Characteristics of membrane transport of methotrexate by cultured human breast cancer cells

Methotrexate, a folic acid analogue, enters cells using a high-affinity carrier system that is shared with naturally occurring reduced folates. Methotrexate transport by MCF-7 cells, a hormonally responsive line of human breast cancer cells, exhibited a high-affinity carrier system that displayed Michaelis-Menten kinetics [K_m = 8.22 ± 0.62 μM; V_max = 12.22 ± 2.8 nmoles · min^?1·(g cell protein)^?1] was competitively inhibited by leucovorin and aminopterin, but not by folic acid; and was temperature-sensitive (Q₁₀ = 2.25 ± 0.32). Initial uptake rates were not affected by ouabain or sodium azide, but efflux of intracellular drug was inhibited markedly by sodium azide, suggesting the presence of an energy-dependent active efflux mechanism. At extracellular methotrexate concentrations in excess of 10 μM, a second, low-affinity uptake component could be identified that may represent a lower affinity membrane carrier or passive diffusion. Examination of hormonal influences on methotrexate transport revealed that growth of MCF-7 cells in serum-free medium induced a significant increase in the transport K_m value (15.93 ± 1.6 μM) compared to the K_m of 8.22 μM for cells grown in fetal calf serum. This change in affinity of the transport carrier could be reversed by the additon of insulin, but not of estradiol, to the culture medium. Methotrexate transport by human breast-cancer cells displayed characteristics that were similar to those of transport reported for human leukemia cells but that have not been documented previously for cells derived from a human solid tumor. In addition, the drug transport carrier was subject to modulation by insulin but not by estrogen.     

Stereospecificity at carbon 6 of fomyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

The unnatural diastereoisomer of l-5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [³H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K_i, were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K_i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.     

Role of transporter-mediated efflux in the placental biodisposition of bupropion and its metabolite, OH-bupropion

Cigarette smoking during pregnancy is a preventable risk factor associated with maternal and fetal complications. Bupropion is an antidepressant used successfully for smoking cessation in non-pregnant patients. Our goal is to determine whether it could benefit the pregnant patient seeking smoking cessation. The aim of this investigation was to determine the role of human placenta in the disposition of bupropion and its major hepatic metabolite, OH-bupropion. The expression of efflux transporters P-gp and BCRP was determined in placental brush border membrane (n = 200) and revealed a positive correlation (p < 0.05). Bupropion was transported by BCRP (K_t 3 μM, V_max 30 pmol/mg protein/min) and P-gp (K_t 0.5 μM, V_max 6 pmol/mg protein*min) in placental inside-out vesicles (IOVs). OH-bupropion crossed the dually-perfused human placental lobule without undergoing further metabolism, nor was it an efflux substrate of P-gp or BCRP. In conclusion, our data indicate that human placenta actively regulates the disposition of bupropion (via metabolism, active transport), but not its major hepatic metabolite, OH-bupropion.     

In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (K_i = 4.98 μM). Cryptotanshinone (K_i = 6.87 μM) and tanshinone I (K_i = 0.42 μM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (K_i = 0.09 μM), and noncompetitive inhibition on CYP4F2 (K_i = 4.25 μM) and CYP4F3B (K_i = 3.08 μM). Salvianolic acid A had competitive inhibition on CYP4A11 (K_i = 19.37 μM), and noncompetitive inhibition on CYP4F2 (K_i = 15.28 μM) and CYP4F3B (K_i = 6.45 μM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (K_i = 5.70 μM) and CYP4F3B (K_i = 18.64 μM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 μM) and salvianolic acid A (20 and 50 μM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.     

Allylglycine, and inhibitor of the uptake of L-leucine and L-proline in rat brain slices

The convulsant allylglycine inhibited the uptake of l-leucine and l-proline in rat brain slices and was without influence on the uptake of acetylcholine, dopamine, l-glutamic acid, glycine and l-lysine under the conditions studied. l-Leucine uptake could also be inhibited by the “large neutral” amino acids l-methionine, l-phenylalanine and l-valine. No kinetic studies of l-leucine uptake were possible due to the inability to measure initial rates of uptake. The time course of l-leucine uptake was not linear under a variety of conditions and appeared to be complicated by a rapid efflux of radioactive label in the form of acidic metabolite(s). l-Proline uptake was mediated by a “high affinity” saturable system (K_m 50 μM), and was non-competitively inhibited by allylglycine (K_i{ca.} 50 μM). l-Leucine competitively inhibited (K_i, 75 μM) l-proline uptake, although l-leucine uptake was unaffected by l-proline. l-Norleucine, DL-pipecolic acid and l-azetidine-2-carboxylic acid inhibited l-proline uptake.     

Transport and interaction of drugs in leukocytes

The accumulation of selected CNS drugs by rat leukocytes was previously reported. This paper presents evidence for the transport into leukocytes of additional drugs. Also studied was the inhibition of the latter processes by various structurally related compounds. The markedly rapid and sodium-independent uptakes into rat leukocytes of amphetamine, codeine, methadone and naloxone fulfilled the basic criteria for active transport. The uptake of morphine was apparently accomplished by more than one process. The affinities of the high capacity transport systems (approximate V_max: 100 nmoles/g cells/5sec) varied considerably as reflected by the two extreme K_m values obtained for methadone (20 μM) and morphine (1.8 mM). A variety of amines inhibited the cellular transport of the drugs. Most potent inhibitors were quinacrine (K_i: 0.5 to 3 μM), desipramine (K_i: 6–20 μM) and methadone (K_i: 18–25 μM). Morphine and tryptamine exhibited inhibition constants higher than 1 mM. The cellular transport processes newly described in rat leukocytes apparently represent a novel addition to the heterogenous biological transport of basic amines. The structural specificity of amine transport in various tissues is discussed.     

Studies on rat kidney 15-hydroxy-prostaglandin dehydrogenase.

Rat kidney 15-hydroxy-prostaglandin dehydrogenase (PGDH) was isolated, and its characteristics and the effects of various drugs upon it were examined. The enzyme was found in the cell cytosol; was labile when unfrozen; and most active at alkaline pH, at 41°, and with the E prostaglandins. Additionally, the enzyme was inhibited by furosemide (K_i = 0.019 mM), ethacrynic acid (K_i = 0.27 mM, phenylbutazone (K_i = 0.16 mM), acetylsalicylic acid (K_i = 3.8 mM), and potassium cyanide (K_i = 1.03 mM). Inhibition of PGDH may play a role in the mechanism of action of the diuretic and anti-inflammatory drugs. Little or no inhibition was seen with amobarbital, hydralazine, alpha-methyldopa, bethanidine and guanethidine. Amobarbital inhibits NADH oxidase (K_i = 0.5 mM), but does not inhibit PGDH. This drug, therefore, may be useful in permitting the use of the fluorometric assay for PGDH in preparations of PGDH contaminated by NADH oxidase.     

Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions

The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this enzyme. The substrate analog, 8-aminoguanine (8-AG), has the lowest dissociation constant (K_i = 0.2–1.2 μM) of any compound reported to date and V_max = 16 per cent (relative to guanine). Other C(8) substituents decreased the affinity for PNP and, with the exception of the methyl and sulfhydryl groups, abolished substrate activity. Halogens or a thiomethyl group at C(5') of inosine resulted in unchanged or improved affinities (K_i = 10–30 μM) and greatly decreased substrate activity (V_max < 1 per cent relative to inosine). The K_i of formycin B was reduced from 100 μM to 10 μM or less by substitution of a halogen at C(5'). Phosphorolysis of purine nucleosides was inhibited significantly by 8-AG in intact human erythrocytes and murine Sarcoma 180, L1210 and L5178Y cells. Although a K_i value of 17 μM was determined for 8-aminoguanosine, it was equally effective in inhibiting PNP activity in intact cells. The nucleoside was cleaved to 8-AG which was not a substrate for guanase or hypoxanthine-guanine phosphoribosyltransferase. Despite its low substrate activity (V_max < 0.2%). 5′-deoxy-5′-iodoinosine was cleaved by intact L1210 and L5178Y cells.     

Sodium-linked transport of ethacrynic acid by rat liver: Possible significance for choleretic action

Uptake, biotransformation and biliary excretion of ethacrynic acid was investigated in the isolated rat liver and related to the choleretic effect of the drug. Ethacrynic acid transport from sinusoidal extracellular space into liver cells is mediated by a saturable, energy-dependent, and partially Na⁺-dependent transport mechanism. Extracellular sodium stimulates translocation of ethacrynic acid across the sinusoidal membrane by increasing the maximal velocity from 0.47 to 0.64 μmoles.min^?1.g liver^?1 without any major effect on the substrate affinity of the carrier. Within the cell, ethacrynic acid is rapidly and almost completely metabolized to its glutathione derivative which, in turn, is excreted into bile canaliculi by a saturable transport system. Canalicular excretion of metabolized ethacrynic acid is the rate-limiting step in hepatic transport of the choleretic drug (V_max0.15 μmoles.min^?1.g liver^?1. Both translocation steps are accompanied by an increase in transmembrane sodium fluxes. At the sinusoidal site, co-transport of Na⁺ with ethacrynic acid and/or inhibition of (Na⁺-K ⁺)-ATPase might be responsible for the net increase in intracellular Na⁺ as observed in liver slices. Excretion of intracellular Na⁺ into bile canaliculi is enhanced during canalicular transport of the ethacrynic acid glutathione adduct. The transepithelial Na⁺ net movement induced by these events might be underlying the stimulatory effect of ethacrynic acid on bile secretion.     

One enzyme for the 5′-deiodination of 3,3′,5′-triiodothyronine and 3′,5′-diiodothyronine in rat liver

Many studies suggest that one enzyme is involved in the phenolic ring deiodination of iodothyronines in rat liver and kidney and another one in the tyrosyl ring deiodination. This study describes some characteristics of the phenolic ring (5′-) deiodination of rT₃ and 3′,5′-T₂ by rat liver microsomes. At pH 7.2 the K_m values of the 5′-deiodination of rT₃ and 3′,5′-T₂ were 0.103 and 0.77 μM, respectively. 3′,5′-T₂ and rT₃ inhibited the respective 5′-deiodination reactions competitively, the K_i values being 1.05 and 0.134 μM, respectively. Several radiographic contrast agents markedly inhibit the 5′-monodeiodination of rT₃ and 3′,5′T-₂, the type of inhibition being competitive. Of these compounds iopanoic acid, ipodic acid and iophenoxic acid are the most potent inhibitors with K_i values of approximately 2 μM for both reactions. The non-iodine containing compound 8-anilino-1-naphthalene sulphonic acid (ANS) appeared to be a very strong competitive inhibitor of both 5′-deiodinations (K_i 4.3–4.7 μM), whereas salicylic acid, which as ANS inhibits the binding of iodothyronines to T₄-binding globulin, inhibited these reactions to a much lesser extent (K_i 300–500 μM). On the other hand, diiodosalicylic acid was a very strong inhibitor. The β-adrenergic blocker d,l-propranolol was a weak noncompetitive inhibitor of both 5′-deiodinations (K_i 0.4–0.7 mM). These reactions were also inhibited by various 2,6-diiodophenol derivatives, triiodophenol being the strongest and diiodotyrosine the weakest inhibitor tested. Comparing the K_i values of various inhibitors for the 5′-deiodination of rT₃ and 3′,5′-T₂, a positive correlation between these values was found (r = 0.97). It was concluded that rT₃ (to 3,3′-T₂) and 3′,5′-T₂ (to 3′-T₁) monodeiodinating activities are very similar to each other and that there may just be one monodeiodinase catalyzing the 5′-deiodination of iodothyronines in rat liver.     

Opiates inhibit paclitaxel uptake by P-glycoprotein in preparations of human placental inside-out vesicles

The use of either methadone or buprenorphine for treatment of the pregnant opiate-dependent patient improves maternal and neonatal outcome. However, patient outcomes are often complicated by neonatal abstinence syndrome (NAS). The incidence and severity of NAS should depend on opiate concentration in the fetal circulation. Efflux transporters expressed in human placental brush border membranes decrease fetal exposure to medications by their extrusion to the maternal circulation. Accordingly, the concentration of either methadone or buprenorphine in the fetal circulation is, in part, dependent on the activity of the efflux transporters. The objective of this study was to characterize the activity of P-gp and its interaction with opiates in the placental apical membrane. Therefore, brush border membrane vesicles were prepared from human placenta. The vesicles were oriented approximately 75% inside-out, exhibited saturable ATP-dependent uptake of P-gp substrate [³H]-paclitaxel with an apparent K_t of 66 ± 38 nM and V_max of 20 ± 3 pmol mg protein⁻¹ min⁻¹. Methadone, buprenorphine, and morphine inhibited paclitaxel transport with apparent K_i of 18, 44, and 90 μM, respectively. Our data indicate that a method has been established to determine the activity of the efflux transporter P-gp, expressed in placental brush border membranes, and the kinetics for the transfer of its prototypic substrate paclitaxel. Furthermore, the method was used to determine the effects of methadone, buprenorphine, and morphine on paclitaxel transfer by placental P-gp and revealed that they have higher affinity to the transporter than its classical inhibitor verapamil (K_i, 300 μM).     

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1′-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC₅₀ = 0.76 μM (29), K_i = 0.68 μM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC₅₀ declining to 0.32 μM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were k_inact = 0.024 min^?1 (30) and K_I = 1.63 μM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC₅₀ and K_i values of 0.057 μM (17) and 0.043 μM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).     

Evidence of competitive inhibition of methotrexate absorption by leucovorin calcium in rat small intestine

The effect of leucovorin calcium on the intestinal absorption of methotrexate in rat small intestine was investigated using an in situ rat gut technique. First, the kinetic absorption in situ parameters for methotrexate in solution were obtained: V_m=21.54 (±2.22) μM/h; K_m=10.51 (±1.08) μM; k_a=0.26 (±0.03) h⁻¹ and AIC=−188.63. The inhibitory effect of leucovorin calcium in methotrexate intestinal absorption has been investigated by perfusing of 10 μM methotrexate isotonic solutions containing increasing concentrations of leucovorin calcium (10–500 μM), and the remaining concentrations of both compounds were measured. A competitive inhibition of methotrexate absorption was detected: the apparent absorption rate constant of the drug decreased as the initial leucovorin calcium concentration increased. Higher leucovorin calcium concentrations, however, did not completely abolish the absorption of the drug (at 500 μM of leucovorin calcium, only 84% inhibition was observed). Apparent parameters characterizing the absorption of leucovorin calcium in the presence of methotrexate 10 μM were: V_mi=14.70 (±1.74) μM; K_mi=9.43 (±1.59) μM; k_ai=0.28 (±0.02) h⁻¹; AIC=−191.53). We can concluded that methotrexate and leucovorin calcium compete for the same intestinal carrier system. This means that since leucovorin calcium, because of its ready conversion to other tetrahydrofolic derivatives (McEvoy, 1996. AHFS Drug Information, Bethesda, MD, pp. 751–758), is administered together with methotrexate in order to prevent the hematopoietic and reticuloendothelial toxic effects of folic acid antagonists, using high leucovorin calcium concentrations, when the urine excretion is decreased, could prevent intestinal drug reabsorption and the drug could then be excreted in the feces, thereby decreasing the risk of poisoning.     

Comparison of dose/conductance curves for gaba and some structurally related compounds at the lobster inhibitory neuromuscular junction

Log-dose/conductance curves for γ-aminobutyric acid (GABA), γ-amino-β-hydroxybutyric acid (GABOB), δ-aminovaleric acid (DAV) and guanidoacetic acid (GuAc) were compared at the lobster inhibitory neuromuscular junction. GABOB and DAV curves paralleled the GABA curves and could be fitted by the same two independent-binding site receptor model (K_GABOB = 275 μM; K_DAV = 945 μM: apparent dissociation constants estimated by double-reciprocal plot). GuAc and GABA curves were non-parallel, and showed different apparent maxima. The normalized GuAc dose/conductance curve could be fitted by a modified form of the general two binding-site model with K₁ =7.4 μM and K₂ = 92 μM. It is suggested that GABA, GABOB and DAV were agonists of comparable ‘intrinsic activity’ but differing receptor affinity whereas GuAc was behaving as a partial agonist.     

Reversible inhibition of acetylcholinesterase by eseroline,an opioid agonist structurally related to physostigmine (eserine) and morphine

The action of eseroline—(3aS, 8aR)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b] indol-5-ol—salicylate was tested on preparations of ChE from different sources and on the longitudinal muscle of guinea-pig ileum. While eseroline is extremely weak-acting on horse serum BuChE (K_i = 208 ± 42 μM), it is a rather strong competitive inhibitor of AChE's, its K_i being 0.15 ± 0.08 μM, 0.22 ± 0.10 μM and 0.61 ± 0.12 μM in electric eel, human RBC and rat brain, respectively. Eseroline inhibitory action on AChE is independent of the duration of pre-incubation and appears fully developed in less than 15 sec. This action is also rapidly reversible; after pre-incubation followed by dilution, maximum enzymic activity is regained within 15 sec. The electrically-evoked contractions of the longitudinal strip were inhibited by concentrations of eseroline in the range 0.2–15 μM, while they were increased by concentrations over 20 μM. In the same preparation, without electrical stimulation, but in the presence of naloxone, eseroline induced contractions at concentrations higher than 5 μM. This effect was antagonized by atropine. The inhibitory activity of eseroline parallels, as regards selectivity, potency and kinetics, that of the phenolic anticurare agent edrophonium, while it differs markedly from that of physostigmine.     

Calcium binding properties of rat heart plasma membrane and inhibition by structural analogues of dl-propranolol

The isolation and characterization of a plasma membrane preparation from rat heart is described. Enzymatic, chemical, and electron microscopic analysis revealed a relative lack of contamination with nuclear, mitochondrial, ribosomal, and sarcoplasmic reticulum membrane. One calcium binding site (K_d) = 265 μM, B_max = 65 $\text{nmoles}\text{mg}$ protein) was detected by equilibrium dialysis. Monovalent metal ions exhibited 10–100-fold less inhibition potency than divalent metal ions when analyzed by competitive inhibition of calcium binding. The range of K_i values found for divalent metal ions was similar to the K_d value for calcium. La⁺³ produced a potent non-competitive inhibition. A large variety of structural analogues of d,l-propranolol, many of which have been shown to lack β-adrenergic blocking activity, were competitive inhibitors of the calcium binding activity, with K_i values ranging from 40–900 μM. Electrophilic, hydrophobic, and diamino substituents greatly increased the inhibitory activity. There was no significant difference between related tertiary and quaternary amines. The experimental antiarrhythmic agent UM 272 had the least ability to inhibit calcium binding to the cardiac plasma membrane preparation (K_i = 795 μM). However, UM 424, another experimental antiarrhythmic agent, had an inhibitory activity similar to dl-propranolol (k_i = 115 μM and 108 μM, respectively).     

Anti-convulsants and brain aldehyde metabolism: Inhibitory characteristics of ox brain aldehyde reductase

The major isoenzyme of aldehyde reductase has been purified from ox brain by affinity chromatography. Carbamazepine (K_i = 7.3 × 10^?4 M) and phenacemide (K_i = 2.5 × 10^?4 M), in common with all other established anti-convulsant drugs tested, have been shown to inhibit the activity of this enzyme. A selection of structural analogues of the anti-convulsant sodium valproate were found to be potent inhibitors of the reductase (K_ivalues in the range 10^?3 M ?5 × 10^?5 M) and these analogues also showed anti-convulsant activity in the mouse maximal electroshock test. A third group of compounds, the flavonoids, constitute the most potent group of aldehyde reductase inhibitors yet reported. Quercetin and morin exhibited K_i values less than 1 μM. The possible relationship between aldehyde metabolism and anti-convulsant action is discussed and structural characteristics pre-disposing to potent inhibition of aldehyde reductase are described.     

Cepharanthine is a potent reversal agent for MRP7(ABCC10)-mediated multidrug resistance

Multidrug resistance protein 7 (MRP7; ABCC10) is an ABC transporter that confers resistance to anticancer agents such as the taxanes. We previously reported that several inhibitors of P-gp and MRP1 were able to inhibit the in vitro transport of E₂17βG by MRP7 in membrane vesicles transport assays. However, compounds that are able to reverse MRP7-mediated cellular resistance have not been identified. In this study, we examined the effects of cepharanthine (6′,12′-dimethoxy-2,2′-dimethyl-6,7-[methylenebis(oxy)]oxyacanthan), an herbal extract isolated from Stephania cepharantha Hayata, to reverse paclitaxel resistance in MRP7-transfected HEK293 cells. Cepharanthine, at 2 μM, completely reversed paclitaxel resistance in MRP7-transfected cells. In contrast, the effect of cepharanthine on the parental transfected cells was significantly less than that on the MRP7-transfected cells. In addition, cepharanthine significantly increased the accumulation of paclitaxel in MRP7-transfected cells almost to the level of control cells in the absence of cepharanthine. The efflux of paclitaxel from MRP7-transfected cells was also significantly inhibited by cepharanthine. The ability of cepharanthine to inhibit MRP7 was analyzed in membrane vesicle assays using E₂17βG, an established substrate of MRP7, as a probe. E₂17βG transport was competitively inhibited by cepharanthine with a K_i value of 4.86 μM. These findings indicate that cepharanthine reverses MRP7-mediated resistance to paclitaxel in a competitive manner.     

Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport

The effects of different fibric acid derivatives (bezafibrate, clofibrate, clofibric acid, fenofibrate, fenofibric acid and gemfibrozil) on human organic anion transporting-polypeptide 1B1 (OATP2, OATP-C, SLC21A6), multidrug resistance protein 2 (MRP2/ABCC2) and MDR1-type P-glycoprotein (P-gp/ABCB1) were examined in vitro. Cyclosporin A (a known inhibitor of OATP1B1 and P-gp), MK-571 (a known inhibitor of MRP2) and cimetidine (an organic cation) were also tested. Bezafibrate, fenofibrate, fenofibric acid and gemfibrozil showed concentration-dependent inhibition of estradiol 17-β-D-glucuronide uptake by OATP1B1-stably transfected HEK cells, whereas clofibrate and clofibric acid did not show any significant effects up to 100?µM. Inhibition kinetics of gemfibrozil, which exhibited the most significant inhibition on OATP1B1, was shown to be competitive with a K_i?=?12.5?µM. None of the fibrates showed any significant inhibition of MRP2-mediated transport, which was evaluated by measuring the uptake of ethacrynic acid glutathione into MRP2-expressing Sf9 membrane vesicles. Only fenofibrate showed moderate P-gp inhibition as assessed by measuring cellular accumulation of vinblastine in a P-gp overexpressing cell-line. Cyclosporin A significantly inhibited OATP1B1 and P-gp, whereas only moderate inhibition was observed on MRP2. The rank order of inhibitory potency of MK-571 was determined as OATP1B1 (IC₅₀: 0.3?µM)?>?MRP2 (4?µM)?>?P-gp (25?µM). Cimetidine did not show any effects on these transporters. In conclusion, neither MRP2- nor P-gp-mediated transport is inhibited significantly by the fibrates tested. Considering the plasma protein binding and IC₅₀ values for OATP1B1, only gemfibrozil appeared to have a potential to cause drug–drug interactions by inhibiting OATP1B1 at clinically relevant concentrations.     

Phosphodiesterase in the liver fluke, Fasciola hepatica.

Phosphodiesterase was found in homogenates of the liver fluke, Fasciola hepatica, and was distributed between a supernatant and particulate fraction after centrifugation at 2000 g. Mg²⁺ was necessary for enzyme activity; Ca²⁺ in the presence of Mg²⁺ did not affect enzyme activity. Enzyme kinetics followed the Michaelis-Menten model with a K_m of 8 μM for cAMP and 300 μM for cGMP as the substrate. The most potent inhibitor tested was 1-ethyl-4-(isopropylidenehydrazino)-1 H-pyrazolo- (3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HC1 (SQ 20009) which had a K_i of 26 μM. The K_i for isobutyl methyl xanthine (IBMX) was 45 μM; for 6,7 dimethyl-4 ethylquinazoline (Quazodine) 75 μM; papaverine. 100 μM; theophylline, 550 μM; and for caffeine or D-lysergic acid diethylamide (LSD), 800 μM. The effects on fluke motility of these phosphodiesterase inhibitors were tested. All phosphodiesterase inhibitors except caffeine stimulated the rhythmical movement of the flukes. None of the inhibitors tested significantly increased the endogenous cAMP concentrations of fluke heads. IBMX potentiated the rise in endogenous cAMP caused by 5-hydroxytryptamine (5-HT) but SQ 20009, LSD, and papaverine prevented it. The latter results could not be explained on the basis of phosphodies-terase inhibition, but might be attributed to interference with the stimulation of adenylate cyclase by 5-HT.