Stereo-dependent inhibition of human platelet function by the optical isomers of trimetoquinol

The stereoisomers of trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6–7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ] were shown to have potent and selective inhibitory effects on human platelet function in vitro. the R(+)-isomer was 12.1-, 12.3-, 39.2-, 82.9- and 36.0-fold more effective than the S(?)-isomer as an inhibitor of aggregation induced by arachidonic acid (AA), collagen, the epoxymethano-PGH2 analogs U44069 and U46619, and thromboxane A; (TxA₂) respectively. the concentrations of the R(+)-isomer that produced 50 percent inhibition (IC₅₀) of platelet aggregation were 4.2, 4.3, 1.4, 0.14 and 0.64 μM using AA, collagen, U44069, U46619, and TxA₂₀ as respective inducers. the graphical approximation of an inhibitory Constant (K_i = 0.13 μM) for the effect of TMQ on U46619-induced aggregation suggested that a competitive-like inhibition was operative. In other experiments, platelet aggregation and serotonin release induced by U46619 were inhibited differentially by the TMQ stereoisomers with nearly identical concentration-response curves. In addition, racemicTMQ blocked the secondary phase of platelet aggregation and serotonin release induced by ADP. These data, together with the ability of the TMQ stereoisomers to selectively inhibit TxA₂-induced aggregation, suggest that TMQ is an inhibitor of endoperoxide or TxA₂ action, e.g. a thromboxane A₂ receptor antagonist.     

Potentiating effects of clofibrate on prostaglandin-dependent and -independent pathways of human platelet activation: evidence for involvement of cyclic AMP

Although clofibrate has been shown to inhibit platelet aggregation that is caused by thrombin, ADP and epinephrine, by blocking the release of arachidonic acid from platelet phospholipids [8], here we have demonstrated that clofibrate enhanced platelet aggregation by arachidonic acid and PLC and reversed the effects of PGE1 on platelet cAMP concentration and on PLC-induced secretion of [14C]-5HT in similar, concentration-dependent manners. Taken together, these findings strongly suggest that the proaggregatory effect of clofibrate is mediated by a lowering of cAMP in platelets.     

Effects of methylphenidate on rat endurance performance and neuromuscular transmission in vitro

The studies were designed to evaluate the effects of methylphenidate on endurance performance in vivo and on neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. Methylphenidate produced a biphasic effect on treadmill endurance performance, increasing running times by 41-61% at 2.5-5 mg/kg, while reducing running times by 35% at 20 mg/kg. A biphasic effect on nerve-stimulated muscle concentrations was also observed, with twitch tension increased by up to 49-106% at low concentrations (0.1-0.3 mM) and blocked at high concentrations (0.6-1.0 mM). Tissues obtained from rats pretreated with alpha-methyl-p-tyrosine or reserpine exhibited no change in twitch height. Methylphenidate failed to protect against irreversible blocking of the twitch by alpha-bungarotoxin and did not modify the resting membrane potential, miniature endplate potential (MEPP) frequency or nerve-stimulated acetylcholine release. High concentrations reduced the amplitudes of the MEPP and endplate potential. Whereas methylphenidate and amphetamine both produced biphasic effects on skeletal muscle contractions in vitro, they act by different neuropharmacological mechanisms. Unlike amphetamine, the biphasic effects of methylphenidate are produced by mechanisms that are independent of cholinergic or adrenergic interactions and may involve direct effects on the muscle.     

Disposition of clofibrate in the rat: Acute and chronic administration

The disposition of 1-[¹⁴C]clofibrate (0.4 $\text{mmole}\text{kg}$) was studied in rats after acute (single dose) and chronic (b.i.d., for 14 days) administration. With a single dose (orally or by intraperitoneal injection) of clofibrate, most (~90 per cent) of the ¹⁴C-dose appeared in the urine within 24 hr and the recovery of ¹⁴C from the urine and feces was nearly quantitative within 72 hr. Little fecal excretion of ¹⁴C (< 5 per cent) occurred after a single or chronic clofibrate administration. Clofibrate was readily absorbed and eliminated, as evidenced by a rapid increase in plasma ¹⁴C level within 90 min and a calculated biological half-life of 4.1 hr. The pharmacokinetic profile of ¹⁴C-elimination in rats was unaffected by pretreatment with cholestyramine. Clofibric acid [2-(4-chlorophenoxy)-2-methylpropionic acid] was identified as the major metabolite in plasma (~97 per cent) whereas the glucuronide of clofibric acid was the main urinary and biliary metabolite (~96 per cent). Clofibric acid, as the free acid and glucuronide form, accounted for 99 per cent of the total ¹⁴C-dose in rats, and unchanged clofibrate was not detected in any of the biological samples. Two unidentified, minor urinary metabolites were also detected. In cannulated bile duct studies, it was found that [¹⁴C]clofibrate, as clofibric acid, was rapidly and efficiently excreted in the bile. The biliary excretion rates of ¹⁴C and of the glucuronide of clofibric acid were also not altered by phenobarbital pretreatment. Chronic treatment with [¹⁴C]clofibrate did not alter the qualitative or quantitative nature of biotransformation in vivo. An increased rate of urinary ¹⁴C-elimination was observed following chronic 1-[¹⁴C]clofibrate treatment, with concomitant reductions in blood and heart ¹⁴C-content and an elevation in ¹⁴C-content of epididymal fat tissue. Subcellular fractionation of liver, from rats given [¹⁴C]clofibrate chronically, indicated an increased distribution of ¹⁴C into mitochondria and peroxisomes. Tissue ¹⁴C-levels, achieved in these in vivo studies, were an order of magnitude lower than those required for the pharmacological activities of clofibrate and clofibric acid in vitro.     

The effect of penicillamine on vitamin B6 function in man

The erythrocyte alanine aminotransferase (Ala-AT) activity, measured in vitro, of rheumatoid arthritis patients under treatment with penicillamine was markedly stimulated by the addition of excess pyridoxal-5′-phosphate (PLP). This indicates that penicillamine can produce a deficiency of vitamin B₆, possibly by reacting chemically with the coenzyme PLP or by inhibiting PLP synthesis. The deficiency, though demonstrable by biochemical tests, was not accompanied by clinical signs of vitamin B₆ lack, and it would not justify the administration of a pyridoxine supplement to penicillamine-treated patients except in those of low nutritional status. In addition to its effect on the coenzyme PLP penicillamine also decreased the concentration of the erythrocyte Ala-AT apoenzyme.     

Effect of three calcium antagonists on platelet secretion and metabolism

Three compounds, 8-(N,N-diethylamino-octyl 3,4,5-trimethoxybenzoate, HCl (TMB-8), 2-propyl-3-dimethylamino-5,6-methylenedioxyindene HCl (2-PIA), and chlortetracycline, were investigated to determine whether their effects on washed human platelets were compatible with a suggested role as calcium antagonists. TMB-8 had little effect on levels of metabolic ATP and IMP, whereas chlortetracycline caused a decrease in metabolic ATP and an increase in IMP; both compounds inhibited thrombin-induced secretion and changed the platelets to spheres. At concentrations up to 0.5 mM, 2-PIA caused a decrease in ATP and an increase in IMP, an induction of secretion, and a centralization of electron-dense material in the platelets, all changes which suggest induction of secretion. The ultrastructural, functional and metabolic effects of TMB-8 and the type of interference by the drug with the effects of thrombin and 2-PIA on the same variables suggest that TMB-8 is mainly a membrane-active drug. Chlortetracycline on the other hand caused changes in platelet metabolism, ultrastructure, and function which, at least partly, indicate an effect on intracellular mechanisms. Both TMB-8 and 2-PIA, and to lesser degree chlortetracycline, caused loss of cytoplasmic nucleotides from the platelets.     

Statistical techniques applied to solubility predictions and pharmaceutical formulations: an approach to problem solving using mixture response surface methodology

Mixture response surface methodology is a group of statistical methods which can generate an empirical equation that can be used to quantitatively define the relationship between some response, such as solubility, and the composition of a system, as for example, different solvent blends. The equation can be used to predict response at any proposed or desired mixture. The term response should be viewed in a very generalized sense to include any property that is affected solely by mixture composition and might include many measurable responses that are of interest to pharmacists: cost, half-life, taste, color, tablet hardness, bioavailability, extraction efficiency, chromatographic resolution, and so on. The method is in no way limited by the number of components in the mixture and thus should be viewed as being general in this sense also. The advantages of mixture response surface methodology and the mechanics involved in its use are illustrated through a prediction of the solubility of diazepam and phenobarbital in solvent blends. The approach is entirely empirical. It is based on rigorous statistical design and data analysis and can lead to excellent prediction of solubility. It also is shown how several responses can be     

Action of phospholipase A2 on bilayers containing lysophosphatidylcholine analogs and the effect of inhibitors

The effects of several lysophospholipid analogs on the phase properties of codispersions with diacylphosphatidylcholine with or without fatty acids were examined. These ternary codispersions were readily hydrolyzed by phospholipase A2, and they underwent a rapid change in turbidity. Nonideal mixing or phase separation in the ternary codispersions is postulated to be responsible for their enhanced susceptibility to pig pancreatic phospholipase A2, as well as for their tendency to undergo spontaneous change in turbidity, presumably due to spontaneous fusion of the vesicles. Both of these processes were inhibited by a variety of structurally unrelated solutes like n-hexanol and mepacrine. These and other observations are interpreted to suggest that structural defects in bilayers of ternary codispersions are a common locus for the binding of phospholipase A2 and are responsible for the process underlying the change in turbidity. The experiments described here suggest that many of the common inhibitors of phospholipase A2 owe their effects to their ability to modify the quality of the substrate interface, rather than to a direct interaction with the enzyme.     

Pharmacological modulation of chemotactic factor-elicited release of granule-associated enzymes from human neutrophils: Effects of prostaglandins,nonsteroid anti-inflammatory agents and corticosteroids

Human neutrophils demonstrated a selective release of granule-associated β-glucuronidase and lysozyme but not of cytoplasmic lactate dehydrogenase during cell contact with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Enzyme discharge was dependent upon treatment of neutrophils with cytochalasin B prior to exposure to FMLP. Prostaglandins (PG) D₂, E₂ and I₂ inhibited enzyme release from cytochalasin B-treated neutrophils incubated with FMLP in phosphate buffered saline, pH 7.4, at 37°. Flurbiprofen, ibuprofen, indomethacin, ketoprofen and benoxaprofen reduced the extrusion of β-glucuronidase and lysozyme from FMLP-stimulated neutrophils; acetylsalicylic acid was inactive. Methylprednisolone sodium succinate, hydrocortisone sodium succinate, prednisolone sodium succinate and triamcinolone acetonide hemisuccinate also demonstrated the capacity to inhibit the selective release of granule-associated enzymes from human neutrophils. Aldosterone hemisuccinate and deoxycorticosterone hemisuccinate were inactive. These studies indicate that certain pharmacological and therapeutic agents may function to alleviate various inflammatory conditions by curtailing the extrusion of degradative enzymes from neutrophils.     

C(2′)-substituted purine nucleoside analogs: Interactions with adenosine deaminase and purine nucleoside phosphorylase and formation of analog nucleotides

Four C(2′)-substituted 2′-deoxyadenosines were examined as substrates for human erythrocytic adenosine deaminase and for formation of intracellular nucleotide analogs in human erythrocytes, lymphocytes and murine Sarcoma 180 cells: 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)adenine, 9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine, 9-(2′-azido-2′-deoxy-β-D-ribofuranosyl)adenine (2′-N₃-riboA) and 9-(2′-azido-2′-deoxy-β-D-arabinofuranosyl)adenine. All four adenosine analogs were substrates of human erythrocytic adenosine deaminase, but the corresponding inosine analogs (synthesized by the adenosine deaminase reaction) were highly resistant to cleavage by human erythrocytic purine nucleoside phosphorylase. Only 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 μM concentration of any studied inosine analog was added to a reaction mixture containing 30 μM inosine (the K_m concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2′-N₃-riboA (K_i=2μM), but the reaction velocity was highest with the F-substituted analogs. All four adenosine analogs formed triphosphate nucleotides after incubation with human erythrocytes, murine Sarcoma 180 cells, or human lymphocytes (tested only with the F analogs) in the presence of deoxycoformycin.     

Effects of hypoxia on atrial muscarinic cholinergic receptors and cardiac parasympathetic responsiveness

Chronic exposure of rats to hypoxia resulted in a lower resting heart rate and a supranormal increase in heart rate in response to parasympathetic blockade by atropine. The density of muscarinic cholinergic receptors labeled by the antagonist [³H]quinuclidinyl benzilate was elevated significantly in the atria of animals kept hypoxic for 2–4 weeks. Chronic hypoxia did not change the affinity of the receptor for [³H]quinuclidinyl benzilate, the weight of the atria, or the amount of protein per atrial pair. Thus, the decrease in resting heart rate may be explained by the increase in the density of atrial muscarinic cholinergic receptors.     

Nonenzymatic glucosylation of human serum albumin and its influence on binding capacity of sulfonylureas

To estimate the functional change occurring in human serum albumin by nonenzymatic glucosylation, glucosylated human serum albumin was prepared by in vitro incubation with glucose. The rate of glucosylation proceeded as a first-order reaction. The binding of sulfonylureas to serum albumin was determined by equilibrium gel filtration. Through this method, it was possible to estimate the binding capacity of a low water solubility drug with a high affinity to protein. The amounts of the sulfonylureas bound to glucosylated HSA decreased by 44% with tolazamide and acetohexamide, 50% with glibenclamide, and 52% with tolbutamide, compared to human serum albumin (HSA). This suggests that a high concentration of glucosylated HSA in diabetic patients may possibly cause an increase in free drug concentration exceeding normal levels. This study shows that the decrease in the binding capacity of sulfonylureas with protein is due to the modification of albumin molecules by the covalent binding of glucose.     

Binding of [3H]quinuclidinyl benzilate to intestinal mucus: An artifact in identification of epithelial cell muscarinic receptors

The widely used muscarinic receptor ligand [³H]quinuclidinyl benzilate ([³H]QNB) was found to bind in a site-specific but artifactual manner to rat intestinal mucus, obscuring specific binding to muscarinic receptors on intestinal epithelial cells. Atropine inhibited [³H]QNB binding to mucus with an apparent IC₅₀ of 2.1 × 10^?7 M, compared to an IC₅₀ of 1.4 × 10^?8 M obtained with a homogenate of intestinal epithelial cells. Unlabeled QNB also inhibited binding of [³H]QNB to mucus but the apparent IC₅₀(4 × 10^?7 M) was about 300-fold greater than the IC₅₀ determined with a control tissue, heart muscle (IC₅₀, 1.2 × 10^?9M). [³H]QNB binding was saturable over the concentration range of 1–7 nM in the heart, with an apparent k_D of 0.76 nM. As expected from the high IC₅₀ for QNB in the mucus binding experiments, binding to mucus was not saturable over the 1–15 nM concentration range. Based on pH profiles and temperature dependency of binding, it seems unlikely that mucin, the primary component of mucus, was responsible for [³H]QNB binding to the mucus. The findings have implications for studies which involve binding of [³H]QNB in particular and other ligands in general to mucus-secreting epithelial tissues.     

Influence of delta 9-tetrahydrocannabinol on expression of histone and ribosomal genes in normal and transformed human cells

The influence of delta 9-tetrahydrocannabinol (delta 9-THC) on the cellular levels of histone mRNAs and ribosomal RNAs was examined in several normal and transformed human cell lines--HeLa S3 cells, WI-38 human diploid fibroblasts, SV40-transformed WI-38 cells, and A549 lung carcinoma cells. RNA sequences were quantitatively assayed by electrophoretic fractionation, transfer to nitrocellulose, and hybridization with cloned genomic human histone or ribosomal DNA sequences. Treatment with delta 9-THC (10-40 microM) for 10 hr resulted in a concentration-dependent decrease in the representation of H2A, H2B, H3 and H4 histone mRNAs without a significant inhibitory effect on the levels of ribosomal RNAs. The cannabinoid-mediated inhibitory effect on histone gene expression was less evident in cells with active drug-metabolizing systems.     

Effects of several antimalarials and phenothiazine compounds on the formation of coat structure from clathrin

We have evaluated the effects of two phenothiazine and several antimalarial drugs on the rates of polymerization of 8S clathrin molecules to 300S coat structures. Most of the drugs investigated have been shown in other studies to inhibit receptor-mediated endocytosis through the coated pit regions of plasma membranes. The two types of drugs were found to accelerate the polymerization rate without having much effect on the size distribution of the polymer species. The activities of the drugs appear to depend on a dibasic moiety and a large, hydrophobic aromatic ring in their structures.     

Effects of lithium on the activities of phosphofructokinase and phosphoglucomutase and on glucose-1,6-diphosphate levels in rat muscles, brain and liver

Incubation of rat diaphragm muscle in the presence of lithium chloride (a drug used widely in the therapy of patients with mental illness), resulted in a sharp decrease in the level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism. This decrease in Glc-1,6-P2, the most potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a marked reduction in the activities of both enzymes, when assayed in the absence of exogenous Glc-1,6-P2 under conditions in which these enzymes are sensitive to regulation by endogenous Glc-1,6-P2. A decrease in Glc-1,6-P2 and the concomitant reduction in the activities of phosphofructokinase and phosphoglucomutase, were also obtained in the rat gastrocnemius and tibialis anterior muscles, as well as in brain, following Li+ injection. In contrast to its effects in muscles and brain, Li+ did not exert any effect on Glc-1,6-P2 level and on the enzymes' activities in the liver. The marked inhibition of brain and muscles phosphofructokinase (the rate-limiting enzyme in glycolysis) induced by Li+, may play an important role in the mechanism of the therapeutic action of this agent in the manic state.     

Effects of copper on the binding of agonists and antagonists to muscarinic receptors in rat brain

Studies were performed to assess the effects of copper treatment in vitro on muscarinic binding parameters in rat brain homogenates. Brainstem, an area low in copper, was found to be insensitive to copper treatment as compared to forebrain, a region of relatively high copper content. Inclusion of 3 microM copper in forebrain homogenates decreased the number of sites seen by [3H]-l-quinuclidinyl benzilate (QNB) by 40-50%. Copper-enhanced displacement of bound QNB was noted for agonists and antagonists. Both ligands showed maximal effects at 6 microM copper, although quantitative differences could be determined at any copper level. At levels of maximal effect, the increase in QNB displacement was greater than or less than 50% for agonists and antagonists respectively. Two-site analyses of carbamylcholine (CCH) binding showed that the addition of 1 microM copper to forebrain homogenates increased the percentage of high affinity sites (alpha) from 42 to 70%. The IC50 decreased from 3.1 to 1.7 microM, but the dissociation constants for the high and low affinity sites were not changed. The effect of added copper on CCH binding to muscarinic receptors was reversible with the addition of the copper-chelating agent triethylene tetramine.