In vitro studies on the mechanism of action of a new antiallergic,Ro 21-7634

The effects of Ro 21-7634 and disodium cromoglycate (cromoglycate) on the in vitro release of mediators of anaphylaxis from rat peritoneal cells and guinea pig lung tissue were compared. Ro 21-7634 was 25 fold more potent than cromoglycate as an inhibitor of antigen-induced histamine release from passively sensitized (IgE) rat peritoneal cells. Ro 21-7634 was also the more potent inhibitor of both compound 48/80- and concanavalin A-induced histamine release from rat peritoneal cells. The two drugs shared the common properties of producing the same maximal level of inhibition in each of the above releasing systems and exhibiting a time and concentration dependent loss of inhibitory activity when added to the cells prior to the releasing agent. Neither drug inhibited ionophore A23187- or ionophore X537A-induced histamine release from these cells. Ro 21-7634 inhibited antigen-induced (IgG₁) histamine and SRS-A release from actively sensitized guinea pig lung fragments, whereas cromoglycate did not. The results indicate that Ro 21-7634 and cromoglycate act through a common mechanism to inhibit allergic mediator release and that Ro 21-7634 is the more potent inhibitor.     

The pharmacological profile and initial clinical evaluation of tiacrilast (Ro 22-3747): a new antiallergic agent

Tiacrilast (Ro 22-3747) is an allergic mediator release inhibitor which has demonstrated potent oral activity in two IgE-mediated animal models of immediate hypersensitivity: the rat passive cutaneous anaphylaxis test (ID₅₀ of 0.65 mg/kg) and a model in which anaphylactic bronchospasm is induced in passively sensitized rats (ID₅₀ of 0.022 mg/kg). In addition to oral efficacy, in the latter model Ro 22-3747 was 23-fold more potent than cromoglycate by the aerosol (nebulization) route of administration.In vitro studies have confirmed that the mechanism of action of Ro 22-3747 in thein vivo models is through allergic mediator release inhibition since Ro 22-3747 was a potent inhibitor of antigen-induced (IgE-mediated) histamine release from passively sensitized rat peritoneal cellsin vitro (IC₅₀ values of 0.25 and 1.5 M for Ro 22-3747 and cromoglycate, respectively), and Ro 22-3747 did not display end organ antagonism to histamine, serotonin, or the leukotrienes. Clinical evaluations of Ro 22-3747 at a 350 mg oral dose have been conducted in patients with allergic asthma and allergic rhinitis. In a limited study in allergic asthmatics, Ro 22-3747 demonstrated significant inhibitory activity relative to placebo in reducing acute airway responses to inhaled pollen extracts in patients with ragweed sensitivity (measured by changes in log PD₂₀ FEV₁ and log PD₃₅ SGaw). The activity seen, however, was less than that observed with cromoglycate (20 mg) administered by inhalation. In a pilot evaluation in patients with allergic rhinitis, Ro 22-3747 administered at a 350 mg oral dose exhibited activity significantly greater than placebo in terms of improvements in the objective measurement of changes in nasal airflow at 2 and 3 hrs after treatment and the subjective overall global evaluation made by the patients at the end of the treatment period. The preclinical pharmacology and initial clinical efficacy of Ro 22-3747 support the continued clinical evaluation of this drug for prophylactic use in the management of asthma and other allergic disorders.     

Thein vivo andin vitro activity of AHR-13268D,a new antiallergic/antihistaminic agent

AHR-13268D (4-[3-[4-[Bis(4-flurophenyl)hydroxymethyl]-1-piperidinyl]propoxy]benzoic acid, sodium salt) is a potent, long-acting water soluble, antiallergic and antihistaminic agent. AHR-13268D protects sensitive guinea pigs from collapse induced by aerosolized antigen; 1, 5, and 24 h ED₅₀s in the test were 0.27, 0.25, 0.93 mg/kg, PO, respectively. AHR-13268D was also active when given as an aerosol, the 1 h ED₅₀=0.29%. In the rat passivefoot anaphylaxis test, AHR-13268D was slightly more active (1.55 times) than AHR-5333B when given orally 1 h prior to challenge and equipotent to cromolyn when given intravenously immediately prior to challenge. AHR-13268D displayed potent, long-acting antihistaminic activity in naive guinea pigs; the 1, 5, and 24 h oral ED₅₀s being in the range of 0.3 mg/kg. AHR-13268D (10 to 20 mg/kg, PO) attenuated the skin responses to ascaris antigen in sensitive dogs and did not alter the EEG pattern or sleep/wake patterns of cats at doses in vast excess of its antihistaminic activity.In vitro, AHR-13268D was a potent inhibitor of histamine release from rat peritoneal mast cells (IC₅₀=0.51 nM) and was as potent as the reference 5-LO inhibitor phenidone in inhibiting antigen-induced contractions of guinea pig ileum in the presence of pyrilamine, atropine, and imidazole (IC₅₀300 M). AHR-13268B was bioavailable (88%) from capsules or from oral solutions.     

The effect of Ro 21-7634 on the antigen-induced production of bronchoconstrictive arachidonic acid metabolites in the guinea pig lung

Ro 21-7634 has previously been shown to inhibit histamine and SRS-A release from actively-sensitized guinea pig lung fragments upon antigen challenge. In the studies described herein, it was observed that Ro 21-7634 does not decrease SRS-A release but instead acts to inhibit the synthesis of this mediator. This was confirmed by studying SRS-A synthesisin vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC₅₀ of 500 M, in comparison with 5,8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC₅₀'s of 2, 100, and 100 M, respectively). When studied at 10^–4 and 10^–3 M in perfused guinea pig lung, Ro 21-7634 inhibited antigen-induced thromboxane A₂ production by 68 and 96%, respectively. In this system, antigen is believed to induce thromboxane A₂ production through the release of histamine and SRS-A from lung tissue. These mediators then interact at receptor sites in the lung parenchyma to induce thromboxane A₂ synthesis. Ro 21-7634 could thus be inhibiting thromboxane A₂ production by preventing the release of histamine and synthesis of SRS-A in the perfused lung system. Such a mechanism is suggested by the fact that although Ro 21-7634 was effective in inhibiting antigen-induced thromboxane production, it was ineffective in inhibiting thromboxane A₂ production induced in the guinea pig lung system by the direct perfusion of histamine or SRS-A through the lung.     

Preventive effect of ketotifen, a new antiallergic agent, on histamine-induced bronchoconstriction in asthmatics

The preventive effect of ketotifen, a new drug with anti-histaminic and antiallergic properties, on histamine-induced bronchoconstriction was studied by open assessment in twenty-four adult patients with extrinsic asthma. A single oral dose of 1 mg ketotifen reduced the post-histamine mean drop in peak expiratory flow from 33 to 16% of the basal values (P<0.001). After a 4 weeks' regimen of 1 mg ketotifen twice daily the post-drug histamine-induced fall in PEF was further significantly reduced (P<0.001). Tests performed after 8 and 12 weeks of treatment showed no additional decrease in bronchial reactivity to histamine. Tests performed 1 week after cessation of treatment showed return of bronchial reactivity to the pretreatment level. The results suggest that a single dose of ketotifen has a marked preventive effect on histamine-induced bronchoconstriction and that this effect is enhanced during continued treatment.     

LG 30435, a new bronchodilator/antiallergic agent,inhibits PAF-acether induced platelet aggregation and bronchoconstriction

LG 30435 is a quaternary phenothiazinic antihistamine, endowed with bronchodilator and antiallergic activity. Since PAF-acether (PAF) is a potential mediator of asthma, LG 30435 was assayed for its ability to counteract PAF-induced platelet aggregation (PA) in rabbit platelet rich plasma and bronchoconstriction (BC) in anaesthetized guinea-pigs, in comparison with other antihistamines. LG 30435 was the most potent and selective inhibitor of PAF-induced PA (IC₅₀66 M), concentrations three and more than fifteen fold higher being needed to inhibit PA induced by collagen and arachidonic acid respectively. The other antihistamines, namely mepyramine, promethazine, mequitazine, thiazinamium methyl sulfate and ketotifen were less potent inhibitors of PAF-induced PA, while interfered at lower concentrations with collagen-induced PA. LG 30435 and thiazinamium, administered intravenously, inhibited dose-dependently PAF-induced BC, starting from the dose of 0.1 mol/kg. The travenously, inhibited dose-dependently PAF-induced BC, starting from the dose of 0.1 mol/kg. The ED₅₀ of LG 30435 was 0.28 mol/kg, while the inhibition obtained with thiazinamium did not reach 50% even at 3 mol/kg. Ketotifen and promethazine were partially active only at 3 mol/kg, while mequitazine and mepyramine were inactive up to this dose. These results show that LG 30435 is endowed with a peculiar anti-PAF action, which may be advantageous in the treatment of asthma.A preliminary report on some of these results was presented at the 2nd World Conference on Inflammation, Montecarlo, March 1986.     

The protective effect of a new antiallergic agent,KP-136 on mast cell activation: A comparison with disodium cromoglycate

The protective effects on mast cell activation were compared between a new antiallergic agent, KP-136 and disodium cromoglycate (DSCG), both of which inhibited the immunological degranulation of rat peritoneal mast cells. The IC₅₀ was 0.03 g/ml for KP-136 and 4.7 g/ml for DSCG. KP-136 predominantly acted on the early stage of mast cell activation processes and inhibited the immunological increase in⁴⁵Ca uptake. KP-136 also inhibited A23187-and heat-induced degranulation and heat-induced hemolysis. In addition, KP-136 was effective on phospholipase A₂-induced degranulation, although the compound did not directly affect the enzyme activity. In all tests for comparison, KP-136 and DSCG had similar profiles of action and the parallel experiments indicated that KP-136 was a more potent inhibitor of mast cell activation than DSCG, having a DSCG-like membrane stabilizing activity.     

Comparative in vitro activity of Ro 40-6890, Ro 41-3399, and other antimicrobial agents against anaerobic bacteria

The in vitro activity of the ester Ro 41-3399 and its free active acid Ro 40-6890 was tested against 189 strains of anaerobic bacteria in comparison to other oral cephalosporins and to antimicrobial agents established in the treatment of anaerobic infections.Prevotella, Porphyromonas, Peptostreptococcus, Fusobacterium andClostridium spp. were susceptible to Ro 40-6890, with few exceptions. Due to its lack of activity against the major pathogens of theBacteroides fragilis group, Ro 40-6890 does not promise to be of major use in the treatment of infections caused by anaerobes.     

Ro 15-1788 is a potent antagonist of benzodiazepines in the olfactory cortex slice

The olfactory cortex slice from the guinea pig has been used to test the benzodiazepine antagonist, Ro 15-1788. Bath application of muscimol has a GABA-mimetic effect on the resting input conductance of these neurones. Benzodiazepines increase the potency of muscimol and increase the duration of postsynaptic inhibitory conductance. To measure the effect of muscimol, the input conductance was measured either directly using intracellular microelectrodes or by measuring its effect on the amplitude of the evoked compound potentials recorded from the slice surface after stimulating the lateral olfactory tract. The potentiation of postsynaptic inhibition produced by benzodiazepines was measured indirectly by their effect on the amplitude of the second of two evoked compound potentials. All of the potentiating effects of diazepam, clonazepam, flurazepam and chlordiazepoxide were blocked by Ro 15-1788 (0.01-10 mumol/l). Ro 15-1788 up to a concentration of 10 mumol/l had no effect on any of the synaptic or electrical responses when applied alone. General anaesthetics which also potentiate inhibition were unaffected by Ro 15-1788. It is concluded that Ro 15-1788 is a highly potent and specific benzodiazepine antagonist in this preparation.     

Determination of olopatadine, a new antiallergic agent, and its metabolites in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry.

A rapid, sensitive and specific assay method has been developed to determine plasma concentrations of olopatadine hydrochloride (A) and its metabolites, M1 (B), M2 (C) and M3 (D), using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Olopatadine, its metabolites, and internal standard, KF11796 (E), were separated from plasma using solid-phase extraction (Bond Elut C18 cartridge). The eluate was dried, reconstituted and injected into the LC-ESI-MS-MS system. The calibration curves showed good linearity over the ranges 1-200 ng/ml for olopatadine and M3, and 2-100 ng/ml for M1 and M2, and the method was thoroughly validated and applied to the determination of olopatadine and its metabolites in plasma collected during Phase I clinical trials. Furthermore, the assay values were compared with those determined by the radioimmunoassay method, which has been routinely used to determine olopatadine in plasma.     

Antivirus agent, Ro 09-0410, binds to rhinovirus specifically and stabilizes the virus conformation

The antiviral mechanisms of Ro 09-0410 (4'-ethoxy-2'-hydroxy-4,6'-dimethoxychalcone), which inactivates rhinovirus exclusively, have been investigated. It was suggested that Ro 09-0410 bound to human rhinovirus type 2 (HRV-2) and made it inactive, since the reduced infectivity was completely restored to original levels by extraction of the agent with chloroform [H. Ishitsuka, Y. Ninomiya, C. Ohsawa, M. Fujiu, and Y. Suhara (1982) Antimicrob. Agents Chemother. 22, 617-621]. This was confirmed using radioactively labeled Ro 09-0410 and HRV-2. HRV-2 was inactive while bound to the agent, whereas a subline of HRV-2 resistant to the agent had no binding site for the agent. Ro 09-0410 appeared to bind to some specific site(s) on the virion of susceptible virus strains. Treatment of rhinovirus at pH 5 or 56 degrees caused a change of the virion size and greatly reduced its infectivity. Ro 09-0410 could no longer bind to HRV-2 after such treatment. On the other hand, when the virion bound with Ro 09-0410 was treated at pH 5 or 56 degrees, the Ro 09-0410 remained bound and the conformational alteration of the virion did not take place. Furthermore, Ro 09-0410 protected HRV-2 from the reduction of infectivity caused by mild acid or heat treatment, as revealed by infectivity measurements after extraction of the agent with chloroform. These results suggest that Ro 09-0410 binds to the HRV-2 virion and prevents viral replication in the cell.     

In vitro activity of temafloxacin,a new difluoro quinolone antimicrobial agent

Temafloxacin, a new difluoro quinolone, inhibited the majority ofEnterobacteriaceae at 1µg/ml. It was 4–8-fold less active than ciprofloxacin and 2-fold less active than ofloxacin. Cefotaxime and imipenem-resistant isolates such asEnterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, andAcinetobacter spp. were inhibited. Temafloxacin inhibitedNeisseria, Branhamella, andHaemophilus species at < 0.25µg/ml. Methicillin-susceptible and methicillin-resistantStaphylococcus aureus, Staphylococcus epidermidis andClostridium spp. were inhibited at concentrations less or equal to that of ciprofloxacin and ofloxacin.     

In vitro activity of BMY 28100, a new oral cephalosporin

BMY 28100, a new oral cephalosporin, demonstrated good in vitro activity against common gram-positive and gram-negative respiratory and urinary tract pathogens. Its activity was shown by microdilution techniques to be generally higher than those of ampicillin, cephalothin and cephalexin, but comparable to those of cefaclor and, except forHaemophilus spp. andBrantiamella spp., to amoxicillin/clavulanate.     

4-epi-Pimaric acid: a phytomolecule as a potent antibacterial and anti-biofilm agent for oral cavity pathogens

The present study focused on the antibacterial and biofilm inhibitory potential of 4-epi-pimaric acid isolated from aerial parts (stem and leaves) of Aralia cachemirica L. (Araliaceae) against oral cavity pathogens. 4-epi-Pimaric acid exhibited minimum inhibitory concentration (MIC) in the range of 4–16 μg/ml and minimum bactericidal concentration (MBC) two- to four-folds higher than MIC. There was significant inhibition in the biofilm formation by Streptococcus mutans on the saliva coated surface (P < 0.05), and confocal microscopy revealed that 4-epi-pimaric acid inhibited the clumping and attachment of S. mutans. At 8 × MIC concentration, it significantly prevented the pH drop and reduced S. mutans biofilms (P < 0.05). Increased propidium iodide staining and leakage of 260- and 280-nm absorbing material by 4-epi-pimaric acid treated cells of S. mutans suggested that it probably causes disruption of the cytoplasmic membrane structure. It also exhibited significant suppression of TNF-α expression in human neutrophils, suggestive of its anti-inflammatory activity. Furthermore, the compound was found to be significantly safe (IC₅₀ >100 μg/ml) in the MTT assay on AML-12 cell lines. In conclusion, 4-epi-pimaric acid showed promising antibacterial, anti-biofilm and anti-inflammatory potency and this compound can be exploited for therapeutic application in oral microbial infections.