EC50 Ratios Research Articles

BGS-2456 Is a Novel Potent Covalent Inhibitor of FLT3 That Highly Discriminates Against KIT and Is Not Toxic Toward Normal Hematopoiesis in Vitro

Introduction: Class III receptor tyrosine kinases include FLT3, KIT, PDGFRA/B and CSF1R. Activating mutations in FLT3 represent the most common genetic anomalies in AML (30%). While there are numerous examples of KIT TKIs that do not inhibit FLT3 (imatinib, avapritinib, dasatinib), to date, all clinically active FLT3 TKIs (quizartinib, gilteritinib, midostaurin, sorafenib) fail to spare KIT inhibition. Preclinical studies suggest that coinhibition of FLT3 and KIT can result in non-specific myelosuppression, which can limit dose intensification and the ability to safely achieve maintenance dosing. A potent FLT3 TKI that is non-myelosuppressive may enable prolonged treatment in conjunction with chemotherapy and thereby substantially improve clinical outcomes, similar to BCR::ABL1 TKIs in Ph+ ALL (Slayton et al, JCO 2018). FF-10101 is a recently described covalent inhibitor to irreversibly bind FLT3. In vitro and in vivo studies have revealed that FF-10101 exhibits substantial activity against FLT3 and resistant mutants F691L and D835V/Y. However, it does not effectively discriminate against KIT inhibition (Yamara et al, Blood 2018). In clinical experience, a majority of responses were associated with persistent myelosuppression (Levis et al, JCO 2021 39:15_suppl, 7008). Further clinical development of FF-10101 has been abandoned. Here we describe a novel covalent inhibitor (BGS-2456) that potently targets FLT3 with outstanding selectivity against KIT. Results: To confirm that BGS-2456 is a covalent FLT3 inhibitor, we tested various Cys→Ser substitutions in FLT3-ITD. Of these, only substitution of C695 resulted in loss of activity, suggesting that this residue is critical for covalent bond formation with BGS-2456, as has also been demonstrated for FF-10101 (Yamara et al, Blood 2018). Additionally, the compound without the covalent warhead (BGS-2457) was found to be unimpacted by the C695S substitution and substantially less potent than BGS-2456 (Figure). To characterize in vitro properties of BGS-2456 and whether it achieves selectively against FLT3 relative to KIT, we performed cell viability assays of the patient-derived FLT-ITD-positive AML Molm14 cell line as well as the gastrointestinal stromal tumor cell line GIST-T1, which contains an activating juxtamembrane mutation in KIT. We compared EC50 ratios with other FLT3 TKIs and found that BGS-2456 is highly potent, with an EC50 value of ~0.2nM. Moreover, BGS-2456 displayed remarkable selectivity toward FLT3, >10-fold greater than all other TKIs tested. Kinome profiling confirmed FLT3 as the kinase that was most highly inhibited by BGS-2456 among the 300 kinases assessed, with BMX being a distant second (Figure). To assess whether the selectivity of BGS-2456 results in less myelosuppression in vitro, we performed CFU assays of healthy bone marrow. Compared to FF-10101 and gilteritinib, BGS-2456 exhibited the least amount of hematologic toxicity, facilitating in vitro proliferation and differentiation of normal hematopoietic progenitor cells even at 100x EC50 concentration against Molm14 cells. Finally, we assessed BGS-2456's ability to retain activity against the known clinically problematic drug-resistant FLT3-ITD mutants D835Y and F691L. Encouragingly, BGS-2456 retained activity against both D835Y and F691L mutants in the low nanomolar range. Conclusion: This is the first description of a potent and exquisitely specific FLT3 inhibitor that spares KIT inhibition and displays no myelosuppression in vitro at >100x EC50 concentration. The potent inhibitory effects of BGS-2456 on both D835Y and F691L mutants support its promise as a best-in-class TKI for the treatment of FLT3-mutant AML. Efforts to molecularly dissect the basis of the high degree of selectivity of BGS-2456 are ongoing.

An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH

Despite the exquisite specificity and high affinity of antibody-based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor FcγRIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc–FcγRIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced FcγRIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ∼2-fold pH-selectivity for FcγRIIIa binding based on the ratio of equilibrium dissociation constants Kd,7.4/Kd,6.5, due to a faster dissociation rate at pH 7.4. Finally, in vitro ADCC assays with human FcγRIIIa-positive natural killer and Her2-positive target cells demonstrated similar activities for anti-Her2 antibodies bearing the wild-type or acid-Fc at pH 6.5, but nearly 20-fold reduced ADCC for acid-Fc at pH 7.4, based on EC50 ratios. This work shows the promise of mammalian cell display for Fc engineering and the feasibility of pH-selective Fc activation to provide a second dimension of selective tumor cell targeting.

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes.

Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7days. The median of the EC50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC50 on day 1 and day 7 (R = 0.985; 95% CI 0.960-0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910-0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955-0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14days, was analyzed. A high correlation coefficient of EC50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798-0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity.

Assessing ecotoxicity and the endocrine potential of selected phthalates, BADGE and BFDGE derivatives in relation to environmentally detectable levels

There is no doubt that the subject area of plastic materials (e.g., production of epoxy resins or polyesters) is inherently connected to issues concerning bisphenol A (BPA) and its analogues. Unfortunately, much less attention has been given to other compounds, which are also used for the production of these materials. Bisphenol A diglycidyl ether (BADGE) is a synthetic industrial compound obtained by a condensation reaction between epichlorohydrin (ECH) and BPA. Similarly, novolac glycidyl ether (BFDGE) is produced in the reaction between novolac and epichlorohydrin. Nevertheless, there is a lack of information on the combined effects of BADGE derivatives at environmentally relevant levels. In the current study, toxicity levels in Microtox® and XenoScreen YES/YAS assays were determined for several analogues alone, then the biological effects of compound pairs mixed in 33, 66 and 100% of each compounds' EC50 ratios were evaluated. The Microtox® test has been chosen as a relevant tool, and the results were referred to the Xenoscreen YES/YAS assay, which has been chosen for the fast determination of the endocrine potential of the compounds tested. The results obtained constitutes the basis for model studies, with Concentration Addition (CA) and Independent Action (IA), followed by Model Deviation Ratio (MDR) interpretation, to evaluate the possible interactions occurring between analytes when present in mixtures. The results indicate that the hydrochloric derivatives of BADGE and BFDGE are of the greatest toxicological and endocrine threat. Thus, their presence in mixtures under certain environmental conditions (including presence in the tissues of living organisms) should be strictly monitored and reported, especially in acidic environments. Strong evidence on the synergic behaviors of these analytes, which expressed high toxicity (EC50 2.69–117.49μg/mL), is demonstrated with Model Deviation Ratio (MDR).

Synthesis and biological activity of mono- and diamides of 2,3-secotriterpene acids

Four types of amide (C3; C28; C3-C28) conjugates based on 2,3-seco-18alphaH-oleanane and 2,3-secolupane mono- and dicarboxylic acids were synthesized. The range of diamide derivatives was supplemented with C3-C3' and C28-C28' dicondensed amides with two A-secotriterpene backbones educed by reacting monocarboxylic A-secoacids with biogenic amino acid lysine. Compounds with inhibitory action against herpes virus reproduction (EC50 8.7 and 4.1 McM) were found among the synthesized mono- and diamide derivatives containing an ethyl-beta-alaninate fragment. It has been ascertained that diamide with ethyl-beta-alaninate fragment combines anti-herpes virus properties and anti-HIV activity (EC50 5.1 McM). For active compounds, the maximum non-toxic concentration (MNTC)/EC50 ratios ranges from 9.7 to 40.8. The synthesized amide conjugates do not exhibit any marked cytotoxic effects against human tumor cell lines rabdomiosarcoma RD TE32, A549 lung carcinoma and melanoma MS.

Transgenerational effects of heavy metals on L3 larva of Caenorhabditis elegans with greater behavior and growth inhibitions in the progeny

Heavy metals are ubiquitous environmental pollutants, and their toxic effects have been widely studied. However, their transgenerational effects between parent and progeny at environmental relevant concentrations need further investigations. Currently, L3 stage of Caenorhabditis elegans was exposed to aqueous metals (Cd, Cu, Pb and Zn) at environmentally realistic concentrations for 96h. The whole exposure time covered the formation of sperm, ovum and eggs. Subsequently the behavior and growth indicators were measured. The parent nematodes were then bleached to gain synchronized eggs, which were cultured under non-toxic conditions to L3 stage when the same indicators were measured in the progeny. The parent suffered concentration-dependent inhibitions on behavior and growth. Based on the median effective concentration (EC50) values, body bending frequency showed relatively higher sensitivity than other behavior indicators. The inhibitions on growth and behavior of progeny were more severe than those of the parent, based on their respective EC50 values. Interestingly, Cd was not the most toxic metal in either parent or progeny according to EC50 values, but its EC50 ratios between parent and progeny (EC50, parent/EC50, progeny) were the most significant, indicating its greatest transgenerational effects. The results demonstrated the higher sensitivity of L3 larva stage of C. elegans in the transgenerational effect studies than other life stages used before. Our findings suggested that parental exposure to heavy metals can multiply their harmful effects in following generations.

Synergy between fipronil and amitraz in a Rhipicephalus sanguineus tick residual contact test

Amitraz, a formamidine acaricide, has been reported to act as a synergist of contact insecticides and acaricides in both agriculture and animal health. A laboratory contact test was therefore conducted to determine whether amitraz at 12.5 ppm could improve the contact potency of fipronil on ticks. A controlled glass vial bioassay was used to assess the efficacy of fipronil alone, amitraz alone, and fipronil plus amitraz on unfed adult Rhipicephalus sanguineus. Assessments of lethality were made at 6, 24, and 48 h after the introduction of the ticks to the vials. No significant mortality was observed in the control treatment or in the amitraz alone treatment. Concentration and time dependent mortality rates were observed in ticks exposed to fipronil alone or fipronil plus amitraz, with higher mortality observed in the latter group. Results from this study gave synergistic EC50 ratios between fipronil alone and fipronil plus amitraz of >7.3, 137 and 97 at 6, 24, and 48 h, respectively. A similar response was seen at the EC90 level. These results indicate that fipronil was synergized by amitraz in this adult tick residual contact study. The addition of amitraz to fipronil also provided a significant improvement in the speed of kill.

Anionic Amphiphilic Dendrimers as Antibacterial Agents

An anionic amphiphilic dendrimer is reported that possesses increased cytotoxicological potency against prokaryotic cells compared to eukaryotic cells. The half-maximal effective concentration (EC50) for the dendrimer against Bacillus subtilis, a Gram-positive bacterial strain, was measured to be 4.1 x 10(-5) M, while that against human umbilical vein endothelial cells (HUVEC) was more than 36x greater at a value of 1.5 x 10(-3) M. EC50 ratios for two commercial amphiphiles, sodium dodecyl sulfate (SDS) and Triton X-100, in addition to a similar synthesized dendritic structure were at most only 3.8x greater. Furthermore, the observed EC50 values appear to be correlated to the critical aggregation constant (CAC) in solution suggesting a mechanism of action for these anionic amphiphilic dendrimers related to their supramolecular structures.

Isoform-selectivity of PKC Inhibitors Acting at the Regulatory and Catalytic Domain of Mammalian PKC-α, -βI, -δ, -η and -ζ

The aim of the present study was to compare the potency of a series of widely used PKC inhibitors acting either at the regulatory (NPC 15437, tamoxifen and d-sphingosine) or at the catalytic domain (Ro 32-0432, chelerythrine and rottlerin) on individual mammalian PKC isoforms of the classical (α and βI), novel (δ and η) and atypical (ζ) PKC families, using the yeast phenotypic assay, in order to determine their isoform-selectivity. The PKC inhibitors studied presented differences in their ability to reduce the effect of the appropriate PKC activator (estimated as EC50 ratios) which was interpreted as an index of PKC inhibitory potency. In general, the more marked inhibition was observed on novel PKC isoforms, particularly on PKC-η. This study indicates promising isoform-selectivity of some PKC inhibitors, namely NPC 15437 for PKC-η or rottlerin for both novel PKC isoforms. It also suggests that the PKC domain involved in the inhibition does not seem to be relevant for the potency and isoform-selectivity of PKC inhibitors.

Studies on the inherent resistance risk to fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10−5. Resistance factors, based on EC50 values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC50 ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is discussed.

Studies of Interaction between Hexamethonium and Lidocaine, alpha-Bungarotoxin or Decamethonium in Vitro

Background: Interactions of neuromuscular blocking agents are antagonistic in a combination of depolarizing and nondepolarizing agents, additive in a combination of relative two compounds or synergistic in a combination of different two nondepolarizing agents. However, the interactions of neuromuscular blocking agents with a different site of action from each other have not been studied clearly. This study was designed to examine the interaction between hexamethonium and lidocaine, α-bungarotoxin or decamethonium with markedly different pre and postsynaptic sites of action. Methods: Square wave, 0.1 Hz supramaximal stimuli or 2 Hz, 0.2 ms train of four (TOF) stimuli, was applied to the rat phrenic nerve-hemidiaphragm preparation, and the twitch height response was recorded mechanomyographically. The cumulative concentration effect and TOF ratio at each point of twitch depression after hexamethonium, lidocaine, α-bungarotoxin or decamethonium given were measured. The EC50 and EC95 of hexamethonium, lidocaine, α-bungarotoxin and decamethonium were calculated using an inhibitory sigmoid E model. In the experiment of each combination of two drugs, three points of the isobole for hexamethonium-lidocaine, hexamethonium-α-bungarotoxin and hexamethonium-decamethonium were established using ratios of 1: 3, 1: 1 and 3: 1 of their EC50. Points on the line of theoretical additivity and 95% canfidence intervals were calculated according to Tallarida et al. TOF ratios were observed at 75, 50 and 25% of the control twitch height value during each combination ratio of their EC50. Results: Significant deviations of points on the isobole from the line of additivity to the left were found at all EC50 ratios of hexamethonium-lidocaine (P<0.05 respectively), that to the right was found at all EC50 ratios of a hexamethonium-α-bungarotoxin and hexamethonium-decamethonium (P<0.05 respectively). The magnitude of TOF fade depended upon the mixed ratios for their EC50. Conclusions: The interaction was found to be synergistic in the combination of hexamethonium-lidocaine, and antagonistic in the combination of hexamethonium-α-bungarotoxin and hexamethonium-decamethonium. (Korean J Anesthesiol 2001; 40: 645~654)

Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656, and AM630.

We have tested our prediction that AM630 is a CB2 cannabinoid receptor ligand and also investigated whether L759633 and L759656, are CB2 receptor agonists. Binding assays with membranes from CHO cells stably transfected with human CB1 or CB2 receptors using [3H]-CP55940, confirmed the CB2-selectivity of L759633 and L759656 (CB2/CB1 affinity ratios = 163 and 414 respectively) and showed AM630 to have a Ki at CB2 receptors of 31.2 nM and a CB2/CB1 affinity ratio of 165. In CB2-transfected cells, L759633 and L759656 were potent inhibitors of forskolin-stimulated cyclic AMP production, with EC50 values of 8.1 and 3.1 nM respectively and CB1/CB2 EC50 ratios of > 1000 and > 3000 respectively. AM630 inhibited [35S]-GTPgammaS binding to CB2 receptor membranes (EC50 = 76.6 nM), enhanced forskolin-stimulated cyclic AMP production in CB2-transfected cells (5.2 fold by 1 microM), and antagonized the inhibition of forskolin-stimulated cyclic AMP production in this cell line induced by CP55940. In CB1-transfected cells, forskolin-stimulated cyclic AMP production was significantly inhibited by AM630 (22.6% at 1 microM and 45.9% at 10 microM) and by L759633 at 10 microM (48%) but not 1 microM. L759656 (10 microM) was not inhibitory. AM630 also produced a slight decrease in the mean inhibitory effect of CP55940 on cyclic AMP production which was not statistically significant. We conclude that AM630 is a CB2-selective ligand that behaves as an inverse agonist at CB2 receptors and as a weak partial agonist at CB1 receptors. L759633 and L759656 are both potent CB2-selective agonists.

Nonanesthetic volatile drugs obey the Meyer-Overton correlation in two molecular protein site models.

Nonanesthetic volatile compounds fail to inhibit movement in response to noxious stimulation at concentrations predicted to induce anesthesia from their oil-water partitioning. Thus they represent tools to determine whether molecular models behave like the targets that mediate in vivo anesthetic actions. The effects of volatile anesthetics and nonanesthetics were examined in two experimental models in which anesthetics interact directly with proteins: the pore of the nicotinic acetylcholine receptor and human serum albumin. Wild-type mouse muscle nicotinic receptors and receptors containing pore mutations (alphaS252I + betaT263I) were studied electrophysiologically in membrane patches from Xenopus oocytes. Patch currents evoked by brief pulses of acetylcholine were measured in the presence of enflurane and two nonanesthetics, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane. Nonanesthetic interactions with human serum album were assessed by quenching of intrinsic protein fluorescence. Both anesthetic and nonanesthetic volatile compounds inhibited wild-type and alphaS252I + betaT263I mutant nicotinic channels but displayed different selectivity for open versus resting receptor states. Median inhibitory concentrations (IC50s) in wild-type nicotinic receptors were 870+/-20 microM for enflurane, 37+/-3 microM for 1,2-dichlorohexafluorocylcobutane, and 11.3+/-5.6 microM for 2,3-dichlorooctafluorobutane. For all three drugs, ratios of wild-type IC50s to mutant IC50mut ranged from 7-10, and ratios of wild-type IC50s to predicted anesthetic median effective concentrations (EC50s) ranged from 1.8-2.3. 1,2-Dichlorohexafluorocyclobutane quenched human serum albumin with an apparent dissociation constant (Kd) of 160+/-11 microM. The ratios of dissociation constants to predicted EC50s for the nonanesthetics were within a factor of two of the dissociation constant:EC50 ratios calculated for halothane and chloroform from previous published results. In two models in which anesthetics bind to protein sites, both anesthetic and nonanesthetic volatile drugs cause similar steady state effects with potencies that are predicted by hydrophobicity. These protein sites do not sterically discriminate between anesthetic and nonanesthetic drugs. However, differential state-selective actions on ion channel targets may underlie the distinct in vivo effects of anesthetics and nonanesthetics.

Interaction of Decamethonium with Hexamethonium or Vecuronium in the Rat

We used isobolographic analysis to investigate the interaction of decamethonium with either hexamethonium or vecuronium in the rat phrenic nerve hemidiaphragm preparation. EC50 values of decamethonium, hexamethonium, and vecuronium were (mean +/- SEM) 47.36 +/- 9.58 microM, 4.27 +/- 0.53 mM, and 5.19 +/- 1.17 microM, respectively. Combinations of drugs in concentrations corresponding to the 1:2, 1:1, and 2:1 ratios of their EC50 values were used to determine three points of each isobole. Decamethonium and hexamethonium showed antagonism: significant deviations from the line of additivity were found at EC50 ratios of 2:1 and 1:1 (P < 0.01 and P < 0.05, respectively) indicating that hexamethonium is a potent antagonist of decamethonium. For decamethonium and vecuronium none of the three points on the isobole was significantly different from the corresponding point on the line of additivity. Hexamethonium is known to be a weak antagonist at the postsynaptic nicotinic acetylcholine receptor but a potent antagonist at the presynaptic nicotinic receptor. Vecuronium is a more potent antagonist at the postsynaptic nicotinic receptor but a much weaker antagonist at the presynaptic site. It was postulated that in the rat the primary site of action of decamethonium is at the presynaptic nerve terminal. Our findings suggest that presynaptic rather than postsynaptic potency of a nondepolarizing drug determines ability to antagonize the effect of a depolarizing drug in the rat.

Progress in the discovery of orally bioavailable inhibitors of HIV protease

The development of inhibitors of HIV protease for the chemotherapy of AIDS has been hampered by the poor pharmacokinetic profile of most inhibitors due to their peptidomimetic nature. Recently, substantial progress in the identification of agents with improved properties has been realized. This Perspective contrasts the preclinical pharmacokinetic parameters of a variety of HIV protease inhibitors with significant oral bioavailability. Inhibitors with high oral Cmax/in vitro EC50 ratios have shown substantial suppression of HIV in vivo. The relationship between the structural and physicochemical features and the pharmacokinetic behavior of peptidomimetic inhibitors is discussed.

Dual action of ranitidine at cholinergic sites in the rat isolated anococcygeus muscle.

1. The effects of ranitidine on contractile responses of the rat isolated anococcygeus muscle to acetylcholine (ACh) and carbachol (CCh) were investigated. 2. Four consecutive cumulative concentration-response curves (CRCs) for ACh or CCh were constructed in the absence or presence of ranitidine. The mean EC50 values were determined from the individual CRCs and used for the calculation of the corresponding EC50 ratios. 3. Ranitidine (0.01, 0.05 and 0.5 mM) produced a progressively leftward shift of the ACh curves; the corresponding EC50 ratios obtained were 3.2 +/- 0.4, 5.7 +/- 0.5 and 9.4 +/- 1.4. Neostigmine 0.5 microM alone or in combination with ranitidine 0.1 mM produced very marked leftward shifts of the ACh curves (EC50 ratios 68.2 +/- 14.3 and 56.3 +/- 9.6 respectively). In contrast, ranitidine (0.05, 0.5 and 2 mM) produced a rightward and parallel shift of the CCh curves (EC50 ratios 1.0 +/- 0.1, 3.4 +/- 0.32 and 12.5 +/- 1.7 respectively). 4. Ranitidine can therefore mediate a dual action at cholinergic sites in the rat isolated anococcygeus muscle, actions which can be attributed to its inherent anticholinesterase and antimuscarinic activity. The relatively marked anticholinesterase action of ranitidine was rather surprising in view of the limited cholinergic innervation present in the rat anococcygeus muscle.

Prediction of the therapeutic dose for β-stimulants based on preclinical data: Application of oral dosage forms and aerosols to asthmatic patients

The therapeutic doses of beta-stimulants in the treatment of bronchial asthma show wide variation among the drugs. In the present study, we tried to construct a system for prediction of the optimum clinical dose of beta-stimulants as oral dosage forms and aerosols using the data from preclinical pharmacological studies and pharmacokinetic data in humans. Values of EC50 (drug concentration that gives the half-maximum bronchodilator effect) in the in vitro studies based on isolated trachea of guinea pigs were collected from the literature. The ratios of the EC50 value of each drug to that of isoproterenol were used as the indicator of in vitro pharmacological activity. There were significant correlations between the EC50 ratios and the oral dose or the maximum plasma concentration after single oral administration, but the correlation coefficients were relatively small (r < .9). On the other hand, a linear log-log relationship was observed between the EC50 ratio and the maximum plasma unbound concentration (Cu(max)) (slope = 0.907, r = .955, p < 0.001). In the case of aerosols, a good correlation was observed between the EC50 ratio and the dose (slope = 0.770, r = .900, p < 0.01). The predicted concentration of beta-stimulants in the lung after aerosol administration tended to be higher than the Cu(max) after oral administration, suggesting the contribution of nonspecific binding in the lung tissue. These findings indicate the possibility of predicting the appropriate dose of beta-stimulants based on the preclinical pharmacological data and the pharmacokinetic data in humans.

Extraluminally applied acetylcholine and substance P on the release of EDRF.

Acetylcholine, substance P and nitroglycerin applied intra- and extraluminally to the perfused dog femoral artery segment with endothelium caused depressor responses. Endothelium denudation abolished the responses to acetylcholine and substance P. EC50 ratios of extra- versus intraluminal acetylcholine and substance P were 43 and 79, respectively, whereas those of nitroglycerin did not differ. Physostigmine potentiated the response to extraluminal acetylcholine. Acetylcholine seems to be degraded partly by cholinesterase in the arterial wall. Acetylcholine and substance P applied extraluminally are expected to reach the endothelium and release endothelium-derived relaxing factor.

Extraluminally Applied Acetylcholine and Substance P on the Release of EDRF

Acetylcholine, substance P and nitroglycerin applied intra- and extraluminally to the perfused dog femoral artery segment with endothelium caused depressor responses. Endothelium denudation abolished the responses to acetylcholine and substance P. EC50 ratios of extra- versus intraluminal acetylcholine and substance P were 43 and 79, respectively, whereas those of nitroglycerin did not differ. Physostigmine potentiated the response to extraluminal acetylcholine. Acetylcholine seems to be degraded partly by cholinesterase in the arterial wall. Acetylcholine and substance P applied extraluminally are expected to reach the endothelium and release endothelium-derived relaxing factor.

Effect of ouabain on norepinephrine-induced contractions of isolated rabbit aorta.

The acute potentiating effect of ouabain on norepinephrine (NE) induced contractions of isolated rabbit aorta was investigated. Ouabain, at concentrations of 3 X 10(-7) to 10(-5) mol/l potentiated the vasoconstrictor effect of NE as demonstrated by a shift to the left of the NE concentration-response curve in a parallel manner and a concentration-dependent increase in the EC50 ratio. A significant increase in maximal contractility was observed by ouabain at 3 X 10(-6) and 10(-5) mol/l. Removal of the endothelium did not alter the effect of 10(-5) mol/l ouabain. When the NE concentration-response curves were again determined after ouabain had been washed out, it was found that the ouabain-induced increase in EC50 ratios returned to control values, while the increase in maximal contractility did not. The slopes of the NE concentration-response curves were not significantly different in the presence of ouabain than control values, but were higher when obtained after ouabain had been washed out.