Molecular cloning and sequence of a novel ommochrome-binding protein cDNA from an insect, Manduca sexta.

An ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta has recently been purified and characterized. A cDNA clone was isolated from a fifth instar larval cDNA expression library utilizing antiserum against OBP. Northern blot analysis of total fat body RNA detected a transcript of approximately 1.2 kilobases in fifth instar wandering larvae RNA. The complete nucleotide sequence of the 905-base pair cDNA insert was determined by the dideoxy chain termination method. The OBP cDNA encodes a polypeptide of 274 residues with a predicted molecular weight of 30,580 and with one consensus N-linked glycosylation site. Comparison of the NH2-terminal sequence of the mature protein and the cDNA sequence revealed a typical signal peptide of 18 amino acids. In wandering stage larvae, the OBP transcript appeared to be at least 250-fold less abundant than ribosomal RNA.

Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Manduca sexta.

A yellow-colored protein (YCP) was isolated from the hemolymph (i.e. blood) of fifth instar wandering stage larvae of Manduca sexta. The molecular mass of YCP was 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography suggested that native YCP was a monomer. The absorbance spectrum of YCP revealed maxima at 278 and 405 nm. Chromophore was released from YCP through denaturation of the protein with methanol and chloroform. In neutral solution and in acid, the released chromophore showed the absorbance characteristics of an ommochrome: ommatin D. In addition, the chromophore was sensitive to treatment with arylsulfatase as would be expected for ommatin D. The amino acid composition and the N-terminal sequence of YCP were determined. The YCP polypeptide chain was found to be glycosylated. Carbohydrate analysis suggested that Man and GlcNAc were present in a 3:1 ratio. Circular dichroism indicated that YCP consisted of 68% beta-pleated sheet with no alpha-helices being detected. An in vitro incubation of larval fat body in the presence of [35S]methionine indicated that this organ was the site of synthesis. Ommochromes arise in insects as end products of the metabolism of tryptophan. It is well-documented that ommochromes occur in both the tissues and the excreta of insects. We propose that in M. sexta, one such tryptophan metabolite is found in the hemolymph associated with a specific protein.

Comparative effects of long term treatment with etodolac, naproxen and ibuprofen on articular and bone changes associated with adjuvant arthritis in rats.

The effect of prolonged treatment with etodolac (8 mg/kg) on the articular and bone pathology at the tibiotarsal joint associated with adjuvant arthritis in the rat has been studied and compared to the effects produced by treatment with naproxen (8 mg/kg) and ibuprofen (50 mg/kg). Drug effects were assessed by radiologic and histopathologic examinations. The effects on hindpaw edema, hindleg function, and body weight gain were also evaluated. Treatment was initiated on day 16 after adjuvant injection and continued for 28, 56 or 84 days. The degree of relapse which occurred after a 28 day period without treatment following 28 or 56 days of treatment was also assessed. Etodolac prevented the progression of the disease. Further, it appeared to diminish the incidence and severity of the lesions already present on day 16 before drug treatment began. All the parameters measured were improved and there was good agreement between the radiologic and histopathologic assessments of joint damage. At the doses used, the onset of drug activity was more rapid with etodolac than with either of the other two drugs. By comparison naproxen and ibuprofen inhibited the progression of joint damage, but neither drug consistently decreased the magnitude of the joint damage below that of day 16. With all three drugs there was less resurgence of disease symptoms when treatment was stopped after 56 days rather than 28 days of drug administration.

Effect of etodolac on articular and bone pathology associated with adjuvant arthritis in rats: a comparison with aspirin and naproxen.

The effect of treatment with the new anti-inflammatory drug etodolac on the articular and bone pathology associated with adjuvant arthritis in rats has been compared to the effects produced by aspirin and naproxen. Five measures of drug effect were made (changes in hind paw edema, body weight, normal hind leg function and articular damage as assessed by radiologic and histopathologic techniques). Drug treatment was initiated 16 days after adjuvant injection when arthritis was already established and continued for either 14 or 28 days. Etodolac produced a dose-related inhibition of all arthritic changes. Results from the radiologic study indicated that etodolac not only prevented the further development of articular damage by arthritis but actually caused a regression of these lesions established before drug treatment began. Similar results were obtained from the histopathologic study. Naproxen prevented the further development of arthritic damage but aspirin, although it decreased hind paw edema and increased body weight gains, had no significant effect on the articular damage produced by arthritis.

The effect of the non-steroidal anti-inflammatory drug Etodolac on macrophage migration in vitro and in vivo.

The possible mode of action of Etodolac, a potent anti-inflammatory drug, has been investigated. The effect of Etodolac on macrophage inflammatory depressed the influx of inflammatory macrophages into peritoneal cavity following stimulation with a sterile irritant. This decrease in macrophage accumulation in vivo correlated with the effect of Etodolac on the macrophage chemotaxis in vitro. Etodolac was also capable of reducing the macrophage ability to migrate towards a chemoattractant. In vivo Etodolac should reduce the amount of damage produced at the site of chronic inflammation since fewer macrophages would migrate to the inflammatory sites.

Resolution of etodolac and antiinflammatory and prostaglandin synthetase inhibiting properties of the enantiomers.

Etodolac, 1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetic acid, a clinically effective analgesic and antiinflammatory agent, has been resolved via a chromatographic separation of its diastereoisomeric esters with (-)-borneol. The effects of the enantiomers were studied in vitro on prostaglandin synthetase and on adjuvant-induced arthritis in rats. The biochemical and pharmacological results show that virtually all of the effects of etodolac are due to the (+) enantiomer.

Brain biogenic amines and pituitary-adrenocortical function in the rat.

Treatment of rats with various doses of the monoamine oxidase inhibitor (MAOI) pargyline failed to alter plasma levels of corticosterone at 18 hours post-dosage even though brain levels of serotonin and norepinephrine were increased by 51 to 95 percent. Single pargyline doses of 17.5 or 25 mg/kg blocked the increased plasma corticosterone response to reserpine. Animals pretreated with two doses of the MAOI showed a time-dependent sedative response to reserpine. In these animals, the plasma corticosterone response to reserpine was blocked, while the responses to cold exposure or chlorpromazine were unaffected.

Comparison in rats of the anti-inflammatory and gastric irritant effects of etodolac with several clinically effective anti-inflammatory drugs.

The anti-inflammatory and gastric effects of etodolac were compared in the rat with those of seven clinically established nonsteroidal anti-inflammatory drugs. The anti-inflammatory potency of etodolac was found to lie between that of sulindac and piroxicam. Etodolac was 2.8 times more active than sulindac and 2.2 times less active than piroxicam. Compared to phenylbutazone it was 12.5 times more potent. The irritation produced on the gastric mucosa was less than that of sulindac, although this difference did not reach statistical significance. However, etodolac was significantly (p less than 0.05) less irritant than piroxicam. Of the drugs studied, etodolac showed the highest ratio between the irritant ED50 and the dose which inhibited inflammation by 50%. From these results etodolac is predicted to be a potent anti-inflammatory drug with a high gastric tolerance. Clinical trials appear to confirm these predictions.

Synthesis and antiallergic properties of some 4H,5H-pyrano[3,2-c][1]benzopyran-4-one, 4H,5H-[1]benzothiopyrano[4,3-b]pyran-4-one, and 1,4-dihydro-5h-[1]benzothiopyrano[4,3-b]pyridin-4-one derivatives.

A series of novel 2-carboxylic acids of the title ring systems has been synthesized from the corresponding 3-acetyl-4H-[1]benzopyran-4-one and benzothiopyran-4-one. These acids were examined for their ability to inhibit the rat passive cutaneous anaphylaxis; the pyridinone carboxylic acids 6 displayed a higher degree of iv and ip anaphylactic activities than their pyranone analogues 5. The potassium salt 5a (R6 = K) was the only compound that exhibited a moderate oral activity.

Inhibition of passive lung anaphylaxis in the rat by AY-25,674.

The antiallergic properties of AY-25,674 were studied in a model of passive lung anaphylaxis. AY-25,674 caused a dose-related inhibition of antigen-induced bronchoconstriction in the passively sensitized rat. AY-25,674 was found to be twice as potent as disodium cromoglycate (DSCG) when injected intravenously. The oral administration of AY-25,674 also effectively inhibited antigen-induced bronchoconstriction. A tachyphylactic effect of repeated administration was found when the rats were pretreated with a large dose of AY-25,674. The compound did not significantly antagonize the bronchoconstriction produced by 5-hydroxytryptamine. These results indicate that AY-25,674 is an orally effective antianaphylactic compound with a mode of action similar to that of DSCG in preventing allergic mediator release.

Effect of ring substitution on the metabolic fate and anti-inflammatory activity of some prodolic acid analogs.

Prodolic acid, 1-n-propyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetic acid, exhibits potent anti-inflammatory activity in adjuvant arthritic rats. The potency of prodolic acid is enhanced by indole ring substitution. This increase correlated well with higher and sustained drug concentrations in the serum of normal animals. Pharmacokinetic studies demonstrated that ring substitution prolonged the serum half-life without affecting the absorption or volume of distribution. Because, in the rat, indole ring hydroxylation is a major pathway for the disposition of prodolic acid, we ascribe the increased pharmacological activity of ring substituted derivatives to the interference of substituents with the hydroxylation reaction.

Inhibition of the immune response by rapamycin, a new antifungal antibiotic.

Rapamycin, a new antifungal antibiotic, was found to inhibit the immune response in rats. It totally prevented the development of two experimental immunopathies (experimental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA)) and the formation of humoral (IgE-like) antibody. It was about half as potent as cyclophosphamide in inhibiting EAE. In AA and on antibody formation, rapamycin and cyclophosphamide were about equipotent, whereas methotrexate was more potent. The immunosuppressant activity of rapamycin appears to be related to inhibition of the lymphatic system.

Reagin-mediated hypersensitivity reaction in the rat hindpaw and its inhibition by antianaphylactic drugs.

Immediate hypersensitivity reactions are usually studied locally by the passive cutaneous anaphylaxis method. In the present experiments reaginic antibody-induced hypersensitivity was produced in the rat hindpaw (passive paw anaphylaxis or PPA). Increased vascular permeability was determined by measuring the increase in paw volume. The reaction in the paw and its inhibition with antianaphylactic drugs was found to be similar to that in the skin. The PPA is fast and easy to perform and can be used to study hypersensitivity reactions in the rat.

Anti-inflammatory and analgesic properties of etodolic acid in rats.

Etodolic acid (1, 8-diethyl-1, 3, 4, 9-tetrahydropyrano(3, 4-b) indole-1-acetic acid) showed potent anti-inflammatory and analgesic properties in rats. In adjuvant arthritic rats etodolic acid was approximately six times more potent than phenylbutazone. In the carrageenin and the analgesic assays (inflamed paw pressure test) its potency was comparable to that of phenylbutazone. The potency of etodolic acid in the adjuvant arthritic rat suggests that this novel anti-inflammatory compound will be effective in the treatment of arthritic conditions of man.

Etodolic acid and related compounds. Chemistry and antiinflammatory actions of some potent di- and trisubstituted 1, 3, 4, 9-tetrahydropyrano[3, 4-b]indole-1-acetic acids.

A series of 37 1-ethyl- and 1-n-propyl-1, 3, 4, 9-tetrahydropyrano[3, 4-b]indole-1-acetic acids bearing one, or two, substituents on the benzene ring has been synthesized via the acid-catalyzed condensation of a substituted tryptophol with ethyl propionylacetate or ethyl butyrylacetate. Antiinflammatory and ulcerogenic effects were examined and the results show that 1, 8-diethyl-1, 3, 4, 9-tetrahydropyrano[3, 4-b]indole-1-acetic acid (etodolic acid, USAN) is a potent agent, particularly active against a chronic rat model of inflammation (ED50 0.7 + 1-0.1 mg/kg po in the adjuvant arthritis model) and which has a relatively low acute ulcerogenic potential in the same species.

Chemistry and antiinflammatory activities of prodolic-acid and related 1,3,4,9-tetrahydropyrano[3,4-b]indole-1-alkanoic acids. 1.

The synthesis and antiinflammatory activities of a series of 23 novel 1,3,4,9-tetrahydropyrano[3,4-b]indole-1-alkanoic acids are described and some relationships between structure and activity are discussed. One of these compounds, 1,3,4,9-tetrahydro-1-propylpyrano[3,4-b]indole-1-acetic acid (prodolic acid, USAN), has been selected for further studies.