Perspective on the relationship between GABAA receptor activity and the apparent potency of an inhibitor

Background : In electrophysiological experiments inhibition of a receptor-channel, such as the GABAA receptor, is measured by co-applying an agonist producing a predefined control response with an inhibitor to calculate the fraction of the control response remaining in the presence of the inhibitor. The properties of the inhibitor are determined by fitting the inhibition concentration-response relationship to the Hill equation to estimate the midpoint (IC50) of the inhibition curve. Objective: We sought to estimate here the sensitivity of the fitted IC50 to the level of activity of the control response. Methods: The inhibition concentration-response relationships were calculated for models with distinct mechanisms of inhibition. In Model I, the inhibitor acts allosterically to stabilize the resting state of the receptor. In Model II, the inhibitor competes with the agonist for a shared binding site. In Model III, the inhibitor stabilizes the desensitized state. Results: The simulations indicate that the fitted IC50 of the inhibition curve is sensitive to the degree of activity of the control response. In Models I and II, the IC50 of inhibition was increased as the probability of being in the active state (PA) of the control response increased. In Model III, the IC50 of inhibition was reduced at higher PA. Conclusions: We infer that the apparent potency of an inhibitor depends on the PA of the control response. While the calculations were carried out using the activation and inhibition properties that are representative of the GABAA receptor, the principles and conclusions apply to a wide variety of receptor-channels.

A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor

A one-step incubation ELISA kit based on monoclonal antibody against dibutyl phthalate (DBP) was developed. After optimizing concentrations of coating antigen, antibody and composition of the assay buffer, an inhibition curve was plotted. The IC50 is 29.6 ng·mL-1, and the detection limit for DBP is 3.6 ng·mL-1. Compared with other ELISA methods, this ELISA kit had a simpler sample preparation, costed less time for detection and could detect more types of sample. The recoveries of DBP in water, beverage and liquor samples were range from 78% to 110.4%, the range of coefficient of variations is 7.7-15.3%. The cross reactivity was very low (&1%) except that for butyl benzyl phthalate (3.9%) and the di-isobutyl phthalate (12.5%). The detection results in liquor showed good correlation with those from GC-MS. All data above indicated that this kit could be used as the fast and high-throughput screening of DBP in water, beverage and liquor.

A Rapid and Sensitive Chemiluminescent Immunoassay of Bisphenol a with NSP-SA-NHS-Labeled

A novel chemiluminescence immunoassay (CLIA) of Bisphenol A (BPA) with the acridinium ester of NSP-SA-NHS-labeled has been developed. In this study, BVA and NSP-SA-NHS had been coupled with BSA, the UV spectrum results indicated the conjugates which were successfully synthesized. Basing on these luminescence data the inhibition curve of BPA was established, then the linear arrang of the curve was between 0.4 ng/ml and 5 ng/ml, the 50% inhibitory concentration (IC50) was 2.3ng/ml, the lowest limit of detection was 0.1ng/ml, which showed it’s an efficient and highly sensitive method.

DNA repair suppression and effect of cisplatin in mesothelioma and lung cancer cell lines.

e13542 Background: Recently we characterized a gene profile with overexpressed DNA double strand repair systems in mesothelioma versus normal tissue, where homologous recombination (HR) and non-homologous end joining (NHEJ) were prominent systems overexpressed. They play a role in resistance to the DNA damaging agent cisplatin, the backbone of both mesothelioma and lung cancer treatment. We hypothesized that inhibition of these systems could increase the effect of cisplatin. Methods: Cisplatin and two natural compounds A and B inhibiting HR and NHEJ respectively were tested as single treatment and various combinations in mesothelioma (MSTO-211H), parental (A549) and cisplatin resistant lung cancer cells (A549/DDP) were the dose-response and time-response was determined. Sterile MTT (0.5mg/ml) was added at 37°C for 4h, then removed and DMSO added for 10 minutes. Spectrometric absorbance at 570nm was measured. The inhibition rate was calculated by the relative inhibition curve. The IC30 doses of cisplatin was combined with IC5 of the experimental compounds to reveal the effects of combining chemotherapy with non-toxic compounds. Results: The IC5 of the experimental compounds A and B were 2μM and 1mM in the MSTO-211H and A549 respectively, and similarly in the A549/DDP cells, 2μM and 0.5mM. The IC30 of cisplatin was 1,5 μg/ml in the MSTO and A549 and 20 μg/ml (13-fold) in the A549/DDP cells.The combination of IC5 of A and B without cisplatin showed a synergic effect of about 50%. IC30 of cisplatin from 24h to 72h without pretreatment showed 20% inhibition at 48h and interestingly the inhibition rate was reduced to 10% at 72h. Pretreatment with A and B for 24h, and then cisplatin for 48h had the same effect as cisplatin together with A and B for 48h, about 30% inhibition. Pretreatment with A and B for 24h, then addition of cisplatin plus A and B for 48h had the strongest effect of >50% inhibition rate. Conclusions: Our results indicate that suppression of HR and NHEJ in both mesothelioma and non-small cell lung cancer cell lines by non-toxic doses increased cisplatin efficacy. The acquired cisplatin resistant A549/DDP remained sensitive to the natural inhibitors. Molecular analyses on DNA damage and apoptosis will be presented at the meeting.

Pixantrone (PIX) Inhibition of Doxorubicinol (DOXOL) Formation in Human Myocardium: Implications for Cardiac Safety in Non-Hodgkin Lymphoma (NHL) Patients with Prior Anthracycline Treatment

Abstract 4966 Background Doxorubicin (DOX) and related drugs are incompletely cleared from the heart, persist for months to years (Stewart et al Anticancer Res 1993), and slowly transform to longer-lived and more toxic secondary alcohol metabolites (eg, DOXOL) that are associated with an increased lifetime risk of congestive heart failure (Minotti et al J Pharmacol Exp Ther 2010). Anthracycline-related cardiotoxicity can be precipitated by enhanced stress or exposure to drugs that increase DOXOL formation. PIX is a novel aza-anthracenedione that is under development in patients with NHL that relapsed or was refractory after DOX-containing combination therapy. In preclinical models, PIX produced substantially less cardiotoxicity than DOX or MITOX even when animals were pretreated with DOX. Compared to anthracyclines and the anthracenedione analogue, mitoxantrone (MITOX), PIX lacks a hydroquinone moiety that binds iron and facilitates cardiotoxic free radical reactions. To further establish that it is safe in patients with NHL and prior DOX exposure, PIX should also have no effect on or diminish DOXOL formation from the cardiac residues of first-line DOX. Aims To characterize the effects of PIX on DOXOL formation in a translational cardiac model of DOX administration followed by PIX administration. MITOX was used as the comparator. Methods Myocardial samples that were routinely discarded during aorto-coronary bypass grafting were dissected into strips and loaded with 10 μM DOX in plasma. After 30 min the strips were subjected to 2h multiple washouts to simulate post-treatment clearance. The strips were then incubated for 1.5h in fresh anthracycline-free plasma w/wo 1 μM PIX or MITOX. After the experiments, DOX(OL), PIX, and MITOX were extracted from the soluble fractions of the strips and assayed by HPLC. Pharmacometabolic interactions of 10 μM or 50 μM DOX with increasing concentrations of PIX or MITOX were also studied in NADPH-supplemented isolated soluble fractions. Results After sequential DOX loading/clearance and PIX or MITOX administration, PIX:DOX ratios were significantly higher than MITOX:DOX ratios in soluble fractions of human myocardial strips. This correlated with inhibition of DOXOL formation by both PIX and MITOX, but PIX decreased DOXOL levels in a significant manner when compared to MITOX (Table). In isolated soluble fractions, both PIX and MITOX, in a concentration-dependent manner, inhibited the metabolism of 10 μM DOX to DOXOL; however, PIX caused stronger inhibition (Figure). Increasing DOX to 50 μM abated inhibition by MITOX but not by PIX. Discussion PIX inhibited DOXOL formation in a translation model of human heart used to simulate sequential DOX and PIX administration. This finding was obtained under pharmacokinetically relevant conditions that probed DOX and PIX at their clinically documented plasma Cmax values of 10 μM or 1 μM, respectively. Studies with isolated soluble fractions suggest that PIX diminished DOXOL formation by noncompetitive inhibition of NADPH-dependent reductases. MITOX was less effective than PIX at diminishing DOXOL formation, which correlated with the lower MITOX:DOX ratios in treated human myocardial strips and with the competitive mode of action of MITOX. By inhibiting formation of toxic and long-lived DOXOL, PIX potentially is a cardiac tolerable therapeutic agent for patients with NHL that failed or relapsed after DOX treatment. Inhibition of DOXOL formation by PIX and MITOX in isolated soluble fractions of human myocardium incubated with NADPH (0.25 mM) and DOX for 4h. Control DOXOL was 0.15–0.19 nmoles/mg of protein at 10 μM DOX and 0.34–0.84 nmoles/mg of protein at 50 μM DOX. Each inhibition curve is the mean and SE of three experiments. Disclosures: Salvatorelli: Cell Therapeutics, Inc: Research Funding. Gonzalez Paz:Cell Therapeutics, Inc: Research Funding. Singer:Cell Therapeutics, Inc: Employment. Menna:Cell Therapeutics, Inc: Research Funding. Minotti:Cell Therapeutics, Inc: Research Funding.

Studies of the Toxicological Potential of Capsinoids, XIII: Inhibitory Effects of Capsaicin and Capsinoids on Cytochrome P450 3A4 in Human Liver Microsomes

This study evaluated potential effects of a number of capsinoids (ie, capsiate, dihydrocapsiate, nordihydrocapsiate) and a single capsaicinoid (ie, capsaicin) on liver microsomal cytochrome P450 3A4-mediated midazolam 1'-hydroxylase activity. Where possible, an inhibition curve was prepared; the concentration at which enzyme activity dropped to 50% was calculated. Capsaicin clearly inhibited cytochrome P450 3A4 activity, losing 50% of the activity at 21.5 μmol/L. No enzyme inhibition was observed in the presence of capsiate, dihydrocapsiate, or nordihydrocapsiate (<100 μmol/L). Preincubation increased the capsaicin inhibitory activity against cytochrome P450 3A4 in a time-dependent manner. Enzyme activity was slightly reduced by capsiate, dihydrocapsiate, and nordihydrocapsiate to the same level as that attained with tolbutamide, the negative control compound. Capsaicin was shown to inhibit cytochrome P450 3A4, probably through a mechanism-based inhibition. In contrast, capsiate, dihydrocapsiate, and nordihydrocapsiate did not inhibit cytochrome P450 3A4 activity and were unlikely to be mechanism-based inhibitors of CYP3A4.

Localization and functional characterization of Na+/H+ exchanger isoform NHE4 in rat thick ascending limbs

The Na+/H+ exchanger NHE4 was cloned from a rat stomach cDNA library and shown to be expressed predominantly in the stomach and less dramatically in the kidney. The role and precise localization of NHE4 in the kidney are still unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification of thick ascending limbs and collecting ducts, respectively. The results indicate that NHE4 is highly expressed in basolateral membranes of thick ascending limb and distal convoluted tubule, whereas collecting ducts from cortex to inner medulla and proximal tubules showed weaker basolateral NHE4 expression. Western blot analysis of NHE4 in membrane fractions prepared from the inner stripe of the outer medulla revealed the presence of a 95-kDa protein that was enriched in basolateral membrane vesicles isolated from medullary thick ascending limbs. The inhibition curve of H+-activated 22Na uptake by 5-( N-ethyl- N-isopropyl)amiloride (EIPA) was consistent with the presence, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 μM. Kinetic analyses showed that the extracellular Na+ dependence of NHE4 activity followed a simple hyperbolic relationship, with an apparent affinity constant of 12 mM. Intravesicular H+ activated NHE4 by a positive cooperative mechanism. NHE4 had an unusual low affinity for intravesicular H+ with a half-maximal activation value of p K6.21. We conclude that NHE4, like NHE1, is expressed on the basolateral membrane of multiple nephron segments. Nevertheless, these two proteins exhibited dramatically different affinities for intracellular H+, suggesting that they may play distinct physiological roles in the kidney.

Evidence for involvement of IgA1 hinge glycopeptide in the IgA1-IgA1 interaction in IgA nephropathy.

The study was performed to investigate the role of the IgA1 hinge region in the IgA1-IgA1 interaction, which was observed previously in IgA nephropathy. The competitive inhibition assays of the IgA1-IgA1 binding were performed using the following candidates for inhibitors: native IgA1 hinge glycopeptide (nHGP), IgA1, IgA2, and IgG. The IgA1-IgA1 binding was definitely inhibited by the nHGP and the IgA1 (maximum of percent inhibition: 66.1 and 60.5%, respectively). There was no obvious inhibition in the IgA2 and the IgG. The inhibition curves of the nHGP and the IgA1 were significantly different from that of the IgG (P < 0.01, respectively). Furthermore, to reveal the detailed binding sites in the interaction, the same inhibition assays were performed using the following substances composing the IgA1 hinge glycopeptide: galactose (Gal), N-acetyl-galactosamine (GalNAc), Gal beta 1-3GalNAc, sialic acid, tetrapeptide PTPS, and synthesized hinge proline-rich peptide PVPSTPPTPSPSTPPTPSPS (sHP). sHP, Gal beta 1-3GalNAc, Gal, and GalNAc inhibited the binding (69.3, 34.1, 14.9, 14.6%, respectively). No obvious inhibition was observed in sialic acid and tetrapeptide PTPS. The inhibition curve of sHP was significantly different from that of the PTPS (P < 0.05). Those of Gal beta 1-3GalNAc, Gal, and GalNAc were also significantly different from that of sialic acid (P < 0.05, respectively). These results suggested that the IgA1-IgA1 interaction could be mediated by the core structure including the peptide and the sugars, except for sialic acid in the hinge region, resulting in the formation of the circulating macromolecular IgA1 in IgA nephropathy.

An immunoassay for the measurement of (1→3)-β-D-glucans in the indoor environment

An inhibition enzyme immunoassay was developed for quantitation of (1→3)-β-D-glucans in the indoor environment. Immunospecific rabbit antibodies were produced by immunization with bovine serum albuminconjugated laminarin.The laminarin calibration curve ranged from 40 to 3000 ng/ml.Another (1→3)-β-D-glucan (curdlan) showed a similar inhibition curve, but was less reactive on a weight basis. Pustulan, presumed to be (1→3)-β-D-glucan, also showed immunoreactivity in the assay. Control experiments indicated that this was due to (1→3)-β-D-glucan structures. Other non-(1→3)-β-D-glucan polysaccharides did not react. (1→3)-β-Dglucan was detectable in dust from a variety of occupational and environmental settings. We conclude that the new assay offers a useful method for indoor (1→3)-β-Dglucan exposure assessment.