Biological effects of α-adrenergic phentolamine on erythrocyte hemeprotein: Molecular insights from biorecognition behavior, protein dynamics and flexibility.

Phentolamine is one of the most representative nonselective α-adrenoreceptor blocking agents, which have been proved to be owned various pharmacological actions. Unfortunately, whether erythrocytes in the veins intervene in biological behaviors of such drug are largely obscured. With the aid of multiple biophysical techniques, this scenario was to detailed explore the potential biorecognition between phentolamine and the hemeprotein in the cytosol of erythrocytes, and the influences of dynamic characters of protein during the bioreaction. Steady-state and time-resolved fluorescence data manifested that the biomolecular recognition of phentolamine by hemeprotein was processed through the biopolymer-drug adduct with a moderate strength of 104M-1. Such procedure causes a reduction in fluorescence intensity of the aromatic tryptophan (Trp) residues, and the R-T transition of the globular protein occurred concurrently. Circular dichroism demonstrated the conclusions of fluorescence essays, viz. biorecognition can induce fairly structural transformation (self-regulation) of protein conformation. Furthermore, one could find that a specific domain for phentolamine is located at the polypeptide chains α1ß2 interface, and hydrogen bonds, π-conjugated and hydrophobic effects are discovered to be held the lowest energy state of the biomacromolecule-drug biosystem, which overtly matches the outcomes of wet experiments. Meanwhile, several crucial residues such as Trp-37 and Arg-40 were confirmed to have directly noncovalent interactions with phentolamine, and the effect of the heme group on the biomolecule-drug recognition is minimal. Further analyses of molecular dynamics simulation supported that the inherent protein flexibility may notably elicit alterations in some key noncovalent bonds between biomacromolecule and drug during the dynamic biointeraction, which might primarily be attributed to the torsion of drug structure and the conformational changes of essential residues. Undoubtedly, this research will not only help to thoroughly unearth the pharmacological profiles of phentolamine, but to elaborate the impacts of the intrinsic features (i.e. dynamics and flexibility) of critically cellular proteins on the biological conducts of active α-adrenergic blockers.

Statistical power analysis of cardiovascular safety pharmacology studies in conscious rats.

UNLABELLED: Cardiovascular (CV) toxicity and related attrition are a major challenge for novel therapeutic entities and identifying CV liability early is critical for effective derisking. CV safety pharmacology studies in rats are a valuable tool for early investigation of CV risk. Thorough understanding of data analysis techniques and statistical power of these studies is currently lacking and is imperative for enabling sound decision-making. METHODS: Data from 24 crossover and 12 parallel design CV telemetry rat studies were used for statistical power calculations. Average values of telemetry parameters (heart rate, blood pressure, body temperature, and activity) were logged every 60s (from 1h predose to 24h post-dose) and reduced to 15min mean values. These data were subsequently binned into super intervals for statistical analysis. A repeated measure analysis of variance was used for statistical analysis of crossover studies and a repeated measure analysis of covariance was used for parallel studies. Statistical power analysis was performed to generate power curves and establish relationships between detectable CV (blood pressure and heart rate) changes and statistical power. Additionally, data from a crossover CV study with phentolamine at 4, 20 and 100mg/kg are reported as a representative example of data analysis methods. RESULTS: Phentolamine produced a CV profile characteristic of alpha adrenergic receptor antagonism, evidenced by a dose-dependent decrease in blood pressure and reflex tachycardia. Detectable blood pressure changes at 80% statistical power for crossover studies (n=8) were 4-5mmHg. For parallel studies (n=8), detectable changes at 80% power were 6-7mmHg. Detectable heart rate changes for both study designs were 20-22bpm. DISCUSSION: Based on our results, the conscious rat CV model is a sensitive tool to detect and mitigate CV risk in early safety studies. Furthermore, these results will enable informed selection of appropriate models and study design for early stage CV studies.

Endogenous Estrogen-Mediated Heme Oxygenase Regulation in Experimental Menopause.

Estrogen deficiency is one of the main causes of age-associated diseases in the cardiovascular system. Female Wistar rats were divided into four experimental groups: pharmacologically ovariectomized, surgically ovariectomized, and 24-month-old intact aging animals were compared with a control group. The activity and expression of heme oxygenases (HO) in the cardiac left ventricle, the concentrations of cardiac interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the myeloperoxidase (MPO) activity in the cardiac left ventricle, and the effects of heme oxygenase blockade (by 24-hour and 1-hour pretreatment with tin-protoporphyrin IX, SnPP) on the epinephrine and phentolamine-induced electrocardiogram ST segment changes in vivo were investigated. The cardiac HO activity and the expression of HO-1 and HO-2 were significantly decreased in the aged rats and after ovariectomy. Estrogen depletion was accompanied by significant increases in the expression of IL-6 and TNF-α. The aged and ovariectomized animals exhibited a significantly elevated MPO activity and a significant ST segment depression. After pretreatment with SnPP augmented ST segment changes were determined. These findings demonstrate that the sensitivity to cardiac ischemia in estrogen depletion models is associated with suppression of the activity and expression of the HO system and increases in the secretion of proinflammatory cytokines and biomarkers.

Cardioprotective effects of voluntary exercise in a rat model: role of matrix metalloproteinase-2.

BACKGROUND: Regular exercise at moderate intensity reduces cardiovascular risks. Matrix metalloproteinases (MMPs) play a major role in cardiac remodeling, facilitating physiological adaptation to exercise. The aim of this study was to examine the influence of voluntary physical exercise on the MMP-2 enzyme activity and to investigate the cardiac performance by measurement of angina susceptibility of the heart, the basal blood pressure, the surviving aorta ring contraction, and the cardiac infarct size after I/R-induced injury. METHODS: Male Wistar rats were divided into control and exercising groups. After a 6-week period, the serum level of MMP-2, basal blood pressure, cardiac angina susceptibility (the ST segment depression provoked by epinephrine and 30 s later phentolamine), AVP-induced heart perfusion and aorta ring contraction, infarct size following 30 min ischemia and 120 min reperfusion, and coronary effluent MMP-2 activity were measured. RESULTS: Voluntary wheel-running exercise decreased both the sera (64 kDa and 72 kDa) and the coronary effluent (64 kDa) MMP-2 level, reduced the development of ST depression, improved the isolated heart perfusion, and decreased the ratio of infarct size. CONCLUSION: 6 weeks of voluntary exercise training preserved the heart against cardiac injury. This protective mechanism might be associated with the decreased activity of MMP-2.

Comparative pathophysiology, toxicology, and human cancer risk assessment of pharmaceutical-induced hibernoma.

In humans, hibernoma is a very rare, benign neoplasm of brown adipose tissue (BAT) that typically occurs at subcutaneous locations and is successfully treated by surgical excision. No single cause has been accepted to explain these very rare human tumors. In contrast, spontaneous hibernoma in rats is rare, often malignant, usually occurs in the thoracic or abdominal cavity, and metastases are common. In recent years, there has been an increased incidence of spontaneous hibernomas in rat carcinogenicity studies, but overall the occurrence remains relatively low and highly variable across studies. There have only been four reported examples of pharmaceutical-induced hibernoma in rat carcinogenicity studies. These include phentolamine, an alpha-adrenergic antagonist; varenicline, a nicotine partial agonist; tofacitinib, a Janus kinase (JAK) inhibitor; and hydromorphone, an opiod analgesic. Potential non-genotoxic mechanisms that may contribute to the pathogenesis of BAT activation/proliferation and/or subsequent hibernoma development in rats include: (1) physiological stimuli, (2) sympathetic stimulation, (3) peroxisome proliferator-activated receptor (PPAR) agonism, and/or (4) interference or inhibition of JAK/Signal Transducer and Activator of Transcription (JAK/STAT) signaling. The evaluation of an apparent increase of hibernoma in rats from 2-year carcinogenicity studies of novel pharmaceutical therapeutics and its relevance to human safety risk assessment is complex. One should consider: the genotoxicity of the test article, dose/exposure and safety margins, and pathophysiologic and morphologic differences and similarities of hibernoma between rats and humans. Hibernomas observed to date in carcinogenicity studies of pharmaceutical agents do not appear to be relevant for human risk at therapeutic dosages.

Local and systemic toxicity of intraoral submucosal injections of phentolamine mesylate (OraVerse).

OraVerse, an injectable formulation of phentolamine mesylate (PM), was recently approved by the U.S. Food and Drug Administration (FDA) for reversal of anesthesia of the lip and tongue and associated functional deficits resulting from an intraoral submucosal injection of a local anesthetic containing a vasoconstrictor. Because PM had not been approved previously for submucosal administration, 2 Good Laboratory Practices (GLP) studies in dogs designed to investigate systemic toxicity and the local effects of single and repeated dosing of OraVerse on the inferior alveolar nerve and branches of the superior alveolar nerve and adjacent soft tissues after local administration were conducted. Systemic toxicity was measured by preinjection and postinjection clinical examinations, clinical chemistry, and gross and microscopic examinations of major organs after necropsy. No evidence of systemic toxicity was detected. Local nerve and adjacent tissue damage was assessed by conventional histopathology. Nerve degeneration was evident in 1 animal. Mild perineural inflammation adjacent to the inferior alveolar nerve and inflammatory exudates were observed in submucosal tissues in several animals. No changes were observed in the nerves at injection sites of dogs from any dose group that were considered directly related to the test articles. These data reveal that single and repeated intraoral administrations of OraVerse are well tolerated in beagle dogs.

Development of hibernomas in rats dosed with phentolamine mesylate during the 24-month carcinogenicity study.

Phentolamine is a reversible competitive alpha-adrenergic antagonist with similar affinities for alphal and alpha2 receptors. It has a long history of safe clinical use, and was developed as a potential therapy for male erectile dysfunction because of its capacity to increase the arteriolar blood flow to the corpora cavernosa. Phentolamine mesylate was administered to rats by oral gavage at daily doses of 10, 50, and 150 mg/kg for 24 months. A dose-related increase in mortality, ascribed to an exaggerated pharmacologic effect, was seen at high doses. Systemic exposure as measured by plasma drug concentration increased with dose and duration of dosing and slight drug accumulation occurred, particularly in high-dose males. In the treated groups, 10 males and 1 female were diagnosed with hibernomas, neoplasms of brown adipose tissue, which appeared in the thoracic cavity or retroperitoneal area as circumscribed, tan to reddish-brown lobulated masses. Histologically, the masses were well circumscribed with variably sized lobules defined by a rich capillary network and consisted of closely apposed oval to polygonal cells with large amounts of cytoplasm and a centrally located nucleus. The cytoplasm's appearance varied from multivacuolated to univacuolated to granular eosinophilic. In a few cases, neoplastic emboli were observed in capsular vessels. Ultrastructurally, the neoplastic cells contained numerous mitochondria with transverse parallel cristae that occupied over 60% of the cytoplasm and lipid droplets. This study documents the previously unreported development of hibernomas in rats treated with phentolamine mesylate.

Cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human corpus cavernosum penis.

OBJECTIVES: To investigate the cytotoxic effect of prostaglandin E(1) (PGE(1)), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the corpus cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. METHODS: Primary endothelial cells were obtained from the corpus cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 microg PGE(1), 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. RESULTS: The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE(1) and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE(1). CONCLUSIONS: These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the corpus cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.

Activation of alpha(1)-adrenergic receptors potentiates the nephrotoxicity of ethylene dibromide.

Ethylene dibromide (EDB) has been used as a model compound for eliciting hepato- and nephrotoxicity. Conjugation with glutathione (GSH) has been shown to play a role in the bioactivation of EDB. The aim of this study was to determine whether activation of alpha(1)-adrenergic receptors, which causes a decrease in cellular GSH levels, could modulate the nephrotoxicity of EDB. For this purpose, male ICR mice were treated with EDB and/or the alpha-adrenergic agonist, phenylephrine (Pe), or the alpha-adrenergic antagonist, phentolamine (Phe). Animals treated with EDB (40 mg/kg, i.p.) had a 9.3-fold increase in urinary gamma-glutamyltranspeptidase (GGTP: EC 2.3.2.2) activity and a 38% decrease in renal non-protein bound sulfhydryl (NPSH) levels; however, animals co-treated with EDB and Pe (50 mg/kg, i.p.) exhibited a 27.8-fold increase in urinary GGTP activity and a 60% decrease in NPSH levels. The enhanced presence of urinary GGTP and decrease in cellular levels of NPSH was nearly blocked by treating animals concomitantly with EDB and Phe (10 mg/kg, i.p.) or EDB, Pe, and Phe. Histopathological examination revealed the enhanced degree of tissue damage and necrosis following treatment with EDB and Pe, and the protective effect of Phe at ameliorating EDB toxicity. These results indicate that factors that can influence alpha-adrenergic receptors may be critical in assessing dose-response data used in the risk assessment process.

Role of sympathetic nervous system in experimental hypertension and diabetes mellitus.

An investigation has been made to elucidate the role of sympathetic nervous system (SNS) in the development of renal hypertension and hyperglycemia in rats and rhesus monkeys using 6-hydroxydopamine (6-OHDA). Development of renal hypertension was blocked in weanling rats (80%), demedullated adult rats with 6-OHDA (75%) but not in adult rats sympathectomised with 6-OHDA. Weanling rats treated with 6-OHDA did not have any detectable catecholamine stores when measured 60 days after treatment with 6-OHDA. Unlike the weanling rats the hearts of the adult rats showed significant refilling of catecholamines 60 days after 6-OHDA treatment. 6-OHDA treatment and adrenalectomy did not modify the development of streptozotocin (STZ) induced hyperglycemia and the hypoglycemic effect of tolbutamide and phenformin. Chemical sympathectomy with 6-OHDA did not show any glucose intolerance in rhesus monkeys or any effect or insulin release. Phentolamine (PHE) and oxprenolol (OXP) pretreatment had no effect on development and maintenance of hyperglycemia. Although there was no effect on the endogenous catecholamines stored in the heart and brain in the diabetic rats, there was a marked increase in the urinary excretion of norepinephrine (NE), epinephrine (E), 3-methoxy-4 hydroxy mandelic acid (VMA) and creatinine. The rate of disappearance of (3H)-NE from rat heart was higher when compared to age matched controls. Diabetic rats showed increase responsiveness to angiotensin II and developed hypertension faster when their renal arteries were clamped as compared to controls. It is concluded that the functional SNS is important in the development of renal hypertension in the rat. Further the SNS in the rats and rhesus monkeys does not play any significant role in the STZ induced hyperglycemia, glucose intolerance and insulin release.

Peptidergic modulation of the sympathetic contraction in the rabbit ear artery: effects of temperature.

1. The effects of neuropeptide Y, endothelin-1, arginine-vasopressin and angiotensin II on the vascular contraction to sympathetic nerve stimulation were studied in isolated segments, 2 mm long, from the rabbit central ear artery, a cutaneous vessel, during changes in temperature (24 degrees -41 degrees C). 2. Transmural electrical stimulation (1-8 Hz, at supramaximal voltage) produced frequency-dependent contraction, and this response, partially blocked by tetrodotoxin (1 microM) and phentolamine (1 microM), was reduced by cooling (30 degrees C -24 degrees C) and was not modified by warming (41 degrees C), as compared to that recorded at 37 degrees C. 3. Pretreatment with neuropeptide Y (10, 30 and 100 nM) increased in a concentration-dependent manner the vascular contraction to sympathetic stimulation at every temperature studied, but this potentiation was greater during cooling (34 degrees C -24 degrees C) than at 37 degrees C or warming (41 degrees C). 4. Pretreatment with endothelin-1 (3 and 10 nM) or vasopressin (0.1, 0.3 and 1 nM) increased in a concentration-dependent manner the vascular contraction to sympathetic stimulation during cooling (34 degrees C -24 degrees C), but not at 37 degrees C or warming (41 degrees C). 5. Pretreatment with angiotensin II (0.1, 0.3 and 1 microM) did not modify the contraction to sympathetic stimulation at any temperature studied. 6. These results suggest that neuropeptide Y, endothelin-1 and vasopressin, but not angiotensin II, modulate the cutaneous vasoconstriction to sympathetic nerve stimulation by potentiating this vasoconstriction during cooling.

Differential effects of alpha and beta adrenergic blockade on glucose and lactate metabolism during acute stress.

In this study we examined the role of alpha and beta blockade on glucose and lactate metabolism during the acute stress of insulin-induced hypoglycemia. Three groups of conscious dogs with chronically fitted catheters in the femoral artery and in the femoral, portal, and hepatic veins were studied after an 18-hr fast. After a 1-hr basal period, hypoglycemia was induced with insulin infusion at 5 mU/kg.min for 3 hr. Group 1 received no other treatment. Groups 2 and 3 received, respectively, phentolamine (8 micrograms/kg.min) and propranolol (4 micrograms/kg.min) beginning 30 minutes before and throughout the experimental period. Despite similar hyperinsulinemia, plasma glucose dropped in Group 1 (from 115 +/- 10 to 40 +/- 3 mg/dl) and in Group 2 (from 110 +/- 4 to 60 +/- 3 mg/dl) but in Group 3 it was maintained at 45 +/- 4 mg/dl by exogenous glucose infusion at a rate of 2.2 +/- 0.4 mg/kg.min. Hepatic glucose production increased 50 +/- 13%, 127 +/- 30%, and 55 +/- 30% in Groups 1, 2, and 3, respectively, within 60 minutes and was 56 +/- 19%, 55 +/- 17%, and -0.04 +/- 12% during the last hour of the experiment. Glucose utilization did not change in Groups 1 and 2 but it increased in Group 3. Plasma lactate increased in Group 1 (from 850 +/- 190 to 1,980 +/- 450 mumol/L) and in Group 2 (985 +/- 180 to 4,785 +/- 500 mumol/L), while in Group 3 there was an early rise (to 695 +/- 120 mumol/L) within 30 minutes that gradually dropped to near basal.(ABSTRACT TRUNCATED AT 250 WORDS)

A nonhuman primate model for evaluating the potential of antihypertensive drugs to cause orthostatic hypotension.

The potential of several classes of antihypertensive drugs to cause orthostatic hypotension in man was evaluated in a conscious cynomolgus monkey model. Supine and erect blood pressure and heart rate were continuously monitored before and after administration of chlorisondamine (a ganglionic blocking agent); phentolamine and prazosin (alpha-adrenergic blocking agents); propranolol (a beta-adrenergic blocking agent); and minoxidil (a vasodilator). Substantial validation of the model was accomplished when it was observed that these drugs evoked cardiovascular responses in the animal model which were similar to those which had been described in the clinical literature.