3,4-Dihydroquinolin-2(1H)-ones as combined inhibitors of thromboxane A2 synthase and cAMP phosphodiesterase.

A series of 1H-imidazol-1-yl- and 3-pyridyl-substituted 3,4-dihydroquinolin-2(1H)-ones was designed and synthesized as combined inhibitors of thromboxane (TXA2) synthase and cAMP phosphodiesterase (PDE) in human blood platelets. A number of structures, e.g. 4b, 7a, 7e, 13a, and 21-25, were superior to dazoxiben 26 as inhibitors of TXA2 synthase in in vitro ADP-induced aggregation experiments with human blood platelets. The TXA2 synthase inhibitory activity was confirmed by measurement of the prostanoid metabolites derived from 14C-labeled arachidonic acid. Three compounds (7a, 7e, and 25) demonstrated in vitro inhibition of human platelet cAMP PDE at micromolar concentrations in conjunction with their TXA2 synthase inhibitory activity. Synergistic enhancement of antiaggregatory and antithrombotic actions was expected when simultaneous stimulation of adenylate cyclase (through increased PGI2 production) and inhibition of platelet cAMP PDE were possible from the same compound. Ex vivo and in vivo experiments were conducted in rats and mice, respectively, to evaluate the effects of compounds 7e and 23 on platelet aggregation and thrombotic events within these animals. Compound 7e, which has a comparable level of TXA2 synthase (IC50 1.2 microM) and human platelet cAMP PDE (IC50 6.4 microM) inhibitory activities, was found to be orally bioavailable with a long duration of action and offered effective protection against mortality in a collagen-epinephrine-induced pulmonary thromboembolism model in mice. Significant blood pressure and heart rate effects were observed for several compounds, e.g. 7e, 9e, 13a, 13d, 18, 20, 21, and 23, when dosed orally in conscious spontaneously hypertensive rats.

Immunoreactivity affects the biodistribution and tumor targeting of radiolabeled anti-P97 Fab fragment.

Hydroxylapatite high performance liquid chromatography was used to prepare two fractions from 125I- Fab 96.5. One fraction (peak 1) had relatively low immunoreactivity (25-38%) and the second fraction (peak 2) had high immunoreactivity (70-81%). Scatchard analysis showed similar affinity constants for the two preparations (2.9 x 10(9) M-1 for peak 1; 3.4 x 10(9) M-1 for peak 2). In biodistribution and imaging studies in athymic mice with human melanoma (FEMX-II) xenografts the high immunoreactivity preparation rapidly cleared from the blood and nontumor organs while retention of radioactivity in the tumor was prolonged. The low immunoreactivity preparation, had slower blood and nontumor organ clearance but faster tumor clearance than the high immunoreactivity fraction. Thus, in these studies highly immunoreactive antibody gave higher tumor to nontumor ratios and enhanced the target to nontarget image contrast.

Differential cellular catabolism of 111In, 90Y and 125I radiolabeled T101 anti-CD5 monoclonal antibody.

We evaluated the differences in catabolism of 125I, 111In and 90Y-T101 monoclonal antibody (anti-CD5) by peripheral blood mononuclear cells (PBMNC), HUT 102, CCRF-CEM and MOLT-4 cells. All cells showed higher retention of 111In than of 125I. PBMNC showed similar retention of 90Y and 111In. The release of 125I was reduced by using metabolic inhibitors, F(ab')2 of T101, pure lymphocytes or incubating at 4 degrees C. Our findings suggest differences in the intracellular catabolism of the radionuclides.

Synthesis and biological evaluation of omega-(N,N,N-trialkylammonium)alkyl esters and thioesters of carboxylic acid nonsteroidal antiinflammatory agents.

A series of novel omega-(N,N,N-trialkylammonium)alkyl ester and thioester derivatives [RCOM(CH2)nNR3+ X-, M = O or S. n = 2-6, X = I or Cl] of 11 nonsteroidal antiinflammatory carboxylic acid agents (naproxen, ketorolac, indomethacin, ibuprofen, sulindac, ketoprofen, flufenamic acid, mefenamic acid, zomepirac, etodolac, and tifurac) was prepared and evaluated for their antiinflammatory, analgesic, and gastrointestinal erosive properties. In general, each prodrug retained the antiinflammatory activity characteristic of the corresponding parent drug but exhibited moderately to greatly reduced gastrointestinal erosive properties and significantly reduced analgetic potencies. This profile is likely due to a combination of factors including the rate of hydrolysis of the esters in the stomach, gut, and plasma, changes in the locus of absorption of the prodrug or nonsteroidal antiinflammatory drug (NSAID), and altered metabolic disposition patterns resulting from these changes. The results obtained from the compounds of this series indicate that esters of this general class may offer a means to modulate both the aqueous/lipid solubility and the hydrolytic/enzymatic cleavage indices of NSAID prodrugs which potentially possess a more favorable therapeutic ratio of antiinflammatory to gastrointestinal erosive activities.

Reduction of background activities by introduction of a diester linkage between antibody and a chelate in radioimmunodetection of tumor.

A diester linkage was added between monoclonal anti-melanoma antibody 96.5 and a diethylenetriaminepentaacetic acid derivative to test if a tumor-to-blood and -to-organ ratio of the injected antibody in nude mice with human melanoma FEM XII xenografts could be increased by the addition of the readily cleavable linkage. Compared to the 111In-labeled antibody DTPA with a peptide linkage, the diester conjugate cleared much faster from the blood and was retained much less in muscle and normal organs such as liver, spleen and kidney over a 48-hr period. On the other hand, the activity retained in the tumor was larger than or similar to that of the peptide conjugate for this time period. This resulted in a 2.5, 2.1, and 2.6 fold increase in a tumor to blood, to liver and to kidney ratio at 48 hr for the diester conjugate as compared to the peptide conjugate. The whole-body biologic half life of the antibody was 36 hr, three times shorter than the peptide conjugate. The external imaging demonstrated a clearly visible tumor at 4 hr and a lower pool activity at 72 hr for the diester conjugate. The peptide conjugate, however, showed a persistant blood-pool activity at 72 hr. The addition of the diester linkage, therefore may be beneficial for imaging tumors in patients at early time intervals after injection.

In vitro complex formation and biodistribution of mouse antitumor monoclonal antibody in cancer patients.

The serum clearance and biodistribution of a murine monoclonal antibody were compared to the in vitro complex formation of the antibody with patients' sera. Iodine-125-labeled 9.2.27, an anti-melanoma antibody, was incubated with sera from ten melanoma patients who had received 9.2.27 in an earlier study. Complexes were observed in all patients using size exclusion high performance liquid chromatography and complex formation was partially blocked by nonspecific murine antibody, suggesting the presence of human anti-murine antibody in serum. All patients subsequently underwent imaging studies with [131I] 9.2.27 given intravenously. The serum levels of the antibody obtained after the second administration were inversely correlated with the level of in vitro complex formation. Patients whose serum formed high levels of complex showed a rapid serum clearance, high hepatic uptake, and accelerated whole body clearance and urinary excretion of 131I. This suggests that in patients who receive repetitive administration of murine antibody the serum clearance rate and biodistribution of intravenously injected antibody are altered by antibody complex formation in the serum.

[(1H-imidazol-1-yl)methyl]- and [(3-pyridinyl)methyl]pyrroles as thromboxane synthetase inhibitors.

Several [(1H-imidazol-1-yl)methyl]- and [(3-pyridinyl)methyl] pyrroles were prepared and evaluated in vitro as thromboxane synthetase inhibitors in human platelet aggregation studies. A number of structures, e.g. 10b,f,g,i (respective IC50 values: 1 microM, 50 nM, 42 nM, 44 nM) showed superior in vitro inhibition of TXA2 synthetase when compared to the standard dazoxiben (1). However, it was found that in vitro potency did not translate into nor correlate with in vivo activity when these compounds were evaluated in mice in a collagen-epinephrine-induced pulmonary thromboembolism model. (E)-1-Methyl-2-[(1H-imidazol-1-yl)methyl]-5-(2-carboxyprop-1-enyl) pyrrole (10b) was found to offer protection against collagen-epinephrine-induced mortality in mice, thereby demonstrating that oral administration is an effective route for absorption of this drug. Additional evidence for the oral effectiveness of 10b in lowering serum TXB2 levels was obtained by performing ex vivo radioimmunoassay experiments with rats. A 13-week study of 10b in rats with reduced renal mass was conducted in order to evaluate the role of TXA2 production in hypertension and renal dysfunction. Although serum and urinary TXB2 levels in rats were found to be lowered during this study by 10b, the levels of urinary protein excretion remained comparable to that of the control group.

Topical nonsteroidal antipsoriatic agents. 1. 1,2,3,4-Tetraoxygenated naphthalene derivatives.

On the basis of previous observations that both 2,3-dihydro-2,2,3,3-tetrahydroxy-1,4-naphthoquinone (oxoline, 1) and 6-chloroisonaphthazarin (2) had demonstrated antipsoriatic activity in vivo, a series of structural derivatives of 2 were prepared and examined in the Scholtz-Dumas topical psoriasis bioassay. Of these six (5, 6, 9a, 10, 11a, 11b), the most effective compound was found to be 6-chloro-1,4-diacetoxy-2,3-dimethoxynaphthalene (RS-43179, lonapalene, 11a). An extensive series of 1,2,3,4-tetraoxygenated naphthalenes (16-74) incorporating variations of the ester, ether, and aryl substituents were prepared as analogues of 11a to examine the structural requirements for activity and were screened in vivo as inhibitors of arachidonic acid induced mouse ear edema, a topical bioassay capable of detecting 5-lipoxygenase inhibitors. Net lipophilicity, hydrolytic stability, and ring substitution play significant roles in determining the observed in vivo activity. Lonapalene (11a) is currently in clinical development as a topically applied nonsteroidal antipsoriatic agent.

Pharmacological investigation of the mechanisms of platelet-activating factor induced mortality in the mouse.

Although the platelets of the mouse are refractory to the direct effects of platelet-activating-factor (PAF), tail vein injection of 10-150 micrograms/kg PAF produces lethal anaphylactic shock. Sensitivity varies with strain and source: Swiss Webster mice show a range of sensitivity and DBA/2 (complement C5-deficient) mice are very resistant. At lethal doses of PAF, animals show labored respiration and general depression; death occurs within 15-45 min. Dexamethasone administered at least 1.5 hr prior consistently protects, whereas the cyclooxygenase inhibitors do not. Antihistamines, adrenergic antagonists, and methysergide have no effect, but cyproheptadine is partially protective at near lethal doses. Calcium entry blockers and calcium chelators, tetracycline and chlortetracycline are partially protective at very high doses consistent with non-specific effects on calcium dependent processes. The arachidonic acid lipoxygenase inhibitors BW755c, phenidone, nordihydroguaiaretic acid and diphenyldisulfide provide nearly complete protection after oral administration of 50-200 mg/kg. Phosphodiesterase inhibitors and dapsone are also effective orally. The leukotriene antagonist FPL55712 administered intraperitoneally (10 mg/kg) 5 min. prior to PAF challenge provides almost complete protection. PAF-induced mortality in the mouse represents a small animal model of systemic anaphylaxis particularly useful for the systemic testing of arachidonic acid lipoxygenase inhibitors and leukotriene antagonists.

The analgesic and anti-inflammatory profile of ketorolac and its tromethamine salt.

Ketorolac tromethamine[(+/-)-5(benzoyl)-2,3-dihydro-1N-pyrrolizine-1-carboxylic acid tris hydroxymethylaminomethane salt] is a highly potent member of a new class of compounds having analgesic and anti-inflammatory activity. When given orally in tests involving underlying inflammation it was a potent analgesic, whereas it was inactive in tests for narcotic activity. It was also highly active orally in rat models of acute and chronic inflammation and pyresis. These properties are mediated primarily via the compound's potent prostaglandin cyclooxygenase inhibitory activity. The agent elicited mild CNS and cardiovascular activity only at doses far in excess of those required for analgesic and anti-inflammatory activity. A single 10 mg tablet given orally to human volunteers following surgery provided pain relief equivalent to that provided by 10 mg of morphine given intramuscularly. When given intramuscularly to rabbits (0.25 ml of a 0.31-5% solution) or man (3 ml of a 1-3% solution), no drug-related irritation or changes in creatine phosphokinase were seen. Solutions (less than or equal to 0.5%) applied to the eyes of animals and man were not irritating. When applied topically in rat and rabbit models of ocular inflammation, less than or equal to 0.5% solutions of ketorolac tromethamine inhibited the inflammatory response.

The analgesic and anti-inflammatory profile of (+/-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid (RS-37619).

RS-37619 showed highly potent analgesic activity when given p.o. in tests utilizing underlying inflammation. It inhibited phenylquinone-induced writhing in the mouse and rat (350 and 180 x aspirin respectively) and the pain induced by flexing the adjuvant-inflamed rat paw (approximately 800 x aspirin). The agent increased the pain threshold of compressed yeast-inflamed rat paws (3-10 x naproxen). RS-37619 did not increase the pain threshold of the non-inflamed paw and was inactive in the mouse hot plate test; therefore it is probably not a centrally acting or morphine-like agent. RS-37619 was also highly active p.o. in rat models of acute and chronic inflammation. It inhibited carrageenan-induced paw edema (36 x phenylbutazone), cotton pellet-induced granuloma (less than or equal to 1 x indomethacin) and in an 18-day test, prevented the development of adjuvant-induced arthritis (approximately 2 x naproxen). RS-37619 exhibited antiphlogistic activity in adrenalectomized rats. It did not have corticosteroid activity. When given p.o., RS-37619 lowered yeast-induced pyrexia (20 x aspirin). Gastrointestinal irritation was seen in the rat with doses greater than or equal to 6.4 mg/kg/day p.o. The agent elicited mild CNS and cardiovascular activity only at doses far in excess of those required for analgesic and anti-inflammatory activity.

Bistability at an electro-optic interface.

We report the first known demonstration of a novel bistable optical device that switches an interface between reflecting and transmitting states. Our results are in agreement with a simple graphical analysis.

Use of a liquid suspension of dielectric spheres as an artificial Kerr medium.

We present the derivation of scaling relationships that describe the behavior of liquid suspensions of dielectric spheres as nonlinear optical media and report results of four-wave mixing experiments that are in good agreement with these predictions.