Inhibitory Effects Of Various Drugs Research Articles

Measurement of Bile Salt Export Pump Transport Activities using a Fluorescent Bile Acid Derivative

A novel fluorescent bile acid derivative, 4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole- conjugated bile acid was synthesized as a probe to develop a rapid screening method for function analysis of bile salt export pump (BSEP, ABCB 11). The transport properties of the synthetic fluorescent bile acid derivative in membrane vesicles obtained from hBSEP-expressing Sf9 cells were examined using the liquid chromatography-electrospray ionization-mass spectrometry method. The Michaelis-Menten constant and maximum uptake rate for the synthetic fluorescent bile acid derivative by hBSEP were 23.1+/-1.6 microM and 623.2+/-22.4 pmol/min/mg protein, respectively. These kinetic parameters of the synthetic fluorescent bile acid derivative were comparable with those of an unlabeled bile acid, taurocholic acid. Moreover, we examined inhibitory effects of various drugs on hBSEP-mediated uptake of the fluorescent bile acid derivative using a fluorescence detection method. The relative uptake activities (percent of control) for the fluorescent bile acid derivative in the presence of an inhibitor were in accordance with previous findings using (3)H-labeled taurocholic acid. Our results suggest that the synthetic fluorescent bile acid derivative may be useful for evaluation of the inhibitory effects of various drugs on hBSEP-mediated uptake.

Investigation of the Inhibitory Effects of Various Drugs on the Hepatic Uptake of Fexofenadine in Humans

Fexofenadine (FEX), an H(1)-receptor antagonist, is eliminated from the liver mainly in an unchanged form. Our previous study suggested that organic anion-transporting polypeptide (OATP) 1B3 contributes mainly to the hepatic uptake of FEX. On the other hand, a clinical report demonstrated that a T521C mutation of OATP1B1 increased its plasma area under the plasma concentration-time curve. Several compounds are reported to have a drug interaction with FEX, and some of this may be caused by the inhibition of its hepatic uptake. We determined which transporters are involved in the hepatobiliary transport of FEX by using double transfectants and examined whether clinically reported drug interactions with FEX could be explained by the inhibition of its hepatic uptake. Vectorial basal-to-apical transport of FEX was observed in double transfectants expressing OATP1B1/multidrug resistance-associated protein 2 (MRP2) and OATP1B3/MRP2, suggesting that OATP1B1 as well as OATP1B3 is involved in the hepatic uptake of FEX and that MRP2 can recognize FEX as a substrate. The inhibitory effects of compounds on FEX uptake in OATP1B3-expressing HEK293 cells were investigated, and the maximal degree of increase in plasma AUC of FEX by drug interaction in clinical situations was estimated. As a result, cyclosporin A and rifampicin were found to have the potential to interact with OATP1B3-mediated uptake at clinical concentrations. From these results, most of the reported drug interaction cannot be explained by the inhibition of hepatic uptake of FEX, and different mechanisms such as the inhibition of intestinal efflux should be considered.

DRUG-DRUG INTERACTION BETWEEN PITAVASTATIN AND VARIOUS DRUGS VIA OATP1B1

It has already been demonstrated that pitavastatin, a novel potent HMG-coenzyme A reductase inhibitor, is taken up into human hepatocytes mainly by organic anion transporting polypeptide (OATP) 1B1. Because OATP2B1 is also localized in the basolateral membrane of human liver, we took two approaches to further confirm the minor contribution of OATP2B1 to the hepatic uptake of pitavastatin. Western blot analysis revealed that the ratio of the band density of OATP2B1 in human hepatocytes to that in our expression system is at least 6-fold lower compared with OATP1B1 and OATP1B3. The uptake of pitavastatin in human hepatocytes could be inhibited by both estrone-3-sulfate (OATP1B1/OATP2B1 inhibitor) and estradiol-17beta-D-glucuronide (OATP1B1/OATP1B3 inhibitor). These results further supported the idea that OATP1B1 is a predominant transporter for the hepatic uptake of pitavastatin. Then, to explore the possibility of OATP1B1-mediated drug-drug interaction, we checked the inhibitory effects of various drugs on the pitavastatin uptake in OATP1B1-expressing cells and evaluated whether the in vitro inhibition was clinically significant or not. As we previously reported, we used the methodology for estimating the maximum unbound concentration of inhibitors at the inlet to the liver (I(u,in,max)). Judging from I(u,in,max) and inhibition constant (K(i)) for OATP1B1, several drugs (especially cyclosporin A, rifampicin, rifamycin SV, clarithromycin, and indinavir) have potentials for interacting with OATP1B1-mediated uptake of pitavastatin. The in vitro experiments could support the clinically observed drug-drug interaction between pitavastatin and cyclosporin A. These results suggest that we should pay attention to the concomitant use of some drugs with pitavastatin.

New Methods to Evaluate Intestinal Drug Absorption Mediated by Oligopeptide Transporter from In vitro Study using Caco-2 Cells

The aim of the present work is to develop a convenient and rapid screening system in vitro for intestinal drug absorption mediated by oligopeptide transporter (PepT1). In this study, (1) Transports of cephalexin (CEX) and L-phenylalanine (L-Phe) across Caco-2 monolayers were measured and compared with those of passively transported drugs, (2) Inhibitory effects of various drugs on the transport of [(14)C]glycylsarcosine (Gly-Sar) across Caco-2 monolayers were measured and correlated with their in vivo permeability to rat small intestine, (3) Intracellular pH-change induced by co-transport of drugs with proton into Caco-2 cells was monitored by using Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices Corp.). Concentration-dependent transport was observed in Caco-2 monolayers for CEX and L-Phe, although their permeability was relatively low compared to those of passively transported drugs. Inhibitory effects of various drugs including beta-lactam antibiotics and angiotensin converting enzyme-inhibitors on the transport of Gly-Sar correlated well with their in vivo permeability to rat small intestine. It was demonstrated that CEX, but not cefazolin, induced gradual decrease in the intracellular pH of Caco-2 cells. The degree of intracellular pH-change caused by various drugs showed a sigmoidal or saturable relationship with their permeability to rat small intestine. These in vitro approaches with Caco-2 cells should be useful to evaluate in vivo intestinal permeability of drugs mediated by PepT1, suggesting a possibility of high throughput screening of drug absorption.

Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte membrane

Phosphatidylserine (PS) containing a 7-nitrobenz-2-oxa-1,3-diazol-4-yl- (NBD-) hexanoyl residue, like native PS, preferentially distributes into the inner membrane leaflet of human erythrocytes. In the case of NBD-PS, this preference results from two opposite active processes, an inward translocation mediated by the aminophospholipid flippase and an outward translocation mediated by an ill-defined floppase. Selective inhibition of this floppase by alkylating reagents or cationic and anionic drugs increases the extent of accumulation of NBD-PS in the inner membrane leaflet from about 70% in control cells to about 90%. Different inhibitor sensitivities of the flippase and the floppase strongly suggest that both represent different entities. The floppase was characterized in further detail by comparing inhibitory effects of various compounds on this translocase with their effects on known primary active transport systems for amphiphilic compounds. The inhibitory effects of various drugs, glutathione conjugates and GSSG on the floppase activity closely correlate with those reported for the active transport by the multidrug resistance protein (MRP) while only poorly going parallel with those for the active transport by the low affinity pump for glutathione conjugates and the multidrug resistance MDR1 P-glycoprotein. The NBD-phospholipid floppase activity of the erythrocyte is thus probably a function of MRP.

Prominent inhibitory effects of tranilast on migration and proliferation of and collagen synthesis by vascular smooth muscle cells

To obtain some ideas about prevention of restenosis after percutaneous transluminal coronary angioplasty (PTCA), we examined the effects of tranilast (anti-allergic agent) on migration and proliferation of, and collagen synthesis by, cultured vascular smooth muscle cells (VSMC) from the thoracic aorta of WKY rats. Tranilast was added to culture medium containing 10% fetal calf serum (FCS). The cultures were pulse-labeled with 3H-thymidine (TdR) or 3 3H-proline (Pro). TdR and Pro uptake into VSMC were measured. The effect of tranilast on migration of VSMC was examined by using culture dishes of an original design. We also examined the inhibitory effects of various drugs, such as a Ca antagonist, an angiotensin converting enzyme (ACE) inhibitor, a phosphodiesterase inhibitor, elastase, colchicine, and mitomycin C, on proliferation and migration of VSMC. Our data showed that the inhibitory effects of tranilast on migration and proliferation of, and collagen synthesis by, VSMC were prominent. Maximal percentage inhibition of proliferation, migration and collagen synthesis was 60.8 ± 2.3%, 52.7 ± 14.7% and 62.1 ± 8.1%, respectively. On the other hand, the inhibitory effects of other drugs, with the exception of colchicine and mitomycin C, on proliferation and/or migration of VSMC were not very strong. Although the inhibitory effects of colchicine and mitomycin C were strong in vitro, their clinical usefulness may be limited by systemic side-effects. These results indicate the potential usefulness of tranilast for prevention of restenosis of coronary arteries after PTCA.

Inhibitory effects of various drugs on phorbol myristate acetate and n−-formyl methionyl leucyl phenylalanine induced O 2− production in polymorphonuclear leukocytes

To clarify the mechanisms of 0 2 − formation by polymorphonuclear leukocytes (PMNs), the effects of clinically employed drugs on PMNs were investigated by measuring changes in membrane potential and rates of O 2 − production. These variables were effectively diminished with antihistaminic agents, adrenergic β-antagonists, and antiarrhythmic drugs when guinea pig peritoneal PMNs were stimulated by either phorbol myristate acetate (PMA) or n-iormyl-methionyl-leucyl-phenylalanine (FMLP). The order of potency of the inhibitory effects of these chemicals on the PMA-induced O 2 − formation was as follows: azelastine (IC 50 = 4.1 μM) < clemastine < d-propranolol < chlorpheniramine maleate < dichlorisoproterenol < quinidine < diphenhydramine < indomethacin (IC 50 > 400 μM). Similar phenomena were observed when FMLP was employed instead of PMA, but the FMLP-stimulated O 2 − production was effectively inhibited by indomethacin. Changes in membrane potential, using the cyanin dye method, also indicated that most of these drugs cancelled functional changes of plasma membrane of PMNs. From these observations, it was demonstrated that changes in membrane potential by the stimuli were essential for the initiation of 0 2 − generation from plasma membrane of PMNs, although the initiation mechanisms were not identical for the two stimuli.

Inhibitory effects of various drugs on dual asthmatic responses in wheat flour-sensitive subjects

In order to investigate the mechanism of late asthmatic response (LAR), inhibitory effects of various drugs for LAR were examined in two wheat flour-sensitive asthmatic subjects who showed immediate and late responses in the allergen provocation test and skin test. Antihistamines did not inhibit the LAR but totally or partially inhibited the immediate response. By contrast, corticosteroids inhibited the LAR but not the immediate response. Disodium cromoglycate inhibited both responses. Diethyl carbamazine citrate, an inhibitor of release of SRS-A, seemed to shorten the duration of the LAR, although it has no effect on the immediate response and/or on the severity of the LAR. Indomethacin and acetyl salicylate, which inhibit prostaglandin synthesis, had no significant effects on either the immediate or the LAR.

Analysis of Inhibitory Effects of Antifungal Drugs on Trichophyton.

The differences of inhibitory effects of various drugs on T. rubrum and T. mentagrophytes were observed. In serial dilution method using Sabouraud's dextrose agar, salicylic acid, merzonin and pentachlorophenol were little effective on the inhibition of mycotic growth to a certain concentration, then suddenly prohibited the growth when the concentration was increased, the reverse cultivation being negative. On the other hand, in griseofulvin and variotin the growth was inhibited gradually as the concentration in the medium was increased, all of the reverse cultivation being always positive. The former three drugs act fungicidally and the latter two antibiotics work fungistatically. Microscopic changes of fungi by these antifungal drugs were not specific for each drug.

A new technique for studying the inhibitory effects of drugs on discriminatory avoidance-escape behaviour in rats

A “brightness discrimination” ☐ was used for inducing typical discriminatoryavoidance habits in rats, and the inhibitory effects of various drugs on this type of behaviour were studied. After about fifty daily training sessions only one-third of the rats are adequately trained. All drugs tested (neuroleptics, CNS-stimulants, analgesics, barbiturates, anti-cholinergics) are selective inhibitors of avoidance. Only typical neuroleptics are capable of blocking avoidance at doses not producing overt signs of neurological impairment. About the same dose levels are necessary for blocking avoidance to a comparable degree in this difficult “discrimination” procedure and in the simple “jumping ☐”.