Constance Kohler

Intravenous arachidonate in the mouse: A model for the evaluation of antithrombotic drugs

Abstract The intravenous administration of arachidonate to mice offers a rapid, convenient, and effective in vivo model for the study of platelet aggregation and thrombosis, as well as the evaluation of potential antithrombotic drugs. Following intravenous administration in the mouse, there is dose-dependent cyanosis and respiratory distress with death at doses about 100 mg/kg. The rabbit is much more sensitive to the effects of intravenous arachidonate with death occurring at doses of 1 mg/kg; the rat shows an intermediate sensitivity. There is a relative specificity for the arachidonate effect: administration of certain other unsaturated fatty acids produces only minimal respiratory distress at equivalent doses. Respiratory distress in the mouse can be correlated with histologic evidence of platelet aggregation in the microvasculature of the lungs, but not in the heart or brain. Several nonsteroidal anti-inflammatory drugs (NSAID) which are known to be inhibitors of collagen-induced platelet aggregation and release, as well as inhibitors of prostaglandin synthetase, are able to block the effect of arachidonate in vivo . ADP inhibitors (dipyridamole, adenosine, and VK 744) are also effective in blocking the effects of arachidonate in vivo . Both the NSAID and the ADP inhibitors also block arachidonate-induced platelet aggregation in vitro .

Synthesis and Structure–Activity Relationships of 3-Cyano-4-(phenoxyanilino)quinolines as MEK (MAPKK) Inhibitors

A series of 3-cyano-4-(phenoxyanilino)cyanoquinolines has been prepared as MEK (MAP kinase kinase) inhibitors. The best activity is seen with alkoxy groups at both the 6- and 7-positions. The lead compounds show low nanomolar IC50s against MAP kinase kinase, and have potent inhibitory activity in tumor cells.

MEK (MAPKK) inhibitors. Part 2: structure-activity relationships of 4-anilino-3-cyano-6,7-dialkoxyquinolines.

A series of 4-anilino-3-cyano-6,7-dialkoxyquinolines with different substituents attached to the 4-anilino group has been prepared that are potent MEK (MAP kinase kinase) inhibitors. The best activity is obtained when a phenyl or a thienyl group is attached to the para-position of the aniline through a hydrophobic linker, such as an oxygen, a sulfur, or a methylene group. The most active compounds show low nanomolar IC(50)s against MEK (MAP kinase kinase), and have potent growth inhibitory activity in LoVo cells (human colon tumor line).

Synthesis and evaluation of 4-Anilino-6,7-dialkoxy-3-quinolinecarbonitriles as inhibitors of kinases of the Ras-MAPK signaling cascade

4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)]anilino-6,7-diethoxy-3-quinolinecarbonitrile (3) was identified as a MEK1 kinase inhibitor with exceptional activity against LoVo cells. The structure-activity relationships of the C-4 aniline substituents were explored, and water-solubilizing groups were added at the C-7 position to improve physical properties. Secondary cellular assays revealed that a compound possessing the appropriate aniline substituents inhibited MEK1 as well as MAPK phosphorylation, thereby acting as a dual inhibitor of the Ras-MAPK signaling cascade.

4-Anilino-3-cyanobenzo[g]quinolines as Kinase Inhibitors

A series of 4-anilino-3-cyanobenzo[g]quinolines was prepared as potent kinase inhibitors. Compared with their bicyclic 4-anilino-3-cyanoquinoline analogues, the tricyclic 4-anilino-3-cyanobenzo[g]quinolines are less active against EGF-R kinase, equally active against MAPK kinase (MEK), and more active against Src kinase. For Src kinase inhibition, the best activity is obtained when both the 7- and 8-positions are substituted with alkoxy groups. Several of these kinase inhibitors show potent growth inhibitory activity in tumor cells.

Studies of the effect of a platelet-activating factor antagonist, CL 184,005, in animal models of gram-negative bacterial sepsis.

The effect of CL 184,005, a potent and specific platelet-activating factor antagonist, has been examined in a variety of animal models relevant to gram-negative bacterial sepsis. Pretreatment of mice with CL 184,005 protected them from the lethal effects of platelet-activating factor. When rats or primates rendered hypotensive with endotoxin were treated with CL 184,005, blood pressure was normalized. Pretreatment of rats with CL 184,005 protected them from the gastrointestinal lesions induced by endotoxin. Pretreatment of rats and mice with CL 184,005 protected them from the lethal effects of endotoxin. Plasma tumor necrosis factor levels in endotoxin-treated mice were lower when the mice were pretreated with CL 184,005. These observations suggest that CL 184,005 may be potentially useful in the treatment of gram-negative bacterial sepsis, and the agent is undergoing clinical evaluation.

Fenbufen and biphenylacetic acid inhibit platelet function and the arachidonate prostaglandin system

Abstract Biphenylacetic acid or BPAA ([1,1-biphenyl]-4-acetic acid) is a metabolite of the non-steroidal anti-inflammatory drug, fenbufen (γ-oxo[1,1-biphenyl]-4-butanoic acid). A 0.12 mM concentration of BPAA produces almost complete inhibition of arachidonate-induced aggregation in vitro comparable to the effect produced by similar concentration of other non-steroidal anti-inflammatory agents. Fenbufen does not inhibit arachidonate-induced aggregation in vitro . These data correlate with the ability of BPAA, but not fenbufen, to inhibit platelet prostaglandin synthetase in vitro . Respiratory distress and histologic evidence of platelet aggregates formed in the pulmonary microvasculature following arachidonate infusion are markedly inhibited by oral doses of both fenbufen and BPAA. The order of efficacy (at 10 mg/kg) for inhibition of respiratory distress is BPAA > fenbufen > aspirin. These data confirm other studies indicating metabolic conversion of fenbufen in vivo to the active metabolite, BPAA. Collagen-induced aggregation is reduced by in vitro addition of either BPAA or fenbufen and in platelet-rich plasma from rats receiving oral doses of these compounds. BPAA added in vitro is more potent than either aspirin or fenbufen as an inhibitor of collagen-induced aggregation. Following in vivo dosing at 10 mg/kg, BPAA is comparable to aspirin in efficacy as an inhibitor of collagen-induced aggregation, while the same dose of fenbufen is less effective. The ability of BPAA to inhibit platelet aggregation appears to be related to intererence with the arachidonate-thromboxane-prostaglandin system. Fenbufens inhibitory action may be dependent on conversion to BPAA or involve another mechanism. Other data suggest, however, that this mechanism is not related to inhibition of serotonin release or inhibition of phosphodiesterase activity in platelets.

Effect of a series of 1-alkyl ether lipids on inhibition of phospholipase A2 activity and PAF responses.

Several 1-alkyl ether lipids were studied for their ability to inhibit PLA2 and antagonize PAF responses. Studies with synthetic micellar substrate (1-stearyl-2-arachidonyl phosphocholine), at concentrations ranging from 0.02 to 1000μM, demonstrate that CL 118326 inhibits porcine pancreatic PLA2 in vitro. As the substrate concentration increases, there is a dose-dependent increase in the IC50 value (IC50 ranges: 1.6–84.6μg/ml or 2.6–137μM). CL 118326 inhibits mammalian pancreatic PLA2, but not snake or bee venom PLA2. CL 118326 inhibits thrombin (IC50 =7.9μM), but not Na arachidonate- (IC50 > 100μM) induced platelet aggregation, indicative of inhibition of cellular PLA2. CL 118326 inhibits other PLA2-dependent processes such as antigen-induced leukotriene (LTC4) release (IC50=2.3μg/ml or 3.8μM) and histamine release (IC50=1.4μg/ml or 2.2μM) in basophil-enriched WBCs. Intradermal coinjection of CL 118326 (10μg) with PLA2 into guinea pig skin inhibits pancreatic PLA2-induced increase in vascular permeability and leakage, but not snake or bee venom PLA2-induced leakage. CL 118326 shows no PAF-like agonist activity in stimulating rabbit platelet-rich plasma. It inhibits PAF-induced aggregation (IC50=5.8μM), but not ADP-induced aggregation. CL 118326 has greater efficacy as a PLA2 inhibitor than as a PAF antagonist since the IC50-substrate concentration ratio for PLA2 inhibition is <- 1.0 at substrate concentrations of 10–1000μM while the IC50-agonist ratio for PAF antagonism is > 100. Results for four other compounds related to CL 118326 are also presented.

A computerized multichannel platelet aggregometer system

Commercially available instrumentation for conducting platelet aggregation studies in clinical and research laboratories consists of one-, two-, or four-channel aggregometers used in conjunction with strip chart recorders. These instruments have limited utility in large-scale drug screening and evaluation of the mode of action of drugs or in the clinical diagnosis of platelet disorders. A new instrument, a computerized multichannel aggregometer system (CMPAS) has been developed to collect, display, and analyze platelet aggregation data. The system is comprised of a 24-channel Born-type aggregometer, interfaced to a Rockwell AIM-65 microcomputer through an analogue-to-digital converter and an Epson dot-matrix printer. Each channel is individually calibrated, and aggregation data can be collected on up to 24 different platelet-rich plasma samples simultaneously. Conversational programs written in BASIC prompt the user for the addition of agonists and inhibitors. The tracings for each channel are displayed simultaneously, and a program automatically analyzes the data to generate the following parameters: baseline optical density, maximum aggregation response, positive and negative slopes, time to peak aggregation, and percentage response. Computerized multichannel aggregometer system data outputs are comparable to data generated by a standard Chronolog aggregometer unit. The advantages of the system include multichannel capability, simultaneous display of all channels allowing relative comparisons between control and experimental groups, and time savings and improved efficiency in conducting and analyzing aggregation experiments.

Automated platelet aggregation analysis using a digitizer.

A method of rapidly entering, reducing, and interpreting data collected in platelet aggregation studies has been developed. The standard aggregometer output is a chart recording of light transmittance (or optical density) as a function of time following the addition of an aggregating agent to a cuvette containing platelet-rich plasma or washed, suspended platelets. Two problems associated with aggregation studies are the proper calibration of the aggregometer and recorder to insure that comparisons of data can be made from experiment to experiment and the need to find a convenient way to analyze and summarize the data generated. In this method, the chart recorder is calibrated using reference cuvettes containing water or a suspension of latex beads of a known optical density. Since the analysis and interpretation of aggregation curves can be a time-consuming task, a standard digitizer has been interfaced to a computer, allowing the X,Y coordinates of the data, and, thus, the time-aggregation history of the sample, to be entered into the computer. The cursor of the digitizer is traced over the aggregation curve and the X,Y coordinates are transferred either at operator-selectable points or at fixed time intervals. A computer program (AGGPAD) calculates and stores several variables (e.g., sample baseline density, the magnitude of the aggregation, time to peak aggregation, maximum aggregation rate, and maximum deaggregation rate) that can be easily retrieved. The system reduces analysis time by a factor of five and allows for automated data storage and retrieval. The method is applicable to any computer and hardware costs are below