Synthesis of [1-sarcosine, 8-O-methylserine]angiotensin II and 1-substituted analogues of [8-threonine]angiotensin II as antagonists of angiotensin II.

[1-N-methylisoleucine,8-threonine]-(I), [1-dimethylglycine,8-threonine]-(II), [1-guanidineacetic acid,8-threonine]-(III), des-1-aspartic acid-[8-threonine]-(IV), and [1-sarcosine,8-O-methylserine]angiotensin II (V) were synthesized by Merrifield's solid-phase procedure to study the effect of (a) substituents in position 1 on the antagonistic activity of [1-sarcosine,8-threonine]angiotensin II, and (b) a change in size and branching in position 8 of [1-sarcosine,-8-O-methylthreonine]angiotensin II. The analogues I-V caused an initial rise in blood pressure (30 min of infusion, 250 ng/kg/min in vagotomized ganglion-blocked rats) of 8.05, 11.7, 3.50, 4.5, and 11.16 mmHg. The pA2 values (rabbit aortic strips) obtained were 7.68, 7.53, 7.23, 7.53, and 9.66, and the dose ratios (in vagotomized ganglion-blocked rats infused at 250 ng/kg/min) obtained were 2.37, 4.49, 1.02, 1.47, and 24.04, respectively. The results obtained indicate that (a) the nature of the substituent in position 1 has an important influence on the biological activity of these peptides, and (b) the potency of antagonists I-IV (all less potent antagonists than [1-sarcosine,8-threonine]angiotensin II) is very much influenced by the length and branching of the side chain in position 8. The in vivo antagonistic activity of [1-sarcosine,8-O-methylthreonine]angiotensin II is reduced considerably by shortening the chain length by one carbon atom as is in V.

Synthesis of angiotensin II antagonists containing N- and O-methylated and other amino acid residues.

[1-N-Methylisoasparagine,8-isoleucine]- (I), [1-sarcosine,4-N-methyltyrosine,8-isoleucine]- (II), [1-sarcosine,5-N-methylisoleucine,8-isoleucine]- (III), [1-sarcosine,8-N-methylisoleucine]- (IV), [1-sarcosine8k-N-methylisoleucine,8-N-methylisoleucine]- (V), [1-sarcosine,8-O-methylthreonine]- (VI), [1-sarcosine,8-methionine]- (VII), and [1-sarcosine,8-serine]angiotensin II (VIII), synthesized by Merrifield's solid-phase procedure, possess respectively 0.8, 0.3, 0.5, 1.0, 0.0, 0.5, 3.7, and 0.7% pressor activity of angiotensin II (vagotomized, ganglion-blocked rats). They caused an initial rise in blood pressure (30 min of infusion, 250 ng/kg/min in vagotomized, ganglion-blocked rats) of 16.57, 9.80, 22.80, 32.00, 7.00, 15.06, 32.50, and 11.42 mmHg and showed secretory activity (isolated cat adrenal medulla) of 1.0, 0.1, 0.01, 0.1, less than 0.01, 0.1, less than 0.01, and 0.05% of angiotensin II. On isolated organs pA2 values (rabbit aortic strips) of 8.74, 7.44, 7.64, 7.85, 7.89, 8.76, 8.63, and 8.08, and pA2 values (cat adrenal medulla of 8.16, 9.16, 9.31, 8.00, 8.00, 7.00, 9.16, and 9.33 were obtained. Dose ratios (ratio of ED20 of angiotensin II during infusion of the antagonist and before infusion of the antagonist) in vagotomized, ganglion-blocked rats, infused at 250 ng/kg/min, were 33.43, 2.14, 3.26, 2.99, 0.62, 62.52, incalculable, and 11.15, respectively. The results obtained suggest that (a) analogs I and VI are potent antagonists of the pressor response of angiotensin II in normal rat, VI being the most potent antagonist thus far synthesized; (b) replacement of position 4 (Tyr) with MeTyr or position 5 and/or 8 (Ile) with Melle in [1-sarcosine,8-isoleucine]angiotensin II reduced the antagonist activity of this peptide (rabbit aortic strips and rats), indicating that steric hindrance imposed due to N-methylation in positions 4, 5, or 8 was not favorable in eliminating the initial pressor activity or prolonging the duration of action of [Sar1, Ile8]angiotensin II without reducing its antagonistic properties; (c) except II, none of the analogs showed any enhanced duration of action, suggesting that N-methylation in positions 5 or 8 did not afford protection against proteolytic enzymes; and (d) perfusion studies in cat adrenals indicated that all of these analogs are only very weak secretagogues. With the exception of [Sar1,Thr(ObetaMe)8]angiotensin II, which gave lower antagonistic properties, all other analogs had either similar antagonistic properties or were better antagonists in adrenal medulla than in smooth muscle.

Studies on the temperature-dependent sensitivity of mouse atria to adrenergic drugs.

The sensitivity of mouse atria (chronotropic response) to beta-adrenergic receptor antagonists was examined at 37 and 26 degrees C under various experimental conditions. When isoproterenol was used as agonist, at 37 degrees C, the pA2 value (from pA2 plots) for propranolol was 8.86 and the slope of the regression line was 0.54. At 26 degrees C, the pA2 value for propranolol was 9.2 and the slope was 0.7. In the presence of tropolone, 10-5 M, the values for pA2 and slope of the regression lines were, respectively, 9.0 and 0.90 at 37 degrees C and 9.17 and 0.98 at 26 degrees C. In other words, decreasing antagonism was prevented by low temperature and tropolone. Isoproterenol was potentiated by tropolone and low temperature, but the effects of low temperature were reduced by tropolone. With sotalol as antagonist, there was greated blockade (dose ratios) of the effects of isoproterenol at 26 than at 37 degrees C. When nylidrin, a non-catecholamine, was used as agonist there was no temperature-dependent sensitivity to sotalol. Furthermore, nylidrin was not potentiated by low temperature. The data show that the observed degree of beta-receptor antagonism can be altered by changing the bath temperature and suggest that this effect is related to COMT activity or an influence of agonist disposition in the tissue.

Influence of the adrenal gland on the pressor effect and antagonistic potency of angiotensin II analogs.

In ganglion blocked vagotomized rats, several 1,8-substituted angiotensin II analogs (250 ng/kg/min, i.v.) antagonized the pressor effect of angiotensin II. Dose ratios measured at the ED20 levels were: [Sar1,Ile8]-28; [Gac1,Ile8]- 19;[MeAla1,Ile8i1- 16;[MeIle1,Ile8]- 10;[sar1,Ala8]- 9;[me2Gly1,Ile8]- 4. Elimination of aspajtic acid in position 1 of [Ile8]-angiotensin II significantly reduced the antagonistic potency of the analog. No antagonistic effect was observed with [Phe4,Ile8] and [Ala4,Ile8]-angiotensin II even when infused at 6 mug/kg/min. During infusion, a partial rise in blood pressure was observed with all the above 1,8-substituted angiotensin II analogs. Phentolamine (100 mug/rat) injected 30 min after the start of the analog infusion reduced and sometimes abolished the pressor effect. However, phenoxybenzamine )Pbz, 2 mg/kg) injected 30 min prior to the analog infusion diminished but did not completely abolish the initial pressor effect. In adrenalectomized rats, the pressor effect was reduced by approximately 50 percent and disappeared completely 15-30 min after start of the infusion. Under these conditions, dose ratios of [Sar1,Ile8]-,[MeAla1,Ile8]- and [Gac1,Ile8]-angiotensin II were significantly reduced. Noradrenaline, 83 ng/kg/min. increased the ED20 value of angiotensin II(ratio 1.79) in normal rats but did not do so in adrenalectomized rats. In these rats no regular correlation was found between the angiotensin II ED20 values and initial blood pressure. These data indicate that under the present experimental conditions, the low pressor effect observed with these angiotensin II antagonists appears to be due to both adrenal catecholamine release and a direct vasoconstrictor effect. Variations in antagonistic activity of angiotensin II analogs, apart from changes introduced in the molecule, may be the manifestation of a complex interaction between angiotensin II, its antagonists, and the sympathoadrenal system.

Clinical and histologic evaluation of an indwelling, inflatable, long-term laryngeal stent in the canine model.

OBJECTIVE: The goal was assessment of a revolutionary approach to laryngeal stenting. A radically designed, atraumatic, inflatable, low-pressure, high-volume, endoscopically inserted laryngeal stent for effective intralaryngeal support or long-term prevention of aspiration was prospectively evaluated in a canine model to reveal any significant short- and long-term anatomic and functional changes in the larynx. METHODS: A self-sustaining, tube-free, long-term flap tracheostomy was performed in 14 dogs, followed by endoscopic laryngeal stent insertion. The stents were inflated, and their intraluminal pressures were monitored daily. The stenting period ranged between 3 and 46 days (mean 27.4 days). The animals were then euthanized, and total laryngectomy was performed. The larynges underwent gross and microscopic examinations, and a grading scale of reactive changes caused by the stent placement was created on the basis of the findings. RESULTS: Dye studies for aspiration were negative in all animals. Gross findings consisted of varying degrees of small ulceration, localized polypoid lesions, and granulomas. Grossly, 9 dogs exhibited small superficial ulceration of the true vocal cords and posterior commissure. Six dogs developed minor polyps or polypoid changes and/or granulomas, respectively. No gross changes were seen in 2 of the dogs. Histopathologically, 10 dogs demonstrated grade I or II histopathologic changes. Only 4 of the dogs exhibited inflammation extending into the underlying cartilages, grade III. One dog did not complete the observation period. CONCLUSION: This new stent demonstrated safety and biocompatibility with minimal local tissue reaction to its extended long-term placement. Good tolerance to the stent was documented, with minimal side effects similar to those that would be observed in human subjects after endotracheal intubation. When followed up in human patients, such minimal lesions have not resulted in significant long-lasting functional impairments. These initial results would indicate that this stent is well tolerated for long-term application and qualify as the preferred device for both management of aspiration and postoperative endolaryngeal bolstering support.

Histopathologic study of alternative substances for vocal fold medialization.

This research investigated the histopathologic and migratory properties of injectable alternatives for vocal fold medialization. Thirteen dogs underwent sectioning of the recurrent laryngeal nerve followed by vocal fold injection with 1 of 4 substances: Teflon, autologous fat, silicone suspension, or hydroxyapatite cement. Six months later, the animals were painlessly sacrificed and histopathologic analysis of the larynx and regional lymph nodes was performed. Although regional lymph node migration was noted, Teflon injection resulted in minimal vocal fold inflammatory reaction. Vocal folds injected with autologous fat exhibited persistence of fat at the injection site without significant inflammation or migration. Silicone suspension caused a localized giant cell reaction without regional lymph node migration, and 1 study subject died secondary to acute inflammation with critical respiratory compromise. Hydroxyapatite cement was well tolerated without inflammation or migration. This pilot study indicates that a wide range of possible substances for vocal fold medialization exist. Many of these may produce results superior to those obtained with Teflon and are thus far untested.

Hypotensive effect of [Sar1,Thr8]angiotensin II in spontaneously hypertensive sodium-depleted rats.

Under inactin anesthesia, intravenous infusion of [Sar1,Thr8]angiotensin II produced a hypotensive effect in young spontaneously hypertensive rats (SHR) treated with furosemide and in mature SH rats fed a low-sodium diet. The angiotensin antagonist also lowered blood pressure of young and mature SH rats receiving a normal diet. Deoxycorticosterone acetate (DOCA) plus saline reversed the hypotensive effect of [Saru,Thr8]angiotensin II in young SH rats, but did not do so in mature SH rats. Plasma renin activity (PRA) was not significantly changed by anesthesia. Furosemide or the low-sodium diet significantly increased PRA in young and mature SH rats. In contrast, DOCA plus saline significantly reduced PRA in both young and mature SH rats. However, there was no correlation between PRA and the action of the angiotensin II antagonist. These data suggest that the renin-angiotensin system is involved in genetic hypertension.

Development of two-kidney Goldblatt hypertension in rats under dietary sodium restriction.

In Sprague-Dawley rats with unilateral renal artery stenosis and an intact contralateral kidney, administration of a low-sodium diet did not prevent the development of hypertension. Despite an elevated blood pressure, hyponatremia, marked activation of the renin-angiotensin system, and increased hematocrit values, only 10% of the rats showed lesions of malignant hypertension. Systolic blood pressures of one- and two-kidney sham-operated rats fed a low-sodium diet were significantly higher than that of normotensive controls fed a normal diet. Uninephrectomy did not reduce plasma renin activity. The low-sodium diet increased plasma renin activity to about the same level in one- and two-kidney normotensive rats. However, the increase in plasma renin activity elicited by dietary sodium restriction was markedly less in one-kidney Goldblatt hypertension. Systolic blood pressure reached similar levels in one- and two-kidney Goldblatt hypertensive rats fed a low-sodium diet. These data indicate that a decrease in sodium intake does not prevent the development of two-kidney Goldblatt hypertension.

In vitro and in vivo studies of (1-sarcosine, 8-threonine) angiotensin II.

The capacity of (1-Sarcosine, 8-Threonine) angiotensin II to block the contractile and pressor effects of exogenous and endogenous angiotensin II was examined. In isolated rabbit aorta, the pA2 value (from pA2 plots) for the analog was 8.75 +/- 0.11. Both the maximum response and the slope of the dose-response curve to angiotensin II were unchanged by the analog. In ganglion-blocked vagotomized rats, infusion of the analog produced a dose-dependent blockage of the angiotensin II pressor effect. In these rats, the analog displayed less agonistic activity than that of (1-Sarcosine, 8-Isoleucine) and (1-Sarcosine, 8-Alanine) angiotensin II. In two-kidney hypertensive rats, the angiotensin II antagonist significantly reduced the arterial blood pressure. The results indicate that (1-Sar, 8-Thr) angiotensin II is a potent antagonist of angiotensin II with less inherent agonistic activity than previously reported analogs.

Use of ethylene vinyl alcohol copolymer for tubal sterilization by selective catheterization in rabbits.

PURPOSE: To assess the efficacy of ethylene vinyl alcohol copolymer (Uryx) in nonsurgically occluding the fallopian tube and achieving tubal sterilization in the rabbit model. MATERIALS AND METHODS: Ten mature virgin female New England rabbits underwent transvaginal selective bilateral fallopian tube cannulation with use of a coaxial catheter system under general anesthesia. Selective salpingography was performed bilaterally to assess patency of the fallopian tubes. Ethylene vinyl alcohol copolymer was injected unilaterally through a microcatheter to completely fill the middle portion of the tube. Three to seven days after injection, each animal was bred. Conception was determined by ultrasonography (US) 7-19 days after effective breeding. If pregnant, the rabbit was killed. Otherwise, it was permitted to rebreed until pregnancy was achieved. Histologic specimens of the fallopian tubes were prepared and analyzed. RESULTS: Patency of the fallopian tubes was demonstrated bilaterally in all animals by the free spillage of contrast material into the peritoneum. The delivery of ethylene vinyl alcohol copolymer into the fallopian tubes was successful in all animals but one, in which most of the plug almost immediately extruded into the uterus. Pregnancy was detected by US in the untreated fallopian tube in the nine rabbits that were receptive to breeding. No pregnancies were detected in the injected side. Histologic analysis demonstrated variable degrees of occlusion, fibrosis, and inflammation, with the majority of specimens demonstrating mild to moderate inflammation and moderate to marked fibrosis. CONCLUSION: Ethylene vinyl alcohol copolymer can reliably be placed nonsurgically via the transvaginal approach into the fallopian tubes with use of a coaxial catheter system. Ethylene vinyl alcohol copolymer appears to result in less fibrosis than previously investigated agents and demonstrates a 100% early sterilization rate in the rabbit model.