Influence of New Ultrashort-Acting Beta-Adrenergic Blockers on Systolic Blood Pressure in Rats

Frydrych M., L. Barto‰ová, T. Flor ian, J . Neãas, L. Barto‰íková, J . Krãmáfi, P. Mokr ̆, V. Bruncl ík: Influence of New Ultrashort-Acting Beta-Adrenergic Blockers on Systolic Blood Pressure in Rats. Acta Vet. Brno 2004, 73: 181-185. Three newly synthesized potential ultrashort-acting beta adrenergic blockers containing metabolically unstable ester functional groups that are easily cleft by plasma esterases were tested. In the experiment, 30 laboratory rats divided into 4 subgroups were used. Agent 42Bu was administered to Group 1 (n = 8), agent 43Bu to Group 2 (n = 8), agent 44Bu to Group 3 (n = 8) and placebo to Group 4 (n = 6). Under general anesthesia the arteria carotis and vena jugularis were exteriorized. The arteria carotis was connected by a cannula to the machine HSE UNIPER UP – 100, a universal perfusion system for isolated organs recording and converting actual values of blood pressure into graphical representation in a computer. Subsequently, 42Bu, 43Bu, 44Bu and placebo were administered into v. jugularis, and systolic blood pressure was recorded within the period of 18 minutes of their administration. All agents were administered at 2.5 mg/kg doses. The systolic blood pressure values after 42Bu, 43Bu, 44Bu administration were compared to those of the placebo Group. The 42Bu caused a statistically significant decrease (p < 0.05) in systolic blood pressure 1 minute after administration, 43Bu 1.5 minute after administration, and 44Bu 9 minutes thereafter. Statistically significant decrease of systolic blood pressure began immediately after administration of all three tested substances. The results of our in vivo testing show that agent 44Bu was the most effective of all three tested agents. Its onset of action was rapid and the hypothesis of ultrashort action was confirmed. Pharmacology, plasma esterases, ester functional group, universal perfusion system, hypotensive effect Beta blockers are one of the main pharmacotherapeutical group in the treatment of cardiovascular diseases (Bar to‰ íková et al. 1998; Neãas et al. 1997). They are indicated in the treatment of hypertension, portal hypertension, angina pectoris, some types of arrhythmias, idiopathic cardiac myopathy and in some non-cardiac disturbances, e.g. hyperthyroidism, glaucoma and some neurological indications (Hynie 1997). In patients dependent on the sympathetic tonus adverse reactions can appear typical of classical beta blockers, e.g. hypotension, bradycardia, heart failure, bronchospasm or peripheral vasoconstriction, whose effect can be manifested for several hours after intravenous administration (Mc Devi t t 1979). For that reason, researchers have developed ultrashortacting beta blockers, whose advantage is in the immediate onset of action after administration, possibility of dose titration, very short duration of action and rapid offset of the action (Barbier et al. 1995). The ultrashort-acting beta blockers are parenteral agents that can be rapidly titrated in clinical situations where immediate beta adrenergic blockade is warranted (Fr ishman et al. 1998). Their efficacy has been shown in specific clinical settings, e.g. in patients with unstable angina, myocardial infarction, atrial fibrillation or flutter and supraventricular tachycardia (Barbier et al. 1995). Esmolol, the prototype drug of this class, has been approved for treatment of supraventricular ACTA VET. BRNO 2004, 73: 181-185 Address for correspondence: PharmDr. Marek Frydrych Department of Human Pharmacology and Toxicology University of Veterinary and Pharmaceutical Sciences Palackého 1-3, 612 42 Brno, Czech Republic Phone: + 420 541 562 895 Fax: + 420 541 240 605 E-mail: marek.fry@atlas.cz http://www.vfu.cz/acta-vet/actavet.htm tachyarrhythmias, but has also potential use in the treatment of patients with perioperative hypertension and acute myocardial ischemia (Fr ishman et al. 1998). Landiolol and flestolol are other agents in the group of ultrashort-acting beta blockers. Three potential ultrashort-acting beta blockers with working names 42Bu, 43Bu and 44Bu have been synthesised at the Department of Chemical Drugs of the Faculty of Pharmacy of the University of Veterinary and Pharmaceutical Sciences in Brno, see Fig. 1 (Mokr ̆ et al. 2002). The tested agents were synthesised by 5-step synthesis, and their structure has been verified by elementary analysis, infrared 1H-NMR and 13C-NMR spectroscopy (Barto‰ová et al. 2002). The tested agents are chemically similar to esmolol, the prototype drug of ultrashortacting beta blockers. The extremely short duration of action of the agents was secured by the incorporation of a metabolically unstable ester functional group into their structure. The presence on the aromatic ring of a metabolically unstable ester functional group, which is very easily cleft by plasma esterases, leads to the ultrashort duration of action of the tested agents. The aim of this in vivo testing was to monitor the influence of three newly synthesised compounds with the working names 42Bu, 43Bu and 44Bu at doses of 2.5 mg/kg of body weight on the values of systolic blood pressure of laboratory rats. Materials and Methods In the experiment, 30 Wistar male rats of the same age (60 days) and comparable body mass (327 ± 25 g) were used. The animals came from a conventional breeding colony (Faculty of Medicine, Masaryk University, Brno). They were placed in PVC cages (n = 3), fed a standard diet (Diet for small laboratory animals SPF M1) and given water ad libitum. After 12 days of acclimatization, the animals were randomly divided into 4 groups: Group 1 (8 rats) was given agent 42Bu, Group 2 (8 rats) was given agent 43Bu, Group 3 (8 rats) was given agent 44Bu and Group 4 was given placebo. In general anesthesia the arteria carotis and vena jugularis were exposed and exteriorized. A. carotis was connected by a cannula to the HSE UNIPER UP – 100 device, which is a universal perfusion system for isolated organs recording and converting blood pressure values into graphical representation in a computer. Subsequently, the tested agents were administered into v. jugularis (approximately 15 min from a. carotis and v. jugularis exteriorization) as a bolus and the values of systolic blood pressure were recorded within the period of 18 min of their administration. The solution for anaesthesia was administered i.m. in the femur area at a dose of 0.5 ml/100 g of body mass and was composed of: xylazinum (Rometar® 2% inj.) and ketamini hydrochloridum (Narkamon® Spofa 1% inj.) in ratio 1:20. All tested agents were administered at doses of 2.5 mg/kg of body mass as a bolus at a 1 ml dose. As a vehicle for tested agents, a 5% solution of dimethylsulfoxide in saline (Infusio natrii chlorati isotonica Infusia®) was used. The placebo group was administered only 1 ml of 5% solution of dimethylsulfoxide in saline. The experiment was always performed in the morning. First, experiment with placebo group was performed followed by the experiments with Group 1, Group 2 and Group 3, respectively. The project was approved and monitored by the local University Ethical Committee. 182 Fig. 1. Chemical structures of the three tested agents OH

Three newly synthesized potential ultrashort-acting beta adrenergic blockers containing metabolically unstable ester functional groups that are easily cleft by plasma esterases were tested.In the experiment, 30 laboratory rats divided into 4 subgroups were used.Agent 42Bu was administered to Group 1 (n = 8), agent 43Bu to Group 2 (n = 8), agent 44Bu to Group 3 (n = 8) and placebo to Group 4 (n = 6).Under general anesthesia the arteria carotis and vena jugularis were exteriorized.The arteria carotis was connected by a cannula to the machine HSE UNIPER UP -100, a universal perfusion system for isolated organs recording and converting actual values of blood pressure into graphical representation in a computer.Subsequently, 42Bu, 43Bu, 44Bu and placebo were administered into v. jugularis, and systolic blood pressure was recorded within the period of 18 minutes of their administration.All agents were administered at 2.5 mg/kg doses.The systolic blood pressure values after 42Bu, 43Bu, 44Bu administration were compared to those of the placebo Group.The 42Bu caused a statistically significant decrease (p < 0.05) in systolic blood pressure 1 minute after administration, 43Bu 1.5 minute after administration, and 44Bu 9 minutes thereafter.Statistically significant decrease of systolic blood pressure began immediately after administration of all three tested substances.
The results of our in vivo testing show that agent 44Bu was the most effective of all three tested agents.Its onset of action was rapid and the hypothesis of ultrashort action was confirmed.

Pharmacology, plasma esterases, ester functional group, universal perfusion system, hypotensive effect
Beta blockers are one of the main pharmacotherapeutical group in the treatment of cardiovascular diseases (Barto‰íková et al. 1998;Neãas et al. 1997).They are indicated in the treatment of hypertension, portal hypertension, angina pectoris, some types of arrhythmias, idiopathic cardiac myopathy and in some non-cardiac disturbances, e.g.hyperthyroidism, glaucoma and some neurological indications (Hynie 1997).In patients dependent on the sympathetic tonus adverse reactions can appear typical of classical beta blockers, e.g.hypotension, bradycardia, heart failure, bronchospasm or peripheral vasoconstriction, whose effect can be manifested for several hours after intravenous administration (M c Devitt 1979).For that reason, researchers have developed ultrashortacting beta blockers, whose advantage is in the immediate onset of action after administration, possibility of dose titration, very short duration of action and rapid offset of the action (Barbier et al. 1995).The ultrashort-acting beta blockers are parenteral agents that can be rapidly titrated in clinical situations where immediate beta adrenergic blockade is warranted (Frishman et al. 1998).Their efficacy has been shown in specific clinical settings, e.g. in patients with unstable angina, myocardial infarction, atrial fibrillation or flutter and supraventricular tachycardia (Barbier et al. 1995).Esmolol, the prototype drug of this class, has been approved for treatment of supraventricular tachyarrhythmias, but has also potential use in the treatment of patients with perioperative hypertension and acute myocardial ischemia (Frishman et al. 1998).Landiolol and flestolol are other agents in the group of ultrashort-acting beta blockers.
Three potential ultrashort-acting beta blockers with working names 42Bu, 43Bu and 44Bu have been synthesised at the Department of Chemical Drugs of the Faculty of Pharmacy of the University of Veterinary and Pharmaceutical Sciences in Brno, see Fig. 1 (Mokr˘et al. 2002).
The tested agents were synthesised by 5-step synthesis, and their structure has been verified by elementary analysis, infrared 1 H-NMR and 13 C-NMR spectroscopy (Barto‰ová et al. 2002).The tested agents are chemically similar to esmolol, the prototype drug of ultrashortacting beta blockers.The extremely short duration of action of the agents was secured by the incorporation of a metabolically unstable ester functional group into their structure.The presence on the aromatic ring of a metabolically unstable ester functional group, which is very easily cleft by plasma esterases, leads to the ultrashort duration of action of the tested agents.
The aim of this in vivo testing was to monitor the influence of three newly synthesised compounds with the working names 42Bu, 43Bu and 44Bu at doses of 2.5 mg/kg of body weight on the values of systolic blood pressure of laboratory rats.

Materials and Methods
In the experiment, 30 Wistar male rats of the same age (60 days) and comparable body mass (327 ± 25 g) were used.The animals came from a conventional breeding colony (Faculty of Medicine, Masaryk University, Brno).They were placed in PVC cages (n = 3), fed a standard diet (Diet for small laboratory animals SPF M1) and given water ad libitum.After 12 days of acclimatization, the animals were randomly divided into 4 groups: Group 1 (8 rats) was given agent 42Bu, Group 2 (8 rats) was given agent 43Bu, Group 3 (8 rats) was given agent 44Bu and Group 4 was given placebo.In general anesthesia the arteria carotis and vena jugularis were exposed and exteriorized.A. carotis was connected by a cannula to the HSE UNIPER UP -100 device, which is a universal perfusion system for isolated organs recording and converting blood pressure values into graphical representation in a computer.Subsequently, the tested agents were administered into v. jugularis (approximately 15 min from a. carotis and v. jugularis exteriorization) as a bolus and the values of systolic blood pressure were recorded within the period of 18 min of their administration.
The solution for anaesthesia was administered i.m. in the femur area at a dose of 0.5 ml/100 g of body mass and was composed of: xylazinum (Rometar ® 2% inj.) and ketamini hydrochloridum (Narkamon ® Spofa 1% inj.) in ratio 1:20.All tested agents were administered at doses of 2.5 mg/kg of body mass as a bolus at a 1 ml dose.As a vehicle for tested agents, a 5% solution of dimethylsulfoxide in saline (Infusio natrii chlorati isotonica Infusia ® ) was used.The placebo group was administered only 1 ml of 5% solution of dimethylsulfoxide in saline.The experiment was always performed in the morning.First, experiment with placebo group was performed followed by the experiments with Group 1, Group 2 and Group 3, respectively.
The project was approved and monitored by the local University Ethical Committee.

Statistics
Statistical evaluation was performed using the Microsoft Excel spreadsheet.The statistical significance of differences between the three tested groups and the placebo group was evaluated using Student's t-test where p < 0.05 was considered as significant and p < 0.01 was considered as highly significant.The obtained values of systolic blood pressure were converted to percentage changes of systolic blood pressure in relation to initial values, where initial values of 100% were assessed before administration of the tested agents (placebo).Values of systolic blood pressure (%) after the administration of tested agents and placebo -0.5 min = time before tested agents and placebo administration 0 min = time of tested agents and placebo administration * = the value is significantly different at p < 0.05 against placebo ** = the value is significantly different at p < 0.01 against placebo blood pressure 9 minutes after administration.We consider both of the above mentioned features of agent 44Bu -immediate effect on blood pressure and rapid fading away of its therapeutical effect -as beneficial and very promising for further laboratory in vivo testing of other pharmacodynamic and pharmacokinetic parameters of agent 44Bu.