Xanthine oxidase/tyrosinase inhibiting,antioxidant, and antifungal oxindole alkaloids from Isatis costata

Phytochemical investigations on the ethyl acetate soluble fraction of the whole plant of Isatis costata Linn. (Brassicaseae) led to the isolation of the oxindole alkaloids costinones A (1), B (2), isatinones A (3), B (4), indirubin (5), and trisindoline (6). Compounds 1–6 displayed significant to moderate inhibition against xanthine oxidase enzyme with IC₅₀ values ranging from 90.3?±?0.06 to 179.6?±?0.04 µM, whereas the standard inhibitor of xanthine oxidase (allopurinol) had an IC₅₀ value of 7.4?±?0.07 µM. Compounds 1 (IC₅₀ 7.21?±?0.05 µM), 2 (IC₅₀ 9.40?±?0.03 µM), 3 (IC₅₀ 11.51?±?0.07 µM), 4 (IC₅₀ 12.53?±?0.06 µM), 5 (IC₅₀ 14.29?±?0.09 µM), and 6 (IC₅₀ 17.34?±?0.04 µM) exhibited pronounced activities when compared with the standard tyrosinase inhibitor l-mimosine (IC₅₀ 3.70?±?0.03 µM), along with DPPH radical scavenging activity with IC₅₀ 226, 270, 300, 320, 401, and 431 µM, respectively. The crude extract and compounds 1, 2, 5, and 6 showed significant antifungal activity against Trichophyton schoen leinii, Aspergillus niger, Candida albicans, Trichophyton simii, and Macrophomina phaseolina.     

Synthesis of novel N-substitutedphenyl-6-oxo-3-phenylpyridazine derivatives as cyclooxygenase-2 inhibitors

Some novel non-ulcerogenic N-substitutedphenyl-6-oxo-3-phenylpyridazines as COX-2 inhibitors have been developed (Supplementary material Appendix 1 ). The novel aldehyde 3 was prepared by reacting 6-phenylpyridazin-3(2H)-one with 4-fluorobenzaldehyde. The aldehyde 3 was reacted with different hydrazines and thiazolidin-4-ones to obtain the novel N-substitutedphenyl-6-oxo-3-phenylpyridazine derivatives. These were assessed for their anti-inflammatory potential and gastric ulcerogenic effects. The molecular docking investigations were also undertaken. The spectroscopic data were coherent with the allocated structures of the compounds. The compounds 4a (IC₅₀ = 17.45 nm; p < .05), 4b (IC₅₀ = 17.40 nm; p < .05), 5a (IC₅₀ = 16.76 nm; p < .05), and 10 (IC₅₀ = 17.15 nm; p < .05) displayed better COX-2 inhibitory activity than celecoxib (IC₅₀ = 17.79 nm; p < .05). These findings were consistent with the molecular docking investigations of 4a , 4b , 5a , and 10 . The in vivo anti-inflammatory profile of 4a , 4b , 5a , and 10 was also superior to celecoxib and indomethacin. The compounds 4b , 5a , and 10 revealed no gastric ulcerogenic effects, wherein the compound 4a produced almost negligible gastric ulcerogenic effects than celecoxib and indomethacin. The compounds 4a , 4b , 5a , and 10 have been postulated as promising non-ulcerogenic COX-2 inhibitors.     

Anti-oxidative and inhibitory activities on nitric oxide (NO) and prostaglandin E<Subscript>2</Subscript> (COX-2) production of flavonoids from seeds of <Emphasis Type="Italic">Prunus tomentosa</Emphasis> Thunberg

Chemical investigation of the 80% Me₂CO extract from the seeds of Prunus tomentosa led to the isolation and identification of six flavonoids: kaempferol (1), kaempferol 3-O-α-L-rhamnopyranoside (2; afzelin), kaempferol 3-O-β-D-(6-acetyl)-glucopyranosyl(1→4)-α-L-rhamnopyranoside (3; multiflorin A), kaempferol 3-O-β-D-glucopyranosyl(1→4)-α-L-rhamnopyranoside (4; multiflorin B), quercetin 3-O-α-L-rhamnopyranoside (5; quercitrin), and quercetin 3-O-β-D-glucopyranosyl (1→4)-α-L-rhamnopyranoside (6; multinoside A). Anti-oxidative and inhibitory activities on nitric oxide (NO) and prostaglandin E₂ production in interferon-γ (INF-γ) and lipopolysaccharide (LPS)-activated RAW 264.7 cells in vitro (COX-2) of the isolated compounds were evaluated. Compounds 1, 5, and 6 exhibited potent anti-oxidative activity in the DPPH radical scavenging assay with IC₅₀ values of 57.2, 59.4, and 54.3 μg/mL respectively. The positive control, ascorbic acid, had an IC₅₀ of 55.5 μg/mL. Compounds 1, 5, and 6 also reduced COX-2 levels in a dose dependent manner with IC₅₀ values of 10.2, 8.7, and 9.6 μg/mL respectively, with the positive control, indomethacin, having an IC₅₀ of 5.1 μg/mL. All six compounds inhibited NO production in a dose dependent manner with IC₅₀ values of 35.1, 42.8, 40.0, 44.8, 43.7, and 43.9 μg/mL respectively, while the positive control, L-NMMA, had an IC₅₀ of 42.1 μg/mL.     

Antidiabetic and antioxidant potency evaluation of different fractions obtained from Cucumis prophetarum fruit

Abstract

Context: Cucumis prophetarum Linn. (Cucurbitaceae) fruit is used for inflammatory-related problems and is proved to be possessing anticancer and hepatoprotective effects.

Objective: The present investigation was to study the effect of different fractions of C. prophetarum on antidiabetic and antioxidant activity.

Materials and methods: Aqueous crude extract (CE) of C. prophetarum fruits was fractionated into water soluble fraction 1 (F1), chloroform fraction 2 (F2), basic fraction 3 (F3), and neutral fraction 4 (F4) by acid–base extraction. CE and its fractions at different doses (0.02–0.1?mg/mL) were subjected to antidiabetic (α-amylase and α-glucosidase inhibition assays) and antioxidant (DPPH, superoxide radical scavenging (SO) and metal chelation) evaluation.

Results: F1 exhibited effective antidiabetic activity (p?<?0.05) with an IC₅₀ value of 20.6 and 59.9?µg/mL. The activity decreased in the order of CE?>?F4?>F3?>?F2, according to α-amylase assay, which were the same, with the exception of the rank order of F4 and CE, as the α-glucosidase assay. Furthermore, F1 (IC₅₀?=?73?µg/mL) showed better reducing ability than CE?>F4?>F2?>?F3 (IC₅₀?=?78–272?µg/mL), according to the DPPH assay. In SO and metal chelation assays, F1 showed the highest activity (IC₅₀?=?101 and 147?µg/mL), respectively; the activity decreased in the order of CE?>F4?>F3?>?F2 (IC₅₀?=?126–469?µg/mL) for SO and 194–944?µg/mL for metal chelation assay.

Conclusion: The results indicate that F1 possesses potent in vitro antidiabetic and antioxidant activities.     

Design and syntheses of diarylisoxazoles: Novel inhibitors of cyclooxygenase‐2 (COX‐2) with analgesic‐antiinflammatory activity

A group of 4,5‐diphenylisoxazoles ( 11a–p ), 3,4‐diphenyl‐5‐trifluoromethylisoxazoles ( 15, 21 ), and 4,5‐diphenyl‐3‐methylsulfonamidoisoxazole ( 23 ) possessing a variety of substituents (H, F, MeS, MeSO, MeSO₂) at the para‐position of one of the phenyl rings were synthesized for evaluation as analgesic, and selective COX‐2 inhibitory antiinflammatory (AI), agents. Although the 4,5‐diphenylisoxazole group of compounds (11a–p) exhibited potent analgesic and AI activities, those compounds evaluated ( 11a, 11b, 11m ) were more selective inhibitors of COX‐1 than COX‐2, with the exception of 4‐(4‐methylsulphonylphenyl)‐5‐phenylisoxazole ( 11n ) that showed a modest COX‐2 selectivity index (SI) of 2.1. In contrast, 3‐(4‐methylsulphonylphenyl)‐4‐phenyl‐5‐trifluoromethylisoxazole ( 15 ), which retained good analgesic and AI activities, was a highly potent and selective COX‐2 inhibitor (COX‐1 IC₅₀ > 500 μM; COX‐2 IC₅₀ < 0.001 μM) with a COX‐2 SI of > 500,000, relative to the reference drug celecoxib (COX‐1 IC₅₀ = 22.9 μM; COX‐2 IC₅₀ = 0.0567 μM) with a COX‐2 SI of 404. The 3‐phenyl‐4‐(4‐methylsulphonylphenyl) regioisomer ( 21 ) was a less potent inhibitor (COX‐1 IC₅₀ = 252 μM; COX‐2 IC₅₀ = 0.2236 μM) with a COX‐2 SI of 1122, relative to the regioisomer ( 15 ). The related compound 4,5‐diphenyl‐3‐methylsulfonamidoisoxazole ( 23 ) exhibited similar (to 21 ) potency and COX‐2 selectivity (COX‐1 IC₅₀ > 200 μM; COX‐2 IC₅₀ = 0.226 μM) with an SI of 752. A molecular modeling (docking) study for the most potent, and selective, COX‐2 inhibitor (15) in the active site of the human COX‐2 enzyme showed the C‐5 CF₃ substituent is positioned 3.37 Å from the phenolic OH of Tyr³⁵⁵, and 6.91 Å from the Ser⁵³⁰ OH. The S‐atom of the MeSO₂ substituent is positioned deep (7.40 Å from the entrance) inside the COX‐2 secondary pocket (Val⁵²³). These studies indicate a C‐5 CF₃ ( 15, 21 ), or C‐3 NHSO₂Me ( 23 ), central isoxazole ring substituent is crucial to selective inhibition of COX‐2 for this class of compounds. Drug Dev. Res. 51:273–286, 2000. © 2001 Wiley‐Liss, Inc.     

Three new C-alkylated flavonoids from Desmodium oblongum

Three new C-alkylated flavonoids, 4′-hydroxy-8-isobutyryl-7-methoxy-6-methyl-flavone (1), 4′,7-dimethoxy-8-isobutyryl-6-methyl-flavone (2), and 4′,7-dimethoxy-8-isobutyryl-flavone (3), together with three know ones luteolin (4), kaemferol (5), and quercetin (6), were isolated from Desmodium oblongum. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D-NMR techniques. Compound 1 showed cytotoxicity against NB4, SHSY5Y, and MCF7 cell lines with IC₅₀ values of 8.5, 6.5, and 7.8 μM; compound 2 showed cytotoxicity against SHSY5Y and PC3 cell lines with IC₅₀ values of 5.0 and 6.8 μM; compound 3 displayed cytotoxicity against SHSY5Y and MCF7 cell lines with IC₅₀ values of 6.4 and 9.4 μM, respectively.     

Fluvoxamine is a More Potent Inhibitor of Lidocaine Metabolism than Ketoconazole and Erythromycin in vitro

Abstract CYP3A4 is generally believed to be the major CYP enzyme involved in the biotransformation of lidocaine in man; however, recent in vivo studies suggest that this may not be the case. We have examined the effects of the CYP3A4 inhibitors erythromycin and ketoconazole and the CYP1A2 inhibitor fluvoxamine on the N-deethylation, i.e. formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine by human liver microsomes. The experiments were carried out at lidocaine concentrations of 5 μM (clinically relevant concentration) and 800 μM. The formation of both MEGX and 3-hydroxylidocaine was best described by a two-enzyme model. At 5 μM of lidocaine, fluvoxamine was a potent inhibitor of the formation of MEGX (IC₅₀ 1.2 μM). Ketoconazole and erythromycin also showed an inhibitory effect on MEGX formation, but ketoconazole (IC₅₀ 8.5 μM) was a much more potent inhibitor than erythromycin (IC₅₀ 200 μM). At 800 μM of lidocaine, fluvoxamine (IC₅₀ 20.7 μM) and ketoconazole (IC₅₀ 20.4 μM) displayed a modest inhibitory effect on MEGX formation, whereas erythromycin was a weak inhibitor (IC₅₀>250 μM). The 3-hydroxylation of lidocaine was potently inhibited by fluvoxamine at both lidocaine concentrations (IC₅₀ 0.16 μM at 5 μM and 1.8 μM at 800 μM). Erythromycin and ketoconazole showed a clear inhibitory effect on the 3-hydroxylation of lidocaine at 5 μM of lidocaine (IC₅₀ 9.9 μM and 13.9 μM, respectively), but did not show a consistent effect at 800 μM of lidocaine (IC₅₀>250 μM and 75.0 μM, respectively). Although further studies are needed to elucidate the role of distinct CYP enzymes in the biotransformation of lidocaine in humans, the findings of this study suggest that while both CYP1A2 and CYP3A4 are involved in the metabolism of lidocaine by human liver microsomes, CYP1A2 is the more important isoform at clinically relevant lidocaine concentrations.     

Inhibition of P-glycoprotein in Caco-2 cells: Effects of herbal remedies frequently used by cancer patients

1. The herbal products Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic were investigated for in vitro inhibitory potential on P-glycoprotein (P-gp)-mediated transport of digoxin (30 nM) in differentiated and polarized Caco-2 cells. 2.?Satisfactory cell functionality was demonstrated through measurements of assay linearity, transepithelial electric resistance (TEER), cytotoxicity, mannitol permeability, and inclusion of the positive inhibition control verapamil. 3.?The most potent inhibitors of the net digoxin flux (IC₅₀) were mistletoe?>?Natto K2?>?Agaricus?>?green tea (0.022, 0.62, 3.81, >4.5 mg ml^?1, respectively). Mistletoe also showed the lowest IC₂₅ value, close to that obtained by verapamil (1.0 and 0.5 µg ml^?1, respectively). The IC₅₀/IC₂₅ ratio was found to be a good parameter for the determination of inhibition profiles. Garlic and noni juice were classified as non-inhibitors. 4.?This study shows that mistletoe, Natto K2, Agaricus and green tea inhibit P-gp in vitro. Special attention should be paid to mistletoe due to very low IC₅₀ and IC₂₅ values and to Natto K2 due to a low IC₅₀ value and a low IC₅₀/IC₂₅ ratio.     

Cytotoxic constituents of<Emphasis Type="Italic">diadema setosum</Emphasis>

5,8-Epidioxycholest-6-en-3-ol (1), cholesterol (2), glycerol 1-palmitate (3) and glycerol 1,3-dioleate-2-stearate (4) were isolated from the methanol extract of the sea urchinDiadema setosum, which was collected from the Halong sea, Vietnam. Chemical structures were established based on extensive 1D, 2D-NMR, FAB-MS, EI-MS spectroscopic data and GC-MS analysis. The NMR spectral data of compound 1 were reassigned by using HMQC and HMBC. Compound1 was found to have strong cytotoxic effect against various cancer cell lines, such as KB (IC₅₀, 2.0 μg/mL), FL (IC₅₀, 3.93 μg/mL), and Hep-2 (IC₅₀, 2.4 μg/mL) byin vitro assay.     

Assessment of specificity of eight chemical inhibitors using cDNA-expressed cytochromes P450

1. The selectivity of eight chemical inhibitors has been extensively evaluated with 10 cDNA-expressed human cytochrome P450 isoforms (CYP). The results indicate that sulphaphenazole, quinidine and α-naphthoflavone are selective inhibitors of CYP2C9 (IC₅₀ = 5 0.5-0.7 μM), CYP2D6 (0.3-0.4 μM) and CYP1A (0.05-5 μM) respectively on the basis of the IC₅₀, which are much lower than those of other P450 isoforms (> 10-fold). 2. Ketoconazole exhibited potent inhibition of both CYP3A4-catalysed metabolism of phenanthrene, testosterone, diazepam (IC₅₀ = 0.03-0.5 μM) and CYP1A1-catalysed deethylation of 7-ethoxycoumarin (0.33 μM). The selectivity of ketoconazole for other P450s was highly related to the concentration used. 3. Diethyldithiocarbamate, orphenadrine and furafylline were shown separately to be less selective inhibitors of CYP2E1, CYP2B6 and CYP1A isoforms by a broad range of IC₅₀ that overlap those observed with other P450 isoforms. 4. Furafylline, quinidine and α-naphthoflavone activated CYP3A4-catalysed phenanthrene metabolism by 1.7-, 2- and 15-fold respectively. 5. The selectivity of orphenadrine and ketoconazole was further examined by using inhibitory monoclonal antibodies (MAb). Inhibitory MAb specific for the individual P450 isoforms may be of greater value than chemical inhibitors.     

Validation of a total IC50 method which enables in vitro assessment of transporter inhibition under semi-physiological conditions

1.?Accurate predictions of clinical transporter-mediated drug–drug interactions (DDI) from in vitro data can be challenging when compounds have poor solubility and/or high nonspecific binding. Additionally, current DDI predictions for compounds with high plasma–protein binding assume that the unbound fraction in plasma is 0.01, if the experimental value is less than 0.01 or cannot be determined. This approach may result in an overestimation of DDI risk. To overcome these challenges, it may be beneficial to conduct inhibition studies under physiologically relevant conditions.

2.?Here, IC₅₀ values, determined in the presence of 4% bovine serum albumin approximating human plasma albumin concentrations, were successfully used to predict DDI for uptake transporters, OATP1B1/1B3, OCT1/2, OAT1/3 and MATE1/2K.

3.?The IC₅₀ values of reference inhibitors with 4% bovine serum albumin, considered total IC₅₀, were comparable to the predicted values based on nominal IC₅₀ values determined under protein-free conditions and unbound fraction in plasma. Calculation of R-total and C_max/IC_50,total values using total plasma exposure and total IC₅₀ values explained the clinical DDI or absence of it for these inhibitors.

4.?These results suggest that IC₅₀ determinations in the presence of 4% albumin can be used, in the context of clinical total exposure, to predict DDI involving uptake transporters.     

Isoflavanones from Desmodium oxyphyllum and their cytotoxicity

Two new isoflavanones, (3R)-7-hydroxy-4′-methoxy-5-methoxycarbonyl-isoflavanone (1) and (3R)-8-hydroxy-4′-methoxy-7-methoxycarbonyl-isoflavanone (2), together with seven known isoflavanones (3–9) were isolated from Desmodium oxyphyllum of the Leguminosae family. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compound 1 showed good cytotoxicity against NB4 and SHSY5Y cell lines with IC₅₀ values of 3.1 and 2.5 μM; compound 2 exhibited cytotoxicity against PC3 cell lines with a IC₅₀ value of 3.6 μM; compound 4 showed cytotoxicity against A549 and SHSY5Y cell lines with IC₅₀ values of 3.6 and 2.8 μM; and compound 5 displayed cytotoxicity against NB4, SHSY5Y, and MCF7 cell lines with IC₅₀ values of 2.6, 3.8, and 2.8 μM, respectively. Other compounds also showed moderate cytotoxicity for some tested cell lines with IC₅₀ values between 5.4 and 8.8 μM.     

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1.?Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.

2.?UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.

3.?UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC₅₀ 0.391?±?0.013?μM; K_i 0.185?±?0.015?μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 2.651?±?0.201?μM; K_i 1.334?±?0.146?μM).

4.?OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC₅₀ 0.336?±?0.013?μM; K_i 0.176?±?0.007?μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 5.468?±?0.697?μM; K_i 6.298?±?0.891?μM).

5.?Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.     

Dillapiole as Antileishmanial Agent: Discovery,Cytotoxic Activity and Preliminary SAR Studies of Dillapiole Analogues

In this paper, the isolation of dillapiole ( 1 ) from Piper aduncum was reported as well as the semi‐synthesis of two phenylpropanoid derivatives [di‐hydrodillapiole ( 2 ), isodillapiole ( 3 )], via reduction and isomerization reactions. Also, the compounds' molecular properties (structural, electronic, hydrophobic, and steric) were calculated and investigated to establish some preliminary structure–activity relationships (SAR). Compounds were evaluated for in vitro antileishmanial activity and cytotoxic effects on fibroblast cells. Compound 1 presented inhibitory activity against Leishmania amazonensis (IC₅₀ = 69.3 µM) and Leishmania brasiliensis (IC₅₀ = 59.4 µM) and induced cytotoxic effects on fibroblast cells mainly in high concentrations. Compounds 2 (IC₅₀ = 99.9 µM for L. amazonensis and IC₅₀ = 90.5 µM for L. braziliensis) and 3 (IC₅₀ = 122.9 µM for L. amazonensis and IC₅₀ = 109.8 µM for L. brasiliensis) were less active than dillapiole ( 1 ). Regarding the molecular properties, the conformational arrangement of the side chain, electronic features, and the hydrophilic/hydrophobic balance seem to be relevant for explaining the antileishmanial activity of dillapiole and its analogues.     

Cinnamic acids derived compounds with antileishmanial activity target Leishmania amazonensis arginase

This study describes the activity of five natural hydroxycinnamic acids and derived compound: caffeic ( 1 ), rosmarinic ( 2 ), chlorogenic ( 3 ), and cryptochlorogenic ( 4 ), acids and isoverbascoside ( 5 ). All compounds inhibited Leishmania amazonensis arginase with IC₅₀ —in range of 1.5—11 μM. Compounds 2 and 5 also showed activity against promastigotes of L. amazonensis with IC₅₀ = 61 (28–133) μM and IC₅₀ = 14 (9–24) μM, respectively. Further computational studies applying molecular docking simulations were performed on the competitive inhibitors to gain insight into the molecular basis for arginase inhibition and could be exploited to the development of new antileishmanials drug targeting parasite arginase.     

Inhibition of DNA topoisomerases I and II and cytotoxicity of compounds from <Emphasis Type="Italic">Ulmus davidiana</Emphasis> var. <Emphasis Type="Italic">japonica</Emphasis>

Twenty five compounds including ten triterpenes (1–3, 5–11), six flavonoids (12–15, 24, 25), five lignans (17, 18, 21–23), two butenyl clohexnone glycosides (19–20), one fructofuranoside (16) and one fatty acid (4) were isolated from the roots of Ulmus davidiana var. japonica. The structures of those compounds were identified by comparing their physicochemical and spectral data with those of published in literatures. All the compounds were evaluated for DNA topoisomerase inhibitory activities and cytotoxicities. Among the purified compounds, 4 and 19 showed more potent inhibitory acitivities (IC₅₀: 39 and 19 μM, respectively) than camptothecin, as the positive control (IC₅₀: 46 μM) against topoisomerase I. Compounds, 4, 10, 12, 19, 24 and 25 showed strong inhibitory activities toward DNA topoisomerase II (IC₅₀: 0.1, 0.52, 0.47, 0.42, 0.17 μM and 17 nM, respectively), which were more potent than that of etoposide as positive control (IC₅₀: 20 μM). In A549 cell line, 5 and 6 showed cytotoxicities (IC₅₀: 4 μM and 3 μM, respectively, with IC₅₀ of camptothecin as positive control: 10.3 μM). In the HepG2 cell line, 3, 5 and 7 showed cytotoxicity (IC₅₀: 4, 3 and 4 μM, respectively, with IC₅₀ of camptothecin: 0.3 μM). Compounds 6, 12 and 23 showed cytotoxicities in the HT-29 cell line (IC₅₀: 19, 19 and 15 μM, respectively, with IC₅₀ of camptothecin: 2 μM).     

Analogues of Carbacholine: Synthesis and Relationship between Structure and Affinity for Muscarinic and Nicotinic Acetylcholine Receptors

A series of acyclic and heterocyclic analogues of carbacholine ( 1 ) was synthesized using N-methylcarbacholine (MCC, 2 ), N,N-dimethylcarbacholine (DMCC, 3 ), and the corresponding tertiary amine ( 4 ) as leads. Whereas nicotinic acetylcholine receptor affinity was determined using [³H]nicotine as the radioactive ligand, [³H]oxotremorine-M ([³H]Oxo-M) and [³H]quinuclidinyl benzilate ([³H]QNB), in some cases supplemented with [³H]pirenzepine ([³H]PZ), were used as radioligands for muscarinic acetylcholine receptors on rat brain membranes. On the basis of receptor binding data, nicotinic/muscarinic (N/M) selectivity factors were determined, and muscarinic receptor efficacy (M agonist index) and M₁ selectivity (M₂/M₁ index) estimated. In most cases, quaternized analogues showed higher affinity than the corresponding tertiary amines for muscarinic and, in particular, nicotinic receptor sites. Among the new compounds, N,N-diethylcarbacholine ( 9e ) (IC₅₀ = 0.046 μM), (S)-1-methyl-2-(N,N-diethyl-aminocarbonyloxymethyl)pyrrolidine ( 17k ) (IC₅₀ = 0.068 μM), and the corresponding quaternized analogue, 18k (IC₅₀ = 0.018 μM) showed the highest nicotinic receptor affinity. The tertiary amine, 17k showed much higher nicotinic receptor affinity than the acyclic analogue, 4 (IC₅₀ = 5.7 μM), and the N/M selectivity factor determined for 17k (150) is an order of magnitude lower than that of nicotine (1400). The N/M selectivity factors for MCC ( 2 ) and DMCC ( 3 ), previously reported to be highly selective nicotinic receptor ligands, were shown to be 6.5 and 60, respectively, the latter value being comparable with that of 18k (89).     

Synthesis and Calcium Antagonistic Activity of 8-[N-[2-(3,4-Dimethoxyphenyl)ethyl]-β-alanyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4]thiazepine Fumarate

KT-362 is an antiarrhythmic and antihypertensive agent with vasodilating activity. Since it carries a homoveratryl group in the side chain, an obvious relation exists to the verapamil-type calcium antagonists. Replacement of the fused aromatic moiety in KT-362 with thiophene provided 8-[N-[2-(3,4-dimethoxyphenyl)ethyl]-β-analyl]-5,6,7,8-tetrahydrothieno[3,2-b][1,4] thiazepine ( 1 ). Compound 1 shows a negative chronotropic activity in spontaneously beating right atria (IC₅₀ = 23 μM, n = 7), and a negative inotropic effect in papillary muscles (IC₅₀ = 2.7 μM, n = 7) and left atria (IC₅₀ = 4 μM, n = 6) of the guinea-pig heart. The decrease of contractility in papillary muscles could be antagonized by increasing the extracellular calcium concentration. Compound 1 was found to affect high (IC₅₀: 70 ± 5 μM) and low (IC₅₀: 129 ± 34 μM) voltage-activated calcium channel currents as well as voltage-activated sodium channel currents (IC₅₀: 80 ± 13 μM) in chick dorsal root ganglion neurons. In addition, nicotine-induced currents were potently inhibited (IC₅₀: 6 ± 0.7 μM) in bovine chromaffin cells.     

Enantiospecific effects of chiral drugs on cytochrome P450 inhibition in vitro

1.?The aim of this work was to examine the differences in the inhibitory potency of individual enantiomers and racemic mixtures of selected chiral drugs on human liver microsomal cytochromes P450.

2.?The interaction of enantiomeric forms of six drugs (tamsulosin, tolterodine, citalopram, modafinil, zopiclone, ketoconazole) with nine cytochromes P450 (CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1A2) was examined. HPLC methods were used to estimate the extent of the inhibition of specific activity in vitro.

3.?Tamsulosin (TAM) and tolterodine (TOL) inhibited CYP3A4 activity with an enantiospecific pattern. The inhibition of CYP3A4 activity differed for R-TAM (K_i 2.88?±?0.12?µM) and S-TAM (K_i 14.22?±?0.53?µM) as well as for S-TOL (K_i 1.71?±?0.03?µM) and R-TOL (K_i 4.78?±?0.17?µM). Also, the inhibition of CYP2C19 by ketoconazole (KET) cis-enantiomers exhibited enantioselective behavior: the (+)-KET (IC₅₀ 23.64?±?6.25?µM) was more potent than (?)-KET (IC₅₀ 66.12?±?12.6?µM). The inhibition of CYP2C19 by modafinil (MOD) enantiomers (R-MOD IC₅₀?=?51.79?±?8.58?µM, S-MOD IC₅₀?=?48.62?±?9.74?µM) and the inhibition of CYP2D6 by citalopram (CIT) enantiomers (R-CIT IC₅₀?=?68.17?±?5.70?µM, S-CIT IC₅₀?=?62.63?±?7.89?µM) was not enantiospecific.

4.?Although enantiospecific interactions were found (TAM, TOL, KET), they are probably not clinically relevant as the plasma levels are generally lower than the drug concentration needed for prominent inhibition (at least 50% of CYP activity).     

In vitro assessment of the roles of drug transporters in the disposition and drug–drug interaction potential of olaparib

1.?In vitro assessments were conducted to examine interactions between olaparib (a potent oral inhibitor of poly[ADP-ribose] polymerase) and drug transporters.

2.?Olaparib showed inhibition of the hepatic drug uptake transporters OATP1B1 (IC₅₀ values of 20.3?μM and 27.1?μM) and OCT1 (IC₅₀ 37.9?μM), but limited inhibition of OATP1B3 (25% at 100?μM); inhibition of the renal uptake transporters OCT2 (IC₅₀ 19.9?μM) and OAT3 (IC₅₀ 18.4?μM), but limited inhibition of OAT1 (13.5% at 100?μM); inhibition of the renal efflux transporters MATE1 and MATE2K (IC₅₀s 5.50?μM and 47.1?μM, respectively); inhibition of the efflux transporter MDR1 (IC₅₀ 76.0?μM), but limited inhibition of BCRP (47% at 100?μM) and no inhibition of MRP2. At clinically relevant exposures, olaparib has the potential to cause pharmacokinetic interactions via inhibition of OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K in the liver and kidney, as well as MDR1 in the liver and GI tract. Olaparib was found to be a substrate of MDR1 but not of several other transporters.

3.?Our assessments indicate that olaparib is a substrate of MDR1 and may cause clinically meaningful inhibition of MDR1, OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K.