Evaluation of cardiovascular parameters of a selenium-based antihypertensive using pulsed Doppler ultrasound.

The pharmacology of selenium is of much interest because selenium deficiency has been linked to cardiovascular diseases, cancer, and arthritis, and selenoenzymes are critical cellular antioxidants. We have previously reported that phenyl-2-aminoethylselenide (PAESe) and its derivatives represent a novel class of selenium-based antihypertensive agents that exhibit unique biochemical and pharmacologic properties. We now report on experiments designed to probe the hemodynamic mechanism of action of these compounds in spontaneously hypertensive rats (SHR). A noninvasive pulsed Doppler ultrasound probe was used to measure peak blood flow velocity in the aortic arch from the right second intercostal space. PAESe was found to increase peak aortic blood flow velocity (+44%), heart rate (+16%), and blood flow acceleration (+105%), while decreasing left ventricular ejection time (LVET) (-37%) concomitant with a decrease in mean arterial pressure (-54%). These results were compared with the known vasodilator hydralazine, which had similar effects on mean arterial pressure (MAP) and peak velocity but caused an increase in LVET (+42%) and a decrease in heart rate (-18%). Taken together, our results suggest that PAESe decreases blood pressure via a decrease in peripheral resistance, which overcomes the initial increase in heart rate and acceleration to give a net decrease in MAP.

Pharmacological evaluation of 1-(carboxymethyl)-3,5-diphenyl-2-methylbenzene, a novel arylacetic acid with potential anti-inflammatory properties.

OBJECTIVE AND DESIGN: 1-(Carboxymethyl)-3,5-diphenyl-2-methylbenzene (CDB), a novel arylacetic acid, was evaluated in vivo for its ability to inhibit acute and chronic inflammation as well as acute pain. MATERIALS AND METHODS: The effects of CDB were evaluated using the following assays: 1) acute inflammation induced by the injection of carrageenan, bradykinin and serotonin into the subplantar region of the hind paw of rats; 2) chronic inflammation produced by the injection of Mycobacterium butyricum into the base of the tail of rats; 3) acute pain induced by the i.p. injection of phenyl-p-quinone into mice resulting in the production of writhes; 4) cyclooxygenase (COX) activity, including COX-1 and COX-2, evaluated using whole blood; and 5) activity of peptidylglycine alpha-monooxygenase (PAM) isolated from Xenopus laevis skin. RESULTS: CDB (10 to 100mg/kg s.c.) produced a dose-dependent inhibition of carrageenan edema (ED50 of 41 mg/ kg at 3 h) which continued for up to 12 h. Using a therapeutic dosing regimen, this compound inhibited hind paw inflammation (>70%) and arthogram scores in rats with adjuvant-induced arthritis. This compound also possessed significant analgesic activity in mice (70% inhibition with 50mg/kg). CDB, however, lacked inhibitory activity on bradykinin and serotonin-induced edema. In addition, CDB significantly inhibited COX-I activity (IC50 approximately = 17 microM) while having only a weak inhibitory activity on both COX-2 and PAM activity. CONCLUSIONS: CDB is an effective anti-inflammatory/analgesic agent whose mechanism of action appears to be associated with inhibition of COX-1 activity.

Selenium-based antihypertensives. Rationale and potential.

Selenium, long recognised as an important 'dietary antioxidant', is now known to be an essential component of the active sites of a number of enzymes, including the glutathione peroxidase selenoenzyme family which scavenge hydroperoxides to prevent cellular damage. Dietary selenium deficiency has been linked to diseases as diverse as cancer, heart disease, arthritis and AIDS, and epidemiological evidence is now emerging for the beneficial effects of selenium supplementation. Thus, the pharmacology, biology and biochemistry of selenium metabolism have become subjects of considerable interest, which are spurring efforts to develop synthetic selenium-containing compounds as potential therapeutic agents. Phenylaminoalkyl selenides were developed in the authors' laboratories as novel, selenium-based pharmacological agents. We demonstrated that these compounds exhibited dose-dependent antihypertensive activity in spontaneously hypertensive rats. Biochemical studies established that as a consequence of the redox properties of their selenium moieties, these phenylaminoalkyl selenides possessed the remarkable property of propagating a cycle of turnover-dependent local depletion of reduced ascorbate when processed by the key enzyme of catecholamine metabolism, dopamine-beta-monooxygenase. On the basis of inductively coupled plasma/mass spectroscopic analyses, corroborated by operant behaviour and locomotor activity investigations, an orally-active phenylaminoalkyl selenide with restricted CNS permeability was successfully developed. To our knowledge, this compound--4-hydroxy-alpha-methyl-phenyl-2-aminoethyl selenide--is the first orally active, selenium-based anti-hypertensive compound ever reported. In the future, we anticipate more widespread efforts to incorporate selenium into rationally designed pharmaceutical agents, with the goal of developing novel compounds which may be of therapeutic benefit toward a variety of human diseases.

An orally active selenium-based antihypertensive agent with restricted CNS permeability.

We report here the first orally active, selenium-based antihypertensive agent, and we demonstrate its restricted CNS permeability using inductively coupled plasma/mass spectroscopy (ICP/MS) and operant behavioral analysis. The biochemistry and pharmacology of selenium are subjects of intense current interest. As a consequence of the redox chemistry of the selenium moiety, phenylaminoalkyl selenides possess the remarkable characteristic of propagating a cycle of turnover-dependent local depletion of reduced ascorbate when processed by the key enzyme of catecholamine metabolism, dopamine-beta-monooxygenase. ICP/MS analysis was used to determine the pharmacokinetic parameters for selenide compounds after i.v. administration to anesthetized rats. Analysis of the data using a two-compartment pharmacokinetic model established very rapid initial clearance and a short beta-elimination half-life from blood. We developed an oxidative procedure for digestion and processing of tissue samples in order to obtain ICP/MS data on the tissue distributions of Se-containing metabolites after the administration of selenide compounds. The results establish that aromatic ring hydroxylation of the selenides results in a marked reduction in brain levels of Se-containing metabolites. The comparative effects of selenide compounds on locomotor activity and operant behavior were then investigated, and the results fully corroborate the ICP/MS analytical results. The novel compound, 4-hydroxy-alpha-methyl-phenyl-2-aminoethyl selenide, exhibits both restricted CNS permeability and oral antihypertensive activity in spontaneously hypertensive rats. This compound is the first orally active selenium-based antihypertensive agent ever reported, and it possesses properties that are highly desirable in pharmacological agents being developed for treatment of chronic diseases such as hypertension.

Antiinflammatory and analgesic activity of an inhibitor of neuropeptide amidation.

4-Phenyl-3-butenoic acid (PBA) has been shown in vitro to be a turnover-dependent inactivator of peptidylglycine alpha-monooxygenase (PAM), the rate-limiting enzyme involved in the formation of amidated neuropeptides from their glycine-extended precursors. In the studies reported herein, we have shown that PBA produces a dose-dependent (50-500 mg/kg s.c.) inhibition of serum PAM activity in normal rats without affecting peptidylamidoglycolate lyase activity. Because amidated neuropeptides such as substance P and calcitonin gene-related peptide are involved in acute inflammation, we evaluated the effects of PBA on carrageenan-induced inflammation in rats. The acute administration of PBA (s.c. or i.p.) produced a dose-related inhibition of edema with maximum inhibition (67%) observed at 2 hr postphlogistic agent. In addition, the continuous administration of PBA to animals over a 7-day period using osmotic pumps not only inhibited hind paw swelling induced by carrageenan but also inhibited serum PAM activity and reduced tissue levels of substance P in hind paws. These results demonstrate for the first time a correlation between the antiinflammatory activity produced by an inhibitor of peptide amidation with its ability to inhibit serum PAM activity and lower endogenous tissue levels of substance P. Moreover, these results confirm our contention that PAM is an excellent pharmacological target for controlling the acute inflammatory response. We also demonstrate the ability of PBA to inhibit phenyl-p-quinone and acetylcholine-induced writhing in mice without affecting the spinally mediated tail immersion assay in rats. Because this analgesic effect was extremely rapid (within 15 min), PBA may be producing this effect by a mechanism other than peptide amidation.

The oral antihypertensive activity of the methylated derivatives of phenyl-2-aminoethyl sulfide.

We have reported previously that phenyl-2-aminoethyl sulfide and its derivatives are excellent substrates for dopamine-beta-monooxygenase and produce an antihypertensive effect in spontaneously hypertensive rats after i.p. administration. In the studies reported herein, we demonstrate that alpha-methyl-phenyl-2-aminoethyl sulfide and 4-hydroxy-alpha-methyl-phenyl-2-aminoethyl sulfide, the methylated and hydroxymethylated derivatives of phenyl-2-aminoethyl sulfide, respectively, decrease mean arterial pressure in conscious, unrestrained spontaneously hypertensive rats after p.o. administration. This antihypertensive effect after p.o. administration occurs without the undesirable transient rise in blood pressure observed previously after i.p. administration. Results using the methodology of food-reinforced operant conditioned behavior are consistent with the interpretation that the ring hydroxylated derivatives, 4-hydroxy-phenyl-2-aminoethyl sulfide and 4-hydroxy-alpha-methyl-phenyl-2-aminoethyl sulfide, do not penetrate into the central nervous system. This finding supports our contention that the primary site of action for the antihypertensive activity of the sulfides may be the peripheral adrenergic nerve ending. In view of the current high degree of interest in chiral development, the enantiomeric specificity of the antihypertensive activity of alpha-methyl-phenyl-2-aminoethyl sulfide was also evaluated. Results from these studies demonstrate that the (S)-enantiomer of alpha-methyl-phenyl-2-aminoethyl sulfide is more effective in lowering blood pressure after p.o. administration than the (R)-enantiomer. The implications of our findings in terms of the mechanism of action of these compounds are discussed.

Inhibitory effect of cyclosporine A on the activity of oral hypoglycemic agents in rats.

Diabetic patients often have a kidney or pancreas allograft requiring cyclosporine A (CsA) to prevent transplant rejection. These patients usually take p.o. hypoglycemic agents to control their diabetes. Because of the reported adverse effects of CsA on glucose metabolism as well as its potential use in Type II diabetics, we were interested in evaluating the in vivo effect of CsA on the activity of p.o. hypoglycemic agents. We also wanted to determine if tolbutamide produced any adverse effects on the pharmacokinetics of CsA. Male, Holtzman rats were administered CsA (p.o.) followed 1 hr later with the hypoglycemic agent. Two hours later, blood samples were obtained to determine blood glucose levels. Animals treated with CsA alone produced a significant hyperglycemia. The hypoglycemic effects produced by tolbutamide and glyburide were inhibited in animals treated concomitantly with CsA. This inhibitory effect was not observed during the first 3 hr of CsA-treatment, could not be overcome by increasing the dose of the hypoglycemic agent and occurred using small doses. CsA did not, however, interfere with the activity of exogenous NPH insulin. Tolbutamide was found to have no effect on the acute pharmacokinetics of CsA. The distribution of CsA was similar to controls in all tissues studied except the liver in which CsA levels were less in tolbutamide-treated animals. These studies demonstrate that CsA interferes with the effects of p.o. hypoglycemic agents and, therefore, blood glucose levels should be monitored closely in Type II diabetic patients taking combinations of these drugs.

The enantiomeric specificity of the antihypertensive activity of 1-(phenylthio)-2-aminopropane, a synthetic substrate analogue for dopamine beta-monooxygenase.

We have found that (R,S)-1-(phenylthio)-aminopropane (4a), a synthetic alternate substrate for the terminal enzyme of norepinephrine biosynthesis, dopamine beta-monooxygenase (DBM), is both an indirect sympathomimetic and a potent antihypertensive agent in spontaneously hypertensive rats. We demonstrate herein that there is a distinct enantiospecific difference in the activities of (R)-1-(phenylthio)-2-aminopropane (4b) and (S)-1-(phenylthio)-2-aminopropane (4c). We find that 4c, the more potent DBM substrate analogue, exhibits both the indirect sympathomimetic activity and the antihypertensive activity previously observed for the racemate and inhibits the active transport of catecholamines at the nerve terminal. In contrast, 4b, which is less potent as a DBM substrate or as an inhibitor of catecholamine uptake, does not exhibit an indirect sympathomimetic effect and is not an effective antihypertensive agent. These results suggest that the greater selectivity of the S enantiomer for both the catecholamine reuptake transporter and the target enzyme DBM accounts for its greater potency as an indirect-acting sympathomimetic agent as well as its activity as an antihypertensive agent. These results are also consistent with the hypothesized mechanism of action of this class of sulfur-containing DBM substrate analogues.

Pharmacokinetic analysis of cyclosporine in adjuvant arthritic rats.

Cyclosporine A (CsA) has been shown to be effective in patients with rheumatoid arthritis and to prevent the development and improve the symptoms of adjuvant-induced arthritis in rats. Since abnormal drug disposition has been reported in inflammatory conditions, we have evaluated the pharmacokinetics of CsA in this animal model of arthritis. We found a statistically significant decrease in the rate of disappearance of blood concentrations of CsA following iv administration to arthritic rats. The plasma half-life of CsA increased with a corresponding decrease in total body clearance. The volume of distribution remained unchanged. This abnormality in CsA kinetics was not observed in these animals until 10 days after adjuvant injection. The administration of CsA (15 mg/kg ip) twice daily to arthritic rats for 8 days produced a 39.8% and 49.5% inhibition of swelling in the right and left hindpaw, respectively. There was also a 63.5% decrease in the arthrogram score. Trough levels of CsA in arthritic animals were initially higher than in controls during this treatment but returned to control values after 8 days of dosing, suggesting reversal of abnormal disposition with improvement of the disease. The addition of indomethacin to the dosing regimen resulted in a significant increase in trough levels of CsA, indicating a drug interaction between these two compounds. Possible mechanisms responsible for these observations with CsA are discussed.

An investigation of the adrenergic uptake specificity of phenyl-2-aminoethyl sulfides.

In earlier work we have demonstrated that a novel series of phenylethylamine analogs (phenyl-2-aminoethyl sulfides) cause a potent antihypertensive effect in spontaneously hypertensive rats. In addition, we have shown in vitro that these compounds are facile substrates for dopamine beta-hydroxylase, the terminal enzyme of norepinephrine synthesis. While the mechanism of action of these derivatives is as yet hypothetical, we have proposed that, if they are capable of gaining entrance into adrenergic nerve endings and neurotransmitter storage vesicles, these compounds may reduce norepinephrine synthesis by competing with dopamine for oxygenation. In this report, we present results of preliminary studies designed to examine this hypothetical mechanism of action. We find that all derivatives of this series are classical indirect-acting sympathomimetics whose initial cardiovascular activity is blocked by cocaine. These results suggest that compounds of this type gain entrance to adrenergic neurons via the normal norepinephrine uptake mechanism on adrenergic nerve endings. We also present data which demonstrate that the methylated derivative was not only the most potent indirect-acting sympathomimetic, but also the only derivative capable of producing a marked tachyphylaxis. In addition, we find these compounds affect a specific pool of intraneuronal norepinephrine, distinct from that affected by tyramine, a well-known indirect-acting sympathomimetic agent.

Demonstration of the antihypertensive activity of phenyl-2-aminoethyl selenide.

The effect of phenyl-2-aminoethyl selenide (PAESe) on blood pressure and heart rate was examined in anesthetized dogs and spontaneously hypertensive rats (SHR) in order to assess its possible utility as an antihypertensive agent. PAESe was shown to be an indirect-acting sympathomimetic whose transient increase in blood pressure was blocked by cocaine. PAESe exhibited potent antihypertensive activity in SHR. This hypotensive activity was dose-dependent, was evident in both acute and chronic assays and occurred after i.v. or i.p. administration or slow release from subdermally implanted osmotic pumps. The hypotensive activity in SHR occurred concomitant with a reduction in heart rate and a reduction in total body weight. Hearts isolated from SHR treated daily for 2 weeks with PAESe were significantly reduced in weight and in total catecholamine content. An investigation of the nature of the body weight loss which accompanied chronic dosing suggested that PAESe also possessed an anorexigenic property distinct from its antihypertensive activity. An examination of plasma electrolytes, enzymes and other metabolites from chronically treated rats showed no obvious toxic effects of such dosing.

Demonstration of the potent antihypertensive activity of phenyl-2-aminoethyl sulfides.

In previous work we established that phenyl-2-aminoethyl sulfide (PAES) and derivatives of this basic structure are novel substrate analogs for the adrenergic synthetic enzyme, dopamine beta-monooxygenase (DBM). We examined the in vivo effects of infusions of PAES and ring-hydroxylated (HOPAES) and/or alpha-methylated derivatives (MePAES, HOMePAES) and observed antihypertensive activity in SHR with HOPAES and HOMePAES using an indirect blood pressure measuring protocol. We now wish to report that by employing a direct blood pressure measuring technique we have been able to demonstrate a potent antihypertensive activity of all these derivatives in conscious, unrestrained spontaneously hypertensive rats (SHR) that persisted beyond a 6-h testing period. We found that MePAES, which displayed only a minor antihypertensive activity in the indirect measurements, was the most potent antihypertensive in the direct measuring protocol. In addition, in this report we demonstrate a potent chronic antihypertensive effect for MePAES over a 2-week period in SHR using continuous infusion with implanted osmotic pumps. From a comparison of the effects of the hydroxylated and alpha-methylated derivatives, we conclude that: (a) the locus of the antihypertensive activity is primarily in peripheral adrenergic sites; (b) alpha-methylation of the basic structure imparts an increased affinity for peripheral adrenergic uptake sites that may be responsible for its increased antihypertensive potency; and (c) monoamine oxidase (MAO) catabolism plays a relatively unimportant role in the termination of the activity of these compounds. These results also demonstrate the importance of direct blood pressure measurements in assaying antihypertensive activity of test compounds that possess indirect sympathomimetic activity. The implications of these findings in terms of the mechanism by which these compounds exert their anti-hypertensive activity is discussed.

Cyclosporine-phenytoin interaction.

Cyclosporine (CsA), an immunosuppressant, is used widely to prevent rejection of transplanted organs. It is extensively metabolized in the liver by hydroxylation and demethylation. Since phenytoin is used extensively post-transplant as an anti-convulsant, we studied the effect of chronic treatment with phenytoin on CsA by determining the disposition of CsA, prior to and after chronic treatment with phenytoin (30 mg/kg, po) for 5 days. CsA was analyzed by HPLC. The clearance (CL) of CsA was significantly enhanced following treatment with phenytoin when compared to the CL values obtained pretreatment (15.46 +/- 0.76 vs. 25.3 +/- 3.11 ml/min/kg). There were no significant changes in the volume of distribution of CsA. From the above data, we conclude that phenytoin probably induces the metabolism of CsA.

Cyclosporine-cimetidine interaction.

Cyclosporine (CsA) is used to prevent rejection of transplanted organs. It is extensively metabolized in the liver by the mixed function oxidase enzyme system by demethylation and hydroxylation. Since cimetidine is a widely prescribed H2 antagonist drug which is known to inhibit the P-450 enzyme system, we studied the effect of chronic treatment with cimetidine on CsA by determining the disposition of CsA (15 mg/kg, iv), prior to and after chronic treatment with cimetidine (60 mg/kg, po), for 5 days. CsA was determined by an HPLC procedure. The clearance (Cl) of CsA was significantly reduced following treatment with cimetidine when compared to the Cl value obtained in the control group (15.46 versus 10.36 ml/min/kg). There were no significant changes in the volume of distribution of CsA, one reason being that cimetidine and cyclosporine bind to different proteins. From the results obtained, we conclude that since cimetidine is an inhibitor of the mixed function oxidase system enzyme system, it inhibits the metabolism of CsA.

Antihypertensive activities of phenyl aminoethyl sulfides, a class of synthetic substrates for dopamine beta-hydroxylase.

Four sulfur-containing analogues of phenylpropylamine were synthesized and evaluated as substrates for dopamine beta-hydroxylase (DBH) and monoamine oxidase (MAO). All four phenyl aminoethyl sulfides were shown to be good substrates for DBH whereas only the two analogues not possessing a methyl group alpha to the terminal amino group were substrates for MAO. All four analogues were tested for acute antihypertensive activity in an animal model for hypertension, the spontaneously hypertensive rat (SHR). Two of the analogues, both of which should partition readily across the blood-brain barrier, did not appreciably reduce systemic blood pressure in the 6-h testing period. However, the two analogues that were designed to be relatively restricted to peripheral sites of action caused a dramatic drop in blood pressure in SHR of 25% within 1-1.5-h postinjection, with the analogue designed to be both restricted to the periphery and MAO inactive, causing a more prolonged antihypertensive activity.

Effects of phenyl-2-aminoethyl sulfide, a novel dopamine-beta-hydroxylase substrate, on the cardiovascular system of the anesthetized dog.

In previous work we have established that phenyl-2-aminoethyl sulfide (PAES) is a novel substrate for dopamine-beta-hydroxylase (DBH) which is stereospecifically oxygenated by the enzyme to the corresponding sulfoxide, (S)-phenyl-2-aminoethyl sulfoxide (PAESO). We now report that PAES possesses very little, if any, direct adrenergic agonist activity, but exhibits indirect sympathomimetic activity at relatively high doses (approximately 4 mg/kg). This assertion, that PAES is a new indirect sympathomimetic, is supported by our finding that pretreatment with cocaine completely abolishes the sympathomimetic activity of PAES. Furthermore, the effects of PAES are diminished with consecutive administration. In addition to its indirect sympathomimetic activity, we have also observed that PAES infusion almost completely blocks the reflex response elicited by hydralazine, a direct vasodilator. In contrast, we have found that PAESO possesses neither direct nor indirect sympathomimetic activity at doses as high as 6 mg/kg. Since PAES should be readily converted in vivo into PAESO, the implications of these findings in terms of potential antihypertensive action of PAES are discussed.

Irreversible inactivation of yeast glucose-6-P dehydrogenase by penicillin G.

Yeast glucose-6-P dehydrogenase is irreversibly inactivated by penicillin G. Kinetic data show that 1 molecule of penicillin G reacts with each active unit when the enzyme is inactivated. The rate of inactivation increases greatly with increasing pH. This irreversible inactivation by penicillin G is largely prevented by pyridoxal-P, a reversible inactivator or this enzyme. Prior treatment of penicillin G with penicillinase totally abolishes its ability to inactivate the enzyme.