Influence of leucine on intestinal baclofen absorption as a model compound of neutral α-aminoacids

The inhibitory effect of the essential α-aminoacid L-leucine on the intestinal absorption of the antispastic drug baclofen was examined by means of an in situ rat gut perfusion technique. When 0.5 mM baclofen solutions were perfused in the presence of increasing concentrations of the aminoacid (5–100 mM), the apparent absorption rate constant of the drug decreased as the initial leucine concentration increased. Higher leucine concentrations, however, did not completely abolish the absorption of the drug (at 100 mM of leucine, only 76% inhibition was observed). The interaction can be mathematically described as a complete competitive inhibition with a second component, K = 0.35 (±0.08)h^?1, K_i = 0.25 (±0.09)mM, AIC= ?97.02. In the light of some of the absorption features of the drug, however, the residual absorption of baclofen in the presence of high leucine concentrations should be attributed to another transport system not used by leucine. Apparent parameters characterizing absorption of leucine in the presence of baclofen (0.5mM) were V_m=61.02 (±5.46)mM h^?1; K_m=8.04 (±0.89)mM, and AIC=62.25. The results indicate that baclofen and leucine share some carriers in the intestinal absorption processes. Since leucine is an essential dietary aminoacid, and therefore a normal food component, this finding could be relevant in preventing interactions that would lead to a reduced oral bioavailability during baclofen therapy.     

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.     

Systemic absorption of ocular pilocarpine is modified by polymer matrices

Systemic absorption of ocularly applied pilocarpine (1.2 mg) was studied after administration in aqueous solution, in hydroxypropylcellulose (HPC) matrix, and in a matrix of n-butyl half-ester of poly(methyl vinyl ether/maleic anhydride) (PVM/MA). In vitro release of pilocarpine from the HPC-matrix deviated slightly and positively from the diffusional square root of time dependence. The rate of drug release was independent of the phosphate buffer concentration of the dissolution medium with an initial pH of 7.4; the rate of release was 10.91 ± 0.59% min^?0.5 in 1.3 mM buffer and 9.91 ±0.37% min^?0.5 in 66.7 mM buffer. A matrix of n-butyl half-ester of PVM/MA released pilocarpine according to zero-order kinetics. The rate of drug release was 0.22 ± 0.02% min^?1 in 1.3 mM phosphate buffer and 0.95 ± 0.06% min^?1 in 66.7 mM phosphate buffer. From the 2% aqueous solution, pilocarpine was absorbed efficiently into the plasma (t_max = 3.6 ± 0.9 min, c_max = 0.384 ± 0.024 μg/ml). Pharmacokinetic analysis of data for drug absorption revealed that the conjunctiva of the eye was the most important site for systemic absorption of pilocarpine. Both the HPC matrix (t_max = 35.0 ± 7.9 min, c_max = 0.256 ± 0.022 μg/ml) and the matrix of n-butyl half-ester of PVM/MA (t_max = 204 ± 17.5 min, c_max = 0.112 ± 0.014 μg/ml) delayed and decreased the peak concentrations of pilocarpine in general circulation. (AUC_{0–6 h}/AUC_{0–6 h,i,v.}) values were 0.72 ± 0.08, 0.67 ± 0.16, and 0.41 ± 0.05 for the aqueous solution, HPC matrix, and n-butyl half-ester of PVM/MA matrix, respectively. During the in vivo study, HPC matrices dissolved in 7–12 min in the tear fluid. n-Butyl half-ester of PVM/MA neither dissolved totally nor released all the drug from the matrix in the tear fluid during 8 h. Besides improving ocular drug absorption, as shown in earlier studies, the pilocarpine concentrations in systemic circulation can be decreased by administering the drug in polymer matrices.     

Regulation of central noradrenergic activity by 5-HT(3) receptors located in the locus coeruleus of the rat

A functional interaction between serotonergic and noradrenergic systems has been shown in the locus coeruleus (LC). Noradrenaline (NA) levels in the prefrontal cortex (PFC) are dependent on the firing rate of LC neurons, which is controlled by α₂ adrenoceptors (α2ADR). The aim of the present study was to investigate the role of 5-HT₃ receptors (5HT3R) in the modulation of central noradrenergic activity. We measured extracellular NA concentrations in the LC and PFC by dual-probe microdialysis in awake rats and the firing rate of LC neurons by electrophysiological techniques in vitro. Administration of the 5HT3R agonists SR57227 (1–100 μM) and m-chlorophenylbiguanide (mCPBG, 1–100 μM) into the LC increased NA in this nucleus (E_max = 675 ± 121% and E_max = 5575 ± 1371%, respectively) and decreased NA in the PFC (E_max = ?49 ± 6% and E_max = ?25 ± 11%, respectively). Administration of the 5HT3R antagonist Y25130 (50 μM) into LC attenuated SR57227 effect in the LC (E_max = 323 ± 28%) and PFC (E_max = ?37 ± 7%). The α2ADR antagonist RS79948 (1 μM) blocked the SR57227 effect in the PFC but it did not change the effect in the LC (E_max = 677 ± 202%). In electrophysiological assays, both mCPBG (1–10 μM) and SR57227 (1–10 μM) reduced the firing rate of about 50% of tested LC neurons (maximal effect = ?37 ± 2% and ?31 ± 4%, respectively); this effect was partially blocked by Y25130 (50 μM). Administration of RS79948 (1 μM) reversed the inhibition induced by mCPBG. Competition radioligand assays against [³H]UK14304 and [³H]RX821002 (α2ADR selective drugs) in the rat brain cortex showed a very weak affinity of SR57227 for α2ADR, whereas the affinity of mCPBG for α2ADR was 17-fold higher than that of SR57227 for α2ADR. The present results suggest that 5HT3R stimulate NA release in the LC, which promotes simultaneously a decrease in the firing rate of LC neurons through α2ADR and then a decrease of NA release in terminal areas such as the PFC.     

Assessing the neurotoxic effects of palytoxin and ouabain,both Na+/K+-ATPase inhibitors,on the myelinated sciatic nerve fibres of the mouse: An ex vivo electrophysiological study

Palytoxin (PlTX) is a marine toxin originally isolated from the zoantharians of the genus Palythoa. It is considered to be one of the most lethal marine toxins that block the Na⁺/K⁺-ATPase. This study was designed to investigate the acute effects of PlTX and ouabain, also an Na⁺/K⁺-ATPase blocker, on the mammalian peripheral nervous system using an ex vivo electrophysiological preparation: the isolated mouse sciatic nerve. Amplitude of the evoked nerve compound action potential (nCAP) was used to measure the proper functioning of the sciatic nerve fibres. The half-vitality time of the nerve fibres (the time required to inhibit the nCAP to 50% of its initial value: IT₅₀) incubated in normal saline was 24.5 ± 0.40 h (n = 5). Nerves incubated continuously in 50.0, 10.0, 1.0, 0.5, 0.250 and 0.125 nM of PlTX had an IT₅₀ of 0.06 ± 0.00, 0.51 ± 0.00, 2.1 ± 0.10, 8.9 ± 0.30, 15.1 ± 0.30 h, and 19.5 ± 0.20 h, respectively (n = 5, 3, 4, 4, 10). PlTX was extremely toxic to the sciatic nerve fibres, with a minimum effective concentration (mEC) of 0.125 nM (n = 5) and inhibitory concentration to 50% (IC₅₀) of 0.32 ± 0.08 nM (incubation time 24 h). Ouabain was far less toxic, with a mEC of 250.0 μM (n = 5) and IC₅₀ of 370.0 ± 18.00 μM (incubation 24.5 h). Finally, when the two compounds were combined – e.g. pre-incubation of the nerve fibre in 250.0 μM ouabain for 1 h and then exposure to 1.0 nM PlTX – ouabain offered minor a neuroprotection of 9.1–17.6% against PlTX-induced neurotoxicity. Higher concentrations of ouabain (500.0 μM) offered no protection. The mouse sciatic nerve preparation is a simple and low-cost bioassay that can be used to assess and quantify the neurotoxic effects of standard PlTX or PlTX-like compounds, since it appears to have the same sensitivity as the haemolysis of erythrocytes assay – the standard ex vivo test for PlTX toxicity.     

Synthesis,in vitro and in vivo giardicidal activity,and pharmacokinetic profile of a new nitazoxanide analog

4-Nitro-N-(5-nitro-1,3-thiazol-2-yl)benzamide (1), a new nitazoxanide analog, was synthesized; its chemical structure was confirmed by ¹H, ¹³C NMR, and HRMS. In this study, we evaluated the in vitro activity of compound 1 against Giardia lamblia trophozoites, as well as its in vivo giardicidal activity in a CD-1 mouse model. A pharmacokinetic study in Wistar rats evaluated compound 1 disposition after intravenous (IV) and oral administration of 3.3 and 150 mg/kg, respectively. Compound 1 inhibited G. lamblia growth in vitro with a median inhibitory concentration (IC₅₀) of 0.78 ± 0.01 μM, and thus was more effective than metronidazole (IC₅₀ = 5.36 ± 0.23 μM), the drug of choice against this parasite. An evaluation of cytotoxicity using VERO cells showed that compound 1 was less cytotoxic than metronidazole (CC₅₀ = 685.98 vs. CC₅₀ = 68 μM, respectively), with a favorable selectivity index (SI = 879). In vivo, we found that 97.2 % of parasite load was eliminated after intragastric administration of compound 1 (75 mg/kg). An analysis of the oral pharmacokinetic profile revealed a double peak of maximum concentration. Pharmacokinetic parameters indicated an absolute bioavailability approaching 33 %, a prolonged half-life, a large distribution volume, and slow clearance. This pharmacokinetic behavior of compound 1 makes it a promising candidate for the treatment of infections caused by both intestinal and systemic parasites.     

Tight binding inhibitors—IX: Kinetic parameters of dihydrofolate reductase inhibited by methotrexate,an example of equilibrium study

Equilibrium studies in the presence of methotrexate (MTX), based on the new theories of tight-binding inhibitors and on classical initial velocity analysis, indicated that the reaction mechanism of dihydrofolate reductase Lactobacillus casei MTX/R is consistent with a rapid equilibrium random bi-bi and that MTX inhibits the enzyme competitively with respect to dihydrofolate but noncompetitively with respect to NADPH. The kinetic parameters determined at pH 7.3 and 23° were: K_m for DHF, 9.8 ± 1.3 μM; K_m for NADPH, 6.0 ± 1.2 μM; K_d for E·DHF, 5.7 ± 0.7 μM; K_d for E·NADPH, 0.037 ± 0.028 μM; K_d for E·MTX, 1.20 ± 0.15 nM; K_d for E·NADPH·MTX → E·NADPH + MTX, 0.19 ± 0.04 nM; and K_d for E·NADPH·MTX → E·MTX + NADPH, 7.6 ± 5.9 nM; the molar equivalency factor was 3.33 ± 0.44 nM per unit/liter of the enzyme, and the catalytic number was 300 min^?.     

Studies on the interaction of five triazole fungicides with human renal transporters in cells

The widespread use of triazole fungicides in agricultural production poses a potential risk to human health. This study investigates the interaction of five triazole fungicides, i.e., tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole with human renal transporters, including OAT1, OAT3, OCT2, OCTN1, OCTN2, MATE1, MATE2-K, MRP2, MDR1, and BCRP, using transgenic cell models. For uptake transporters, triticonazole was the substrate of OAT1 and OAT3 and the inhibitor of OCT2. Tebuconazole and penconazole inhibited OCTN2 (100 μM), while tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole inhibited MATE1 (100 μM). Tebuconazole and hexaconazole inhibited MATE2-K (100 μM). All five triazole fungicides were not substrates or strong inhibitors of MRP2, MDR1, and BCRP efflux transporters. Penconazole inhibited OCT2 with IC₅₀ = 1.12 μM. Penconazole and uniconazole inhibited MATE1 with IC₅₀ = 0.94 μM and 0.87 μM. Tebuconazole and hexaconazole inhibited MATE2-K with IC₅₀ = 0.96 μM and 1.04 μM, indicating that triazole fungicides may inhibit renal drug transporter activity at low concentrations. Triticonazole was transported by OAT1 and OAT3, and the K_m values of triticonazole were 5.81 ± 1.75 and 47.35 ± 14.27, respectively. Tebuconazole and uniconazole were transported by OAT3, and the K_m values of tebuconazole and uniconazole were 30.28 ± 7.18 and 87.61 ± 31.70, respectively, which may induce nephrotoxicity.     

Effects of ropinirole on action potential characteristics and the underlying ion currents in canine ventricular myocytes

In spite of its widespread clinical application, there is little information on the cellular cardiac effects of the dopamine receptor agonist ropinirole. In the present study, therefore, the concentration-dependent effects of ropinirole on action potential morphology and the underlying ion currents were studied in enzymatically dispersed canine ventricular cardiomyocytes using standard microelectrode, conventional whole-cell patch clamp, and action potential voltage clamp techniques. At concentrations?≥?1 μM, ropinirole increased action potential duration (APD₉₀) and suppressed the rapid delayed rectifier K⁺ current (I _Kr) with an IC₅₀ value of 2.7?±?0.25 μM and Hill coefficient of 0.92?±?0.09. The block increased with increasing depolarizations to more positive voltages, but paradoxically, the activation of I _Kr was accelerated by 3 μM ropinirole (time constant decreased from 34?±?4 to 14?±?1 ms). No significant changes in the fast and slow deactivation time constants were observed with ropinirole. At higher concentrations, ropinirole decreased the amplitude of early repolarization (at concentrations?≥?10 μM), reduced the maximum rate of depolarization and caused depression of the plateau (at concentrations?≥?30 μM), and shortened APD measured at 50% repolarization (at 300 μM) indicating a concentration-dependent inhibition of I _to, I _Na, and I _Ca. Suppression of I _Kr, I _to, and I _Ca has been confirmed under conventional patch clamp and action potential voltage clamp conditions. I _Ks and I _K1 were not influenced significantly by ropinirole at concentrations less than 300 μM. All these effects of ropinirole were fully reversible upon washout. The results indicate that ropinirole treatment may carry proarrhythmic risk for patients with inherited or acquired long QT syndrome due to inhibition of I _Kr—especially in cases of accidental overdose or intoxication.     

Impaired Clearance of Methotrexate in Organic Anion Transporter 3 (Slc22a8) Knockout Mice: A Gender Specific Impact of Reduced Folates

Purpose

To elucidate the role of the renal basolateral transporter, Oat3, in the disposition of methotrexate.

Materials and Methods

Chinese hamster ovary cells expressing mouse Oat3 were used to determine kinetics and specificity of inhibition of methotrexate transport. Methotrexate clearance was then examined in vivo in wildtype and Oat3 knockout mice.

Results

NSAIDs, ß-lactams, and uremic toxins inhibited mOat3-mediated methotrexate uptake by 70–100%, while folate, leucovorin, and 5-methyltetrahydrofolate inhibited transport by 25–50%. A K _m of 60.6?±?9.3 μM for methotrexate transport was determined. Oat3 knockout mice exhibited reduced methotrexate-to-inulin clearance ratios versus wildtype. Male wildtype mice, but not knockouts or females, demonstrated significantly accelerated methotrexate clearance in response to reduced folates. Reduced folates also markedly inhibited hepatic methotrexate accumulation in males, but not females, and the response was independent of Oat3 function.

Conclusions

Oat3 contributes to methotrexate clearance, but represents only one component responsible for methotrexate’s elimination. Therefore, in patients, dysfunctional hOAT3 polymorphisms or drug competition for hOAT3 transport may severely impact methotrexate elimination only when redundant means of methotrexate removal are also compromised. Furthermore, the present findings suggest that reduced-folate administration only influences methotrexate disposition in males, with the renal reduced-folate response influenced by OAT3 function.     

Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (K_m and V_max) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.     

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 cells

Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 μM [³H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (K_i = 2.5 μM), 4-biphenylacetic acid (K_i = 5.3 μM), and flurbiprofen (K_i = 5.2 μM), whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (K_i = 0.08 μM), ethacrynic acid (K_i = 0.52 μM), and 1-chloro-2,4-dinitrobenzene (K_i = 0.77 μM). Bilirubin ditaurate was a potent inhibitor of both transport components (K_i = 1.5 and 0.17 μM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 μM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave K_m values of 170 and 250 μM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.     

Amelioration of Tumor Targeting and In Vivo Biodistribution of 99mTc-Methotrexate-Gold Nanoparticles (99mTc-Mex-AuNPs)

Gold nanoparticles (AuNPs) represent very attractive and promising drug delivery carriers due to their unique dimensions, adjustable surface functions, and controllable drug release. Therefore, AuNPs are used to overcome the limitations of conventional chemotherapy, for example methotrexate (Mex), one of the first-generation chemotherapy drugs for cancer treatment, whose usefulness has been restricted due to drug resistance and dose-dependent side effects. In the present study, the AuNPs drug delivery system was synthesized and loaded with technetium-99 m radiolabeled Methotrexate (^99mTc-Mex) to produce new potential nanoradiopharmaceutical for tumor targeting and further imaging. The Methotrexate loaded gold nanoparticles (Mex-AuNPs) successfully prepared in small spherical particle size (20.3 nm), polydispersity index PDI (< 0.5) and a zeta potential (−17.6 mV) with loading efficiency% (93 ± 1.2%) of methotrexate at 30 min as an optimum stirring time and showed strong absorption peak for Mex-AuNPs at λ_max, 525 nm. The in vitro release profile of Mex-AuNPs showed high release percent of methotrexate at pH 5; the Q_0.5 h and Q_8h were 21.2 ± 1.5% and 92.9 ± 3.4%, respectively. The in vitro cytotoxicity was investigated at different concentrations (0.024–50 μl/100 μl) of Mex-AuNPs (1 mg/ml) against MCF-7 (Michigan Cancer Foundation-7) breast cancer cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay technique. Mex-AuNPs showed higher anticancer activity with low inhibitory concentration (IC₅₀ = 0.098 μl/100 μl) that was three times lower than the inhibitory concentration (IC₅₀) of methotrexate (IC₅₀ = 0.3 μl/100 μl). ^99mTc-Mex complex prepared by direct reduction method at maximum radiochemical yield (RCY)% ̴ 98.3 ± 1.09 % was loaded in AuNPs to form ^99mTc-Mex-AuNPs with loading efficiency% (93 ± 1.2 %) at 30 min of stirring time. ^99mTc-Mex-AuNPs showed convenient in vitro stability in mice serum up to 24 h with RCY% > 90 %. The preclinical biodistribution studies of ^99mTc-Mex-AuNPs were performed in 3 experimental groups A (intravenous (I.V.) injected normal mice), B and C (I.V. and intratumor (I.T.) injected tumor bearing mice, respectively). The ^99mTc-Mex-AuNPs achieved highest tumor uptake (93 ± 0.39 %ID/g) and highest Target/NonTarget (T/NT) ratio (58.1 ± 0.91) with high Tumor/Blood (T/B) ratio (25.8 ± 0.11) at 10 min post I.T. injection and retained high tumor uptake (79 ± 0.65 %ID/g) up to 60 min post I.T. injection before escaping into blood stream. Consequently, ^99mTc-Mex-AuNPs can be considered as new potential nanoradiopharmaceutical in tumor diagnosis.     

MDR1 and OAT1/OAT3 Mediate the Drug-Drug Interaction between Puerarin and Methotrexate

Purpose

To conduct in vivo and in vitro experiments to investigate puerarin (PUR), an isoflavone C-glyoside, and elucidate its ability to alter methotrexate (MTX) transport and pharmacokinetics.

Methods

In vivo absorption studies, in vitro everted intestinal sac preparation, kidney slices in rats and bi-directional transport assay with mock-/MDCK-MDR1 cells, uptake studies in HEK293-OAT1/3 cells were employed to evaluate the interaction.

Results

In vivo and in vitro MTX absorption in rats were enhanced in combination with PUR. PUR inhibited digoxin efflux transport in MDCK-MDR1 monolayers with an IC₅₀ value of 1.6?±?0.3 μM, suggesting that the first target of drug interaction was MDR1 in the intestine during the absorption process. MTX renal clearance decreased significantly after simultaneous intravenous administration. MTX uptake in rat kidney slices and HEK293-OAT1/3 cells were markedly inhibited by PUR, suggesting that the second target of drug interaction was OATs located in the kidney. Moreover, concomitant administration of PUR reduced renal MTX accumulation and plasma levels of creatinine and BUN.

Conclusions

Co-administration of PUR enhanced MTX exposure by inhibition of intestinal MDR1 and renal OAT1/3. Although the renal damage of MTX was improved by PUR, the high level exposure of MTX should be cautious in the clinical usage.     

Synthesis and screening of 2-(2-(4-substituted piperazine-1-yl)-5-phenylthiazol-4-yl)-3-aryl quinazolinone derivatives as anticancer agents

Synthesis of novel quinazolinone derivatives was performed from the reaction of N-benzoyl substituted piperazine-1-carbothioamide with 2-chloromethyl quinazolinone derivatives and screened for their in vitro cytotoxic activity by MTT assay. The cell lines used were NCI (human lung cancer cell), MCF 7 (Breast cancer cell), and HEK 293 (Normal epidermal kidney cell). Result of screening on cell line showed moderate to good anticancer activity for all the compounds. Compound 3d (IC₅₀ = 1.1 ± 0.03 μM) was found to be the most active compared to standard methotrexate (IC₅₀ = 2.20 ± 0.18 μM) and 5-florouracil (IC₅₀ = 2.30 ± 0.49 μM). Structure activity relationship of synthesized analogs suggested that the presence of NH linker with aryl moiety at the third position of quinazolinone ring was important for potent anticancer activity. Electron donating group on phenyl ring at the third position of quinazolinone ring gave better anticancer activity then unsubstituted phenyl and electron withdrawing group. Activity by substituted piperazine at 2nd position of thiazole linked with quinazolinone scaffold gave better activity in the order of H > CH₃ > CO–C₆H₅. Our findings may impart new direction to medicinal chemists and biochemists for further investigations of quinazolinone-thiazole containing anticancer agents.     

Urate and antioxidants inhibit Na+-adenosine transport in rat renal brush border membranes

The effects of urate and antioxidants were evaluated on sodium-dependent [³H]adenosine transport (Na⁺/ADO) in rat renal brush border membrane vesicles (BBMV). Na⁺/ADO was estimated for a range of adenosine concentrations of 1–10 μmol/l in BBMV preincubated with urate (0.5–50 μmol/l). Michaelis-Menten kinetics showed a significant increase in K_m values from 2.48±0.49 μmol/l in control to 20.58±4.56 μmol/l with 50 μmol/l urate; V_max (243±15 pmol/mg protein×min) was not modified. Menadione (10 μmol/l) significantly increased the Na⁺/ADO activity, from 17.57±5.50 in the control, to 27.70±7.60 pmol/mg prot.×min (a 1.60 times increase, p<0.05). This stimulation was prevented when BBMV were preincubated with either 1 μmol/l α-tocopherol (trolox) or urate. Similarly conjugated dienes and malonaldehyde were stimulated in a dose-dependent fashion by menadione and the effect was inhibited with 10 μmol/l trolox. The antioxidants probucol, captopril and allopurinol inhibited in a concentration-dependent manner the Na⁺/ADO (IC₅₀ were 79±8, 100±9 and 89±9 nM, respectively). This effect might be specific on K_m of the Na⁺/ADO, since 1 μmol/l trolox (IC₅₀=1000±20 nM), inhibited V_max but not K_m of the Na⁺/glucose transport. Our results suggest that the Na⁺/ADO in BBMV is modified by agents that affect the redox status of the membranes.     

Mechanism of action of relaxant effect of Agastache mexicana ssp.mexicana essential oil in guinea-pig trachea smooth muscle

Context: Agastache mexicana ssp. mexicana (Kunth) Lint &; Epling (Lamiaceae), popularly known as ‘toronjil morado’, is used in Mexican traditional medicine for the treatment of several diseases such as hypertension, anxiety and respiratory disorders.

Objective: This study investigates the relaxant action mechanism of A. mexicana ssp. mexicana essential oil (AMEO) in guinea-pig isolated trachea model.

Materials and method: AMEO was analyzed by GC/MS. The relaxant effect of AMEO (5–50?μg/mL) was tested in guinea-pig trachea pre-contracted with carbachol (3?×?10?^??⁶?M) or histamine (3?×?10?^??⁵?M) in the presence or absence of glibenclamide (10?^??⁵?M), propranolol (3?×?10?^??⁶?M) or 2′,5′-dideoxyadenosine (10?^??⁵?M). The antagonist effect of AMEO (10–300?μg/mL) against contractions elicited by carbachol (10?^??¹⁵–10?^??³?M), histamine (10?^??¹⁵–10?^??³?M) or calcium (10–300?μg/mL) was evaluated.

Results: Essential oil composition was estragole, d-limonene and linalyl anthranilate. AMEO relaxed the carbachol (EC_50?=_?18.25?±?1.03?μg/mL) and histamine (EC_50?=_?13.3?±?1.02?μg/mL)-induced contractions. The relaxant effect of AMEO was not modified by the presence of propranolol, glibenclamide or 2′,5′-dideoxyadenosine, suggesting that effect of AMEO is not related to β₂-adrenergic receptors, ATP-sensitive potassium channels or adenylate cyclase activation. AMEO was more potent to antagonize histamine (pA₂′?=??1.507?±?0.122) than carbachol (pA₂′?=??2.180?±?0.357). Also, AMEO antagonized the calcium chloride-induced contractions.

Conclusion: The results suggest that relaxant effect of AMEO might be due to blockade of calcium influx in guinea-pig trachea smooth muscle. It is possible that estragole and d-limonene could contribute majority in the relaxant effect of AMEO.     

Effect of Three Lipophilic Methotrexate Derivatives Upon Mediator Release by Lipopolysaccharide-stimulated Rat Peritoneal Macrophages

Abstract— The ability of methotrexate and three lipophilic derivatives (methotrexate-γ-dimyristoylphos-phatidylethanolamine (MγD), methotrexate-α-dimyristoylphosphatidylethanolamine (MαD) and metho-trexate-α-γ-di-dimyristoylphosphatidylethanolamine (MαγD) to modulate mediator release by lipopolysaccharide-stimulated rat peritoneal macrophages was investigated. At nontoxic concentrations, approximately 10 nmol/10⁵ cells, MαD and MγD produced 11·06±1·0 and 75·6 ± 5·2%, respectively, inhibition of tumour necrosis factor (TNF) release (mean ± s.e.m., n = 4). At this same dose MαγD resulted in 68·8 ± 2·1 % inhibition of TNF but cellular ATP levels were reduced by 80%. The inhibitory activity of all three derivatives was dose-dependent. Non-derivatized methotrexate at a concentration of 25 nmol/10⁵ cells had no inhibitory effect upon TNF release (14·7 ±0·8%, n = 3). Determination of prostaglandin E₂ (PGE₂) levels in the same samples demonstrated that all three conjugates were powerful inhibitors of prostaglandin release. At a quarter of the conjugate concentrations described above the monoamides MαD (31 nmol/10⁵ cells) and MγD (2·5 nmol/10⁵ cells) maintained their effects on PGE₂ production with 73±2·3 and 71±2·0% (n=4) inhibition, respectively. At this lower concentration, however, the diamide MαγD (3·1 nmol/10⁵ cells) was less effective in reducing the amount of PGE₂ released from the macrophages (29 ± 18%, n = 4). Maximal PGE₂ inhibition by each of the conjugates was attained at approximately 5 nmol/10⁵ cells. Unconjugated methotrexate (range of 2·5–20 nmol/10⁵ cells) did not inhibit the release of PGE₂ from lipopolysaccharide-stimulated macrophages.     

Functional differences in nucleoside and nucleobase transporters expressed on the rabbit corneal epithelial cell line (SIRC) and isolated rabbit cornea

The purpose of this study was to investigate the expression of nucleoside/nucleobase transporters on the Statens Seruminstitut rabbit corneal (SIRC) epithelial cell line and to evaluate SIRC as an in vitro screening tool for delineating the mechanism of corneal permeation of nucleoside analogs. SIRC cells (passages 410–425) were used to study uptake of [³H]thymidine, [³H]adenine, and [³H]ganciclovir. Transport of [³H]adenine and [³H]ganciclovir was studied across isolated rabbit cornea. Uptake and transport studies were performed for 2 minutes and 120 minutes, respectively, at 34°C. Thymidine uptake by SIRC displayed saturable kinetics (K _m=595.9±80.4μM, and V _max=289.5±17.2 pmol/min/mg protein). Uptake was inhibited by both purine and pyrimidine nucleosides but not by nucleobases. [³H]thymidine uptake was sodium and energy independent but was inhibited by nitrobenzylthioinosine at nanomolar concentrations. Adenine uptake by SIRC consisted of a saturable component (K _m=14.4±2.3μM, V _max=0.4±0.04 nmol/min/mg protein) and a nonsaturable component. Uptake of adenine was inhibited by purine nucleobases but not by the nucleosides or pyrimidine nucleobases and was independent of sodium, energy, and nitrobenzylthioinosine. [³H]ganciclovir uptake involved a carrier-mediated component and was inhibited by the purine nucleobases but not by the nucleosides or pyrimidine nucleobases. However, transport of [³H]adenine across the isolated rabbit cornea was not inhibited by unlabeled adenine. Further, corneal permeability of ganciclovir across a 100-fold concentration range remained constant, indicating that ganciclovir permeates the cornea primarily by passive diffusion. Nucleoside and nucleobase transporters on rabbit cornea and corneal epithelial cell line, SIRC, are functionally different, undermining the utility of the SIRC cell line as an in vitro screening tool for elucidating the corneal permeation mechanism of nucleoside analogs.     

Monoamine oxidase in human platelets: Kinetics and methodological aspects

The kinetic properties of human platelet monoamine oxidase (MAO) were examined in 20 apparently healthy controls. The mean value (±S.D.) of the maximum velocity (V) was found to be 5.36 ± 1.97 pmoles of product formed/10° platelets/min and the Michaelis-Menten constants were for phenethylamine (K^PEA_m) 14.6 ± 8.20 μM and for oxygen (K_m^O₂)254 ± 125 μM, when assayed in 0.1 M phosphate buffer, pH 7.4. The relation between the value of the corresponding apparent constants was studied. Inhibition of the enzyme activity was seen at 20 μM of PEA and 180 μM of oxygen. The enzyme kinetics were also studied at different pH. Two pK values were found, pK₁ = 6.65 and pK₂ = 6.95. The influence of homogenization on the MAO activity was compared with the activity in the undisrupted platelet. At PEA concentrations below 10 μM higher MAO activities were found in the intact cell. A 15 per cent loss of activity was detected in platelet samples after storing at ?20° for three and a half years.