, and osazone formation to glucose and energy depletion was highlighted. Hydralazine, with its potential for osazone formation and great susceptibility to acetylation and free radical formation, was judged most capable of giving rise to these side effects, in agreement with reported toxicity. Triamterene was judged less susceptible than hydralazine to acetylation and free radical formation, and hydrochlorothiazide even less so. Propanolol is immune to any of these consequences.

Effect of cimetidine on renal and hepatic drug elimination: studies with triamterene.

A chronic-dosing pharmacokinetic study was carried out in six healthy subjects to examine the potential for cimetidine to reduce the CLR and CLH of triamterene. Blood and urine samples were collected frequently for 24 hours after dosing with triamterene alone (100 mg/day) for 4 days and concomitant cimetidine (400 mg twice daily) for an additional 4 days. Cimetidine significantly reduced the clearance of triamterene by hydroxylation by 32% (P less than 0.016) and the CLR of triamterene by 28% (P less than 0.063), with no change in its protein binding. The CLR of the active sulfate conjugate of triamterene was not altered by cimetidine. There was a reduced recovery of triamterene and its metabolites in urine after cimetidine, suggesting a decreased absorption. These results are consistent with cimetidine inhibiting cytochrome P-450 enzymes in the liver and also competing with triamterene for renal tubular secretion. Despite the pharmacokinetic interaction, cimetidine caused minimal alteration to the natriuretic and antikaliuretic effects of triamterene.

Absorption and disposition of two combination formulations of hydrochlorothiazide and triamterene: influence of age and renal function.

In this study we compared the absorption and disposition of two commonly used combination formulations of hydrochlorothiazide and triamterene (Dyazide and Maxzide) in 48 patients with essential hypertension after dosing with each formulation to steady state. Interdose AUC and urinary recovery of hydrochlorothiazide, triamterene, and the major metabolite of triamterene, hydroxytriamterene sulfate (adjusted for dose), documented marked impairment in the absorption of hydrochlorothiazide (approximately two third as bioavailable) and triamterene (about half as bioavailable) from Dyazide in comparison to Maxzide. The study also demonstrated a reduction in the clearance of triamterene, hydrochlorothiazide, and hydroxytriamterene sulfate with increasing age. Linear correlation analyses suggested that this effect was a result of the reduction in renal function that occurs with increasing age.

[Relation between the hypotensive effect of triampur and its pharmacokinetics in patients with hypertension]. / Zavisimost' gipotenzivnogo effekta triampura ot ego farmakokinetiki u bol'nykh gipertonicheskoi bolezn'iu.

The hypotensive effect of triampur was studied in 39 patients with first- and second-stage essential hypertension, as was the pharmacokinetics of triamterin, the main constituent of triampur. A relationship is demonstrated between the curve area, blood triamterin peak, clearance and cumulative excretion, on the one hand, and the hypotensive effect, on the other. Triamterin pharmacokinetic studies may be used for the screening of hypertensive patients as potential candidates for triampur treatment. Alongside other causes, low blood concentrations of the drug in the course of treatment may be associated with its low efficiency.

Metabolic fate and solubility of triamterene--not an explanation for triamterene nephrolithiasis.

In an attempt to explain the triamterene stone diathesis, we studied the excretion and solubility of triamterene, 1, and its metabolite, the sulfate ester of the hydroxy derivative of triamterene, 3. The urinary excretion pattern and metabolism in stone formers was the same as in other chronic users of triamterene or healthy volunteers. The solubility of triamterene in urine was approximately one-half of its solubility in buffer solution, whereas the sulfate ester, 3, was nearly twice as soluble in urine as in the buffer solution. In the majority of the subjects studied, we found concentrations of 3 which approached or exceeded apparent solubility limits in urine. This was not true for triamterene where most measured urine concentrations were less than the apparent solubility as determined by equilibration. Alteration in the metabolism of triamterene is probably not a causative factor for triamterene nephrolithiasis. The saturation of urine with triamterene and especially with the sulfate ester, 3, may be related to stone formation, but other physical factors play a role in determining the relative amounts of drug found in calculus material.

The true composition of kidney stones passed during triamterene therapy.

The potassium-sparing agent triamterene and its metabolites have been identified as components of kidney stones passed by patients receiving that drug. We have analyzed 66 such kidney stones by use of quantitative thin-layer chromatography. The stones contained not only the unchanged triamterene but also significant amounts of an intermediate metabolite, hydroxytriamterene, and large amounts of the sulfate ester of hydroxytriamterene. Of the stones 49 per cent contained less than 5 per cent triamterene and its metabolites, and no stone comprised 100 per cent triamterene-derived material. Of all the triamterene-related material unchanged triamterene dominated. However, the relative fractions of triamterene and hydroxytriamterene in the kidney stones exceeded the relative fractions of these 2 components in the urine.

Oral triamterene disposition.

Earlier studies of triamterene (T) disposition in man have reported hydroxytriamterene (T-OH) and hydroxytriamterene sulfate (T-O-SO3H) conjugate to be the major metabolites. We describe T kinetics through use of an HPLC method and confirm that after hydroxylation, T is rapidly converted to T-O-SO3H. The intermediate T-OH metabolite could not be detected in urine or plasma. Plasma concentrations of T-O-SO3H exceeded those of T by sevenfold to 26-fold, whereas concentrations of that metabolite in the urine were fourfold to thirteenfold higher than those of the parent. Renal clearance of T was 314.5 +/- 121.6 ml/min, exceeding that of the metabolite, which was 206.1 +/- 93.6 ml/min. Coadministration of hydrochlorothiazide increased urine flow and urinary pH, but it did not affect renal clearance of the parent drug or the metabolite. T bioavailability from capsules was poorer and more variable than that from a suspension. Hydrochlorothiazide did not influence the bioavailability of T.

Triamterene calculus.

Triamterene therapy is an unusual cause of nephrolithiasis and, when this agent is found in a stone, generally it is deposited in minor amounts. We report a renal calculus consisting mostly of triamterene and its 2 major metabolites in a patient taking a triamterene-containing drug, and discuss some implications.