Effect of the novel synthetic protease inhibitor furoyl saccharin on elastase-induced emphysema in rabbits and hamsters.

Furoyl saccharin, a novel heterocyclic acylating agent which has been previously found to possess a potent inhibitory capacity in vitro for elastase and other serine proteases, has been investigated in vivo in two acute animal models of emphysema. In hamsters, intratracheal (i.tr.) administration of 0.1 mg porcine pancreatic elastase resulted seven days later, in a 42% increase of the mean linear intercept (Lm). Addition of 0.3 mg to 0.3 mg furoyl saccharin to elastase exhibited a partial, not dose-related, but statistically significant inhibition of the increase of LM. Addition of 1 mg furoyl saccharin (equivalent to a dose of 12.5 mg/kg) completely abolished the increase in Lm. In the rabbit i.tr. instillation of 3.7 mg porcine pancreatic elastase induced within seven days, a 48% increase of the Lm, a 27% decrease of the internal surface area (ISA) of the lungs and a 33% decrease of the ISA corrected to an arbitrary total lung volume of 70 ml (ISA70). Furoyl saccharin given i.tr. 15 min prior to elastase at the doses 3, 10 and 20 mg prevented significantly in a dose-related manner, the changes in Lm, ISA and ISA70. The highest furoyl saccharin dose (equivalent to a dose of 10.8 mg/kg) completely protected against the emphysematous lesion. Additionally furoyl saccharin (20 mg i.tr.) prevented in the rabbit model the depletion in lung insoluble elastin and the increase in salt soluble collagen induced by the elastase administration. These results show that furoyl saccharin also in vivo has a marked antielastase activity.

The effect of furoyl saccharin, a novel non-peptidic acylating protease inhibitor, on experimental emphysema in the hamster.

Furoyl saccharin was evaluated for its ability to prevent the development of emphysematous lesions produced in hamsters by the exposure to aerosolized papain (3% for 3 h). Pretreatment with intratracheal furoyl saccharin (at the doses of 0.3, 1, 3 mg) reduced the appearance of papain-induced emphysema as evaluated by both physiologic (static compliance) and histologic (mean linear intercept and internal surface area of the lungs) methods. Inhibition was dose-related with maximal reduction of changes in static compliance (74%), mean linear intercept (84%) and internal surface area (65%) observed after a dose of 3 mg. This is the first time that a non-peptide acylating inhibitor of serine proteases is reported to be affective in preventing the development of experimental emphysema.

On the pharmacology of L-645,151: a topically effective ocular hypotensive carbonic anhydrase inhibitor.

L-645,151 [(2-sulfamoyl-6-benzothiazolyl)-2,2-dimethylpropionate] is the O-pivaloyl ester of L-643,799 (6-hydroxybenzothiazole-2-sulfonamide), topically administered L-645,151 being a substrate for ocular esterases with the resultant liberation of the active moiety, L-643,799, during penetration of the ocular surface. The minimum concentrations of topically administered suspensions (1 drop of 50 microliters into both eyes) of L-645,151, L-643,799, dichlorphenamide and methazolamide significantly lowering the elevated intraocular pressure (IOP) of the alpha-chymotrypsinized rabbit eye were 0.25, 2, 10 and 5%, respectively. IOP was not significantly decreased by 10% suspensions of acetazolamide or ethoxzolamide. The IOP lowering action of L-645,151 was local as the unilateral instillation of L-645,151 (0.25%) into the contralateral eye was devoid of effect in alpha-chymotrypsinized rabbits. L-645,151 (2%) decreased aqueous humor inflow in both the conscious rabbit and the anesthetized dog. Outflow facility in the conscious rabbit was unaltered by a 10% suspension of L-645,151. Low peak levels (0.52 microgram/g) of L-643,799 were present in rabbit renal cortex after the instillation of L-645,151 (2%) into both eyes; this treatment did not induce diuresis in the conscious rabbit. The corresponding maximum concentration in the iris + ciliary body was 4.01 micrograms/g. These preclinical studies reveal that L-645,151 is the most potent, topically effective ocular hypotensive carbonic anhydrase inhibitor described to date.

Cyclobenzaprine: effect on tonic vibration reflexes in local tetanus cat preparations.

Local tetanus was induced by the injection of toxin into the gastrocnemius-soleus muscle of cats. After 48 hr, longitudinal vibration of the muscle was used to elicit a reflex contraction (tonic vibration reflex, TVR). In other experiments, electrical stimulation of various regions of the central nervous system served to elicit contraction of the muscle alone or to facilitate a vibration (150 mu at 300 Hz)-induced tonic vibration reflex. In contrast with normal animals, tonic vibration reflex responses in local tetanus preparations of decerebrate cats were augmented after transection of the spinal cord at Cl. In decerebrate local tetanus preparations, a dose-related reduction of tonic vibration reflex responses, induced at all frequencies and amplitudes of muscle vibration, was observed after doses of 0.5 to 3.5 mg/kg (i.v.) of cyclobenzaprine. Muscle contractions induced by stimulation of the medial reticular formation and facilitation of tonic vibration reflex responses were more sensitive to the action of cyclobenzaprine than were similar responses activated through stimulation of Deiters' lateral vestibular nucleus. In spinal preparations, tonic vibration reflex responses were only moderately reduced after similar doses of cyclobenzaprine. Contractions induced by stimulation of the spinal cord (T6) and facilitated tonic vibration reflex responses were only moderately reduced, whereas the post-stimulus-induced facilitations were considerably attenuated. Thus, experiments in local tetanus preparations support further the concept that the major site of action of cyclobenzaprine is supraspinal, whereas its action upon spinal structures contributes to its overall skeletal muscle-relaxant activity.

Studies on L-640,035: a novel antagonist of contractile prostanoids in the lung.

The effects of L-640,035 (3-hydroxymethyl-dibenzo [b,f] thiepin-5,5-dioxide) have been studied on pulmonary smooth muscle contraction in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-640,035 produced significant shifts in the dose-response curves to a prostaglandin (PG) endoperoxide analogue (U-44069) (pA2 7.0), PGF2 alpha (pA2 5.9) and PGD2 (pA2 6.5). L-640,035 produced no significant shift in the dose-response curves to leukotriene D4 or histamine and produced a small but statistically significant shift in the dose-response curve to 5-hydroxytryptamine (5-HT) (pA2 5.2). With the exception of PGF2 alpha, Schild analysis did not in general indicate competitive inhibition. The main metabolite of L-640,035, L-636,499, also produced significant parallel shifts in the dose-response curves to U-44069 (pA2 6.0) and PGF2 alpha (pA2 6.0), but with some reduction in the maximal contraction. When L-640,035 was administered intravenously to guinea-pigs, significant inhibition of increases in pulmonary resistance or insufflation pressure induced by U-44069 (ED50 0.16 mg kg-1), leukotriene D4 (ED50 0.25 mg kg-1) and 5-HT (ED50 3.4 mg kg-1) but not histamine (ED50 greater than 10 mg kg-1) was observed. When L-640,035 was administered intravenously to dogs a significant inhibition of increases in pulmonary resistance induced by U-44069 (ED50 0.85 mg kg-1) but not histamine (ED50 greater than 30 mg kg-1) was observed. 5 When L-640,035 was administered by the intraduodenal route to dogs at doses of 3 and 10 mg kg- significant inhibition of increases in pulmonary resistance induced by sodium arachidonate (3 mgkg1 i.v.) was observed with a duration of action of > 255 min. 6 It is concluded that L-640,035 is a novel, relatively selective, and orally active antagonist of the actions of contractile prostanoids on pulmonary smooth muscle.

R-enantiomer of timolol: a potential selective ocular antihypertensive agent.

Various studies were conducted to evaluate the effects of timolol, an S-enantiomer, relative to its R-enantiomer upon intraocular pressure and related ocular systems in the rabbit. The R-enantiomer was about one-third as potent as timolol in displacing 3H-dihydroalprenolol binding to iris-ciliary body tissue, reducing aqueous humor formation, and lowering intraocular pressure of alpha-chymotrypsin hypertensive eyes. In contrast, the R-enantiomer was 50 to 90 times less potent than timolol in antagonizing the effects of isoproterenol on pulmonary and atrial beta-adrenergic receptors. The data indicate that the R-enantiomer may lower intraocular pressure in man at concentrations less likely than timolol to block extraocular beta-adrenergic receptors. Finally, to account for the differential effect of the R-enantiomer upon ocular as opposed to extraocular beta-adrenergic receptors, it is tentatively suggested that this agent may also act upon a population of ocular beta-adrenergic receptors showing relatively poor stereoselectively.

Beta-adrenergic receptor subtypes in iris-ciliary body of rabbits.

Direct comparison of drug interaction with intraocular relative to extraocular beta-adrenergic receptors was used to test the hypothesis that the receptors in ocular tissues are of the beta 2-subtype. Potencies for the displacement of 3H-dihydroalprenolol radioligand binding to membranes obtained from rabbit-iris ciliary bodies by beta-adrenoceptor agonists was in the order: isoproterenol greater than or equal to epinephrine greater than salbutamol greater than terbutaline greater than tazolol greater than norepinephrine. Similar radioligand displacement by beta-adrenoceptor antagonists was in the order of potency: alprenolol greater than or equal to bupranolol = oxprenolol = timolol = propranolol greater than ICI 118,551 = IPS 339 much greater than betaxolol greater than metoprolol. Finally, for the above beta-adrenoceptor antagonists, a highly significant correlation was obtained for displacement of radioligand to ocular tissues (pKi values) relative to antagonism (pA2 values) of isoproterenol-induced guinea-pig tracheal relaxation but not guinea-pig atrial chronotropic effects. Consequently, the above three observations reinforce the notion that rabbit-iris ciliary bodies contain beta-adrenergic receptors predominantly of the beta 2-subtype.

Intracerebral substance P in mice: behavioral effects and narcotic agents.

Acute intracerebral injection of the undecapeptide, substance P, in mice induced a unique reciprocal hindlimb scratching response whose intensity was dose-related. Similar intracerebral dose-response curves were obtained by the structurally related undecapeptides, physalaemin and eledoisin, but not by several unrelated peptides (TRH, neurotensin, bradykinin, somatostatin), prostaglandins E2 and F2a, dibutyryl cyclic AMP or dibutyrylcyclic GMP. Analgesic narcotic agents with predominant agonist activity administered i.p. prevented the reciprocal hindlimb scratching response induced by intracerebral substance P (0.625 microgram/mouse = ED 95). In this in vivo assay their action was stereospecific and exhibited a rank order of potency similar to that reported for analgesic activity and binding to opiate receptors in vitro. Narcotic agents with mixed agonist-antagonist activity were inactive while the narcotic antagonist, naloxone, completely reversed the action of morphine. Higher doses of naloxone alone potentiated substance P-induced reciprocal hindlimb scratching which may explain why partial narcotic agonists failed to abolish the response. There is now considerable evidence in support of a sensory neurotransmitter/modulator role for substance P within the central nervous system, and one of its actions may be associated with nociception. This concept is supported by observations in the present study which indicate that the substance P-induced reciprocal hindlimb scratching response involves nociceptive pathways within the central nervous system.

Urinary metabolites of timolol from humans and laboratory animals. Syntheses and beta-adrenergic blocking activities.

Syntheses are reported for three metabolites (2-4) of timolol (1) formed by oxidative metabolism of the morpholine ring. GLC-MS comparisons are presented which establish that the two metabolites whose structures were previously in question are identical with their synthetic counterparts 2 and 3. In 2, metabolic oxidation of the 4-morpholinyl group of 1 had occurred at the carbon next to oxygen to give the 2-hydroxy-4-morpholinyl moiety, whereas in 3, the morpholine of 1 has been oxidized one step further and then ring opened to produce the N-(2-hydroxyethyl)glycine substituent. Biological testing of synthetic samples of the three major metabolites from human urine (3, 4, and 6) indicated that only 4, in which the morpholine moiety has been degraded to a 2-hydroxyethylamino group, had significant beta-adrenergic blocking activity (one-seventh that of timolol in anesthetized dogs).

The respiratory response of sensitized rats to challenge with antigen aerosols.

An experimental procedure was developed to evaluate the effects of challenging sensitized conscious rats with antigen aerosols. The challenge resulted in changes in the respiratory patterns which were antigen specific and mediated by IgE antibodies. The response was inducible in non-sensitized rats by passive administration of IgE-rich serum. Sprague-Dawley rats were heterogeneous with respect to the respiratory response. A proportion (20--70%) of each group had continuous dyspnoea and other symptoms similar to asthma; the other had only episodes of apnoea. Wistar and Long-Evans rats resembled Sprague-Dawley rats; Fischer 344 rats had apnoea only, even though they produced IgE antibodies. The type of response did not correlate with serum IgE levels. The respiratory responses were reduced by dexamethasone, disodium cromoglycate, epinephrine and theophylline. Rats that respond with dyspnoea may provide a useful experimental model of allergic asthma.

beta-Adrenergic blocking agents with acute antihypertensive activity.

Modification of the pharmacological profile of the vasodilating/beta-adrenergic blocking agent 2-[4-[3-(tert-butylamino)-2-hydroxypropoxy]phenyl]-4-(trifluoromethyl)imidazole (1) has been investigated. Introduction of selected substitutents onto the imidazole ring, in place of the trifluoromethyl group, has yielded highly cardioselective beta-adrenergic blocking agents such as 7, 17, and 18. The placement of alkyl or chloro groups onto the aryl ring of 1, as illustrated by 33, has produced a class of compounds characterized as antihypertensive beta-adrenergic blocking agents. In these examples, the acute antihypertensive activity does not appear to be due to either vasodilating or beta 2-agonist properties.

Kojic amine--a novel gamma-aminobutyric acid analogue.

A series of compounds containing the 3-hydroxy-4H-pyran-4-one nucleus has been synthesized and tested as potential skeletal muscle relaxants. Reduction of 2-(azidomethyl)-5-hydroxy-4H-pyran-4-one (4) with HBr in HOAc--phenol yielded 2-(aminomethyl)-5-hydroxy-4H-pyran-4-one (kojic amine, 3) in 81% yield. Reaction of 2-[(tosyloxy)-methyl]-5-(benzyloxy)-4H-pyran-4-one (5) with NH3 gave a 40% yield of the O-benzyl ether of kojic amine, which was N-acylated with a series of carbobenzyloxy-protected amino acids. Complete deprotection with HBr--HOAc gave the following amino acid amides of kojic amine: glycyl (23), alpha-alanyl (24), beta-alanyl (25), gamma-aminobutyryl (26), and glycylglycyl (27). Among the analogues of kojic amine prepared was a series of one-carbon homologues: 2-[(methylamino)methyl]-5-hydroxy-4H-pyran-4-one (7a), 2-(1-aminoethyl)-5-hydroxy-4H-pyran-4-one (8), 6-(aminomethyl)-3-hydroxy-2-methyl-4H-pyran-4-one (12), and 2-(2-aminoethyl)-5-hydroxy-4H-pyran-4-one (16). Kojic amine (3) has been found to possess certain of the properties to be expected in a gamma-aminobutyric acid mimetic agent, notably skeletal muscle relaxant activity. In the chronic spinal cat preparation, ED70 values for reduction of flexor spasms of 2.2 and 4.0 mg/kg by iv and po routes of administration, respectively, were observed for kojic amine, which was the most potent of the various hydroxypyrone derivatives investigated.

Effect of human alpha-antitrypsin in papain-induced emphysema in the hamster.

The possibility that human alpha1-antitrypsin could effectively prevent development of emphysematous lesions produced in hamsters 7 days after exposure to aerosolized papain (3% for 3 hours) was investigated. Pretreatment with intratracheal human alpha1-antitrypsin prevented the appearance of these lesions in a dose-dependent manner. On the other hand, systemic administration of large doses of human alpha1-antitrypsin failed to prevent papain-induced pulmonary lesions, despite a significant increase in serum trypsin inhibitory capacity. These results suggest that intra-alveolar rather than serum concentrations of human alpha1-antitrypsin are critical for preventing the development of pulmonary emphysema in this animal model. It is interesting that although the mechanism by which human alpha1-antitrypsin prevented the papain-induced lesions is unknown, intratracheally administered human alpha1-antitrypsin similarly prevented the development of pulmonary emphysematous lesions induced by 0.1 mg of porcine pancreatic elastase given intratracheally.

Timolol maleate, a new beta-adrenergic receptor blocking agent.

Some pharmacodynamic properties of an oxypropanolamine substituted novel heterocyclic compound are described. Initial studies involve comparison of the racemic mixture, dl-timolol maleate, with the beta-adrenergic receptor blocking agent propranolol in the rat, dog and cat. dl-Timolol maleate is shown to be a potent inhibitor of cardiovascular beta-adrenergic receptors activated by isoproterenol or adrenergic nerve stimulation. Blockade of alpha-adrenergic receptors is not observed even after extremely high doses. The compound is approximately 3 times more potent than propranolol in suppressing isoproterenol-induced cardioacceleration by the i.v. route of administration. dl-Timolol maleate is also extremely well absorbed when given orally, being then about 10 times more active than propranolol. Unlike propranolol, neither dl-timolol maleate nor its optical isomers possess demonstrable local anesthetic activity. Similar to propranolol and other beta-adrenergic receptor blocking agents, activity of the compound resides predominantly in the l-isomer (timolol maleate.