Effects of erythropoietin on advanced pulmonary vascular remodelling.

Erythropoietin (EPO) mobilises endothelial progenitor cells and promotes neovascularisation in heart failure. The present authors studied the effects of EPO on pulmonary vascular and cardiac remodelling in a model for flow-associated pulmonary arterial hypertension (PAH). PAH was induced in adult male Wistar rats by the injection of monocrotaline combined with an abdominal aortocaval shunt 1 week later (PAH or experimental group). Immediately afterwards, rats were randomised into those who received treatment with EPO (PAH+EPO group) and controls. Pulmonary and systemic haemodynamics, and right ventricular and pulmonary vascular remodelling were evaluated 3 weeks later. Vascular occlusion of the intra-acinar pulmonary vessels (13.4+/-0.7 versus 16.7+/-1.3% in PAH+EPO and PAH, respectively) and medial wall thickness of the pre-acinar arteries (wall-to-lumen ratio 0.13+/-0.01 versus 0.17+/-0.01 in PAH+EPO and PAH, respectively) decreased after treatment with EPO. Moreover, right ventricular capillary density was increased by therapy (2,322+/-61 versus 2,100+/-63 capillaries x mm(-2) in PAH+EPO and PAH, respectively). Increased mean pulmonary arterial pressure and decreased right ventricular contractility in the model were not altered by EPO treatment. In this rat model of flow-associated pulmonary arterial hypertension, erythropoietin treatment beneficially affected pulmonary vascular and cardiac remodelling. These histopathological effects were not accompanied by significantly improved haemodynamics.

Is time to peak effect of neuromuscular blocking agents dependent on dose? Testing the concept of buffered diffusion.

BACKGROUND AND OBJECTIVES: For neuromuscular blocking agents, an inverse relationship between potency and time to peak effect has been observed. To test the hypothesis that this relationship is due to buffered diffusion, we investigated the influence of dose on time to peak effect. Pharmacokinetic-pharmacodynamic simulations were performed to support the expected relationships between potency, dose, peak effect and time to peak effect. METHODS: Pigs (20-28 kg body weight) were anaesthetized with ketamine and midazolam, followed by pentobarbital and fentanyl intravenously. Neuromuscular block was measured by stimulating the peroneal nerve supramaximally at 0.1 Hz and measuring the response of the tibialis anterior muscle mechanomyographically. After an initial dose to establish the individual ED90 of a neuromuscular blocking agent (rocuronium, vecuronium, pipecuronium or d-tubocurarine), five different doses of the same compound were administered to each animal, aiming at 20%, 40%, 60%, 75% or 90% block, in a random order. Doses were given 45 min after complete recovery of the twitch response. RESULTS: For rocuronium and pipecuronium, time to peak effect increased with dose, whereas dose did not affect time to peak effect of vecuronium and d-tubocurarine. Simulations predict that time to peak effect decreases with dose if buffered diffusion is taken into account. CONCLUSIONS: The results suggest that buffered diffusion does not play a dominant role in the time to peak effect of neuromuscular blocking agents. Therefore it is unlikely that the observed inverse relationship between potency and time to peak effect of neuromuscular blocking agents in the clinical range is due to buffered diffusion.

Effective treatment of verapamil intoxication with 4-aminopyridine in the cat.

To study the value of 4-aminopyridine as an antidote to verapamil intoxication, we subjected 12 adult cats to verapamil poisoning by administering doses of 4.0-25.0 mg/kg verapamil by intravenous infusion. Six animals were given 4-aminopyridine 2 X 0.5 mg/kg i.v. after the verapamil infusion was stopped and the other six animals (the control group) were not. Verapamil caused profound cardiovascular depression and also partial neuromuscular block, both of which were completely reversed by 4-aminopyridine within 50 min, in spite of extremely high serum verapamil concentrations (ranging between 3,700 and 13,500 ng/ml). The six animals that received 4-aminopyridine survived the verapamil intoxication, whereas four of the six animals in the control group died. The results suggest that 4-aminopyridine may be useful in the treatment of verapamil intoxication.

A method for studying the pharmacodynamic profile of neuromuscular blocking agents on vocal cord movements in anaesthetized cats.

1. A new in vivo experimental method is described whereby the neuromuscular blocking effects of muscle relaxants can be investigated on the intrinsic laryngeal muscles of anaesthetized cats. The peripheral tibialis anterior muscle preparation is employed in the same animal to compare the blocking effect on both preparations. 2. The intrinsic laryngeal muscles react with different sensitivities to the neuromuscular blocking agents when compared to the tibialis anterior muscle. 3. The neuromuscular response in both muscle preparations is similar with steroidal agents but appeared to be different after suxamethonium or isoquinoline analogues. 4. It is concluded that this preparation may become a useful tool for studying new muscle relaxants developed to facilitate rapid intubation conditions.

A new method for studying the relationship between hepatic uptake of drugs and their pharmacodynamic effects in anaesthetized cats.

1 A new in vivo experimental method is described whereby the liver can be temporarily excluded from the general circulation by means of a portocaval shunt operation. The influence of this manoeuvre upon the effects of pancuronium and Org 6368 was investigated using the tibialis muscle preparation of anaesthetized cats. 2 The procedure also allowed intraportal injections of the drugs to be made so that the effect of first-passage uptake by the liver could be compared with hapatic exclusion in the same animal. 3 Hepatic exclusion greatly increased the duration of action of both drugs. Whereas intraportal injection did not significantly alter the effect of pancuronium on the tibialis muscle, the effect of Org 6368 was greatly diminished when given by this route. 4 The liver appears to tolerate short periods of hepatic exclusion and it is concluded that this technique may become a useful tool for studying the handling of drugs by this organ.

A comparison of the pharmacological actions of 4-aminopyridine and two of its derivatives in the monkey.

The neuromuscular, cardiovascular and central nervous system stimulating effects of 4-aminopyridine (4-AP), 2,4-diaminopyridine (2,4-DAP) and LF-14 were investigated in the monkey. All these compounds were shown to reverse the stable neuromuscular blockade produced by the intravenous infusion of pancuronium bromide. The doses producing 50% antagonism (ED50) of the pancuronium-induced neuromuscular block were 0.50, 0.54 and 0.71 mg/kg for LF-14, 2,4-DAP and 4-AP respectively. The compounds had only slight cardiovascular effects. In contrast to 4-AP, LF-14 and 2,4-DAP did not reduce the duration of ketamine/diazepam-induced anesthesia, suggesting minimal if any central nervous system effects of these two compounds.

The isolated heart-lung preparation in the cat. An in situ model to study the role of the lungs in the disposition of drugs.

In the search for drugs with an extreme short time course of action, compounds should be developed that are rapidly distributed to and temporarily stored in well-perfused organs. Since the lungs receive the complete cardiac output and have the ability to temporarily store drugs, we have developed an in situ, isolated lung preparation in the cat to study the contribution of the lungs to the disposition of drugs. The cat's own heart perfuses the lung in situ with autologous blood. The circulation between the left ventricle and the right atrium is short-circuited via an aorta-caval shunt. The right forelimb is added to study pharmacodynamics simultaneously (only for muscle relaxants). Validation of the model for 180 min of perfusion showed complete isolation of the organs without major biochemical changes or edema and a stable muscle response. In pilot experiments with two structurally related muscle relaxants, initial muscle relaxation was followed by spontaneous recovery of neuromuscular function and a gradually decreasing plasma concentration, indicating partial disposition by the lungs. This was confirmed by direct concentration measurements in the lung. The present model may provide a powerful experimental tool to elucidate the role of the lungs in the disposition of drugs.

Pharmacokinetics and pharmacodynamics of 2,4-diaminopyridine in the cat.

The pharmacokinetics, antagonistic effects, and cardiovascular effects of 2,4-diaminopyridine (2,4-DAP) were studied in 7 anaesthetized cats. Cats received a pancuronium infusion at a constant rate to cause a 90% block of contraction of the anterior tibialis muscle, stimulated through the sciatic nerve. After steady state was reached, 2,4-DAP (750 micrograms/kg IV) was administered. Plasma, urine, and bile were collected over 8 h and analyzed by means of an HPLC assay. Plasma concentrations decreased biexponentially with half-lives of 9.0 +/- 5.7 min and 140 +/- 36 min, respectively. The volume of the central compartment was 0.85 +/- 0.27 L/kg, and the volume of distribution in the steady state was 3.1 +/- 1.1 L/kg. Total plasma clearance was 18 +/- 5 ml/kg/min. Ninety percent of the administered dose was recovered in the urine and 0.1 percent in the bile in 8 h. The antagonism of the pancuronium-induced steady-state block was 98% +/- 5%, with onset and duration of 3.5 +/- 2 min and 165 +/- 40 min, respectively.

Decreased number of acetylcholine receptors is the mechanism that alters the time course of muscle relaxants in myasthenia gravis: a study in a rat model.

BACKGROUND: In myasthenic patients, the time course of action of non-depolarizing neuromuscular blocking agents is prolonged and the sensitivity is increased. We used our antegrade perfused rat peroneal nerve anterior tibialis muscle model to investigate if this altered time course of effect and sensitivity can be explained by the decreased acetylcholine receptor concentration that is caused by the disease. METHODS: Functional acetylcholine receptors were reduced by administration of alpha-bungarotoxin or by injecting monoclonal antibodies against rat acetylcholine receptors (experimental autoimmune myasthenia gravis). After induction of anaesthesia, the model was set up and perfusion of the tibialis anterior muscle with blood was started. After stabilization of the twitch, rocuronium or pancuronium were infused until 90% block was obtained. Twitch data and infusion data were recorded and used to calculate the time course of effect and potency. RESULTS: The potency of neuromuscular blocking agents was increased and the offset of the neuromuscular block was prolonged in both the alpha-bungarotoxin groups and the experimental autoimmune myasthenia gravis groups compared to controls. CONCLUSION: This study shows that the increased sensitivity to neuromuscular-blocking agents in myasthenia gravis can be accounted for by a decreased number of acetylcholine receptors. It also shows that the antegrade perfused rat peroneal nerve anterior tibialis muscle model is a suitable model to study the effects of myasthenia gravis on the time course of effect of neuromuscular blocking agents.

Effects of hepatic uptake of vecuronium bromide and its putative metabolites on their neuromuscular blocking actions in the cat.

The neuromuscular blocking effects of equipotent doses of the three putative metabolites of vecuronium (3-hydroxy vecuronium, 17-hydroxy vecuronium and 3,17-dihydroxy vecuronium) were compared with those of an equipotent dose of vecuronium. Results obtained with the liver excluded from the circulation and after intraportal injection were compared with those following i.v. injection, in the cat. Following injection i.v., and with the liver excluded from the systemic circulation, there was a marked prolongation of duration of action (which averaged 100%) with all four compounds. After intraportal injection the only significant results were shortening of the duration of action and a decrease in the maximum blockade achieved with the 3-hydroxy metabolite. It is concluded that, in the cat, the hepatic removal of these compounds from the circulation is a major determinant of their duration of action. This process seems to be of importance for all four compounds, although the 3-hydroxy derivative appeared the most sensitive.

Does intravenous infusion of nitroglycerin potentiate pancuronium- and vecuronium-induced neuromuscular blockade?

The dose requirement for 95% depression of twitch tension and the time course of the neuromuscular blocking effects of the ED95 of pancuronium bromide and vecuronium bromide were studied during intravenous infusion of glucose, 5%, and nitroglycerin, 1 microgram X kg-1 X min-1, in 20 cats anesthetized with pentobarbital. Nitroglycerin administered continuously starting 1 hr before the administration of the ED95 and maintained during at least five maintenance doses of either pancuronium or vecuronium did not significantly potentiate the action of the neuromuscular blocking drugs, nor did it alter their time course of action. A tendency for a decrease (statistically not significant) rather than an increase in the duration of action of maintenance doses of both pancuronium and vecuronium was apparent during the treatment with nitroglycerin. These findings indicate a lack of interaction between pancuronium or vecuronium and nitroglycerin, provided that moderate doses are used.

Structure-pharmacokinetics relationship of series of aminosteroidal neuromuscular blocking agents in the cat.

To obtain more insight in the relationship between physicochemical properties of neuromuscular blocking agents (NMBAs) and their pharmacokinetic characteristics, a series of 12 aminosteroidal NMBAs, supplemented with data on five related NMBAs from the literature, was investigated in anaesthetized cats. After i.v. bolus injection, plasma concentration decreased very rapidly, showing a biphasic pattern, with half-lives ranging from 0.4 to 1.4 min, and from 3 to 10 min, respectively. Clearance was in the range from 24 to 58 ml. min(-1). kg(-1). Compounds containing an acetyl-ester group at position 3 were partly metabolized to the 3-OH derivative. The urinary excretion of the parent drug and metabolites amounted to <10% for each of the compounds. The parent drugs were excreted in large amounts into bile, along with smaller amounts of 3-OH derivatives. The terminal half-life of the urinary and biliary excretion rate were markedly longer than the apparent terminal half-life in plasma, ranging from 11 to 40 min, and from 119 to 489 min in urine and bile, respectively. Lipophilicity of the NMBAs, expressed as the partition coefficient octanol/Krebs (log P), was found to be correlated positively with unbound plasma clearance and unbound initial plasma clearance, and negatively with plasma half-life, volume of distribution at steady state, and mean residence time. The increase of the unbound plasma clearance with increasing lipophilicity is counteracted by the concurrent increase in plasma protein binding.

Interaction of 2,4-diaminopyridine with cholinesterase inhibitors.

The interaction between 2,4 diaminopyridine (2,4-DAP) and neostigmine or edrophonium was studied in vivo in the rat sciatic nerve-tibialis anterior muscle preparation using the constant infusion technique of pancuronium. The ED50 value (dose of drug which produces 50% antagonism) decreased from 17 to 7 micrograms/kg with neostigmine and from 70 to 20 micrograms/kg with edrofonium, when these 2 compounds were co-administered with 40 micrograms/kg 2,4-DAP. The ED50 value of 2,4-DAP decreased from 160 to 90 micrograms/kg when 3.5 micrograms/kg neostigmine was added. From these experiments we conclude that 2,4 DAP acts synergistically with both neostigmine and edrophonium. In experiments using the train of four (T4) stimulation, we compared the antagonistic potencies of the drugs alone and in combination with each other. Neostigmine proved to be the most potent. 2,4-DAP given alone was not able to restore the muscle contraction completely (T4 ratio greater than 70%) up to a dose of 400 micrograms/kg. The doses of neostigmine and edrophonium could be halved, when they were combined with a small amount of 2,4-DAP without apparent lose of antagonistic potency.

The effect of single twitch and train-of-four stimulation on twitch forces during stable neuromuscular block.

OBJECTIVE: We investigated whether the response to a single twitch (ST) stimulus or the first response (T1) to a train-of-four (TOF; 4 stimuli at 2 Hz) stimulus following a stimulus interval of 10 s (i.e., the time between two consecutive ST or TOF stimuli) is influenced by the preceding stimulus in the presence of a stable 50% neuromuscular block. In addition, we determined whether ST and TOF stimulation yield different results under these circumstances. METHODS: Twitch forces were measured in both tibialis anterior muscles of six cats. In the presence of a stable 50% neuromuscular block the stimulation pattern (ST or TOF) or stimulus interval (3.3, 10 or 30 s) was varied every 30 min. A linear mixed model was used for statistical analysis. RESULTS: ST forces with a stimulus interval of 3.3 s were 10.3% (95% CI: 7.3-13.3%) smaller than those with a stimulus interval of 10 s. For T1 forces this effect was 15.2% (95% CI: 12-18.4%). There was no significant difference between twitch forces with stimulus intervals of 30 and 10 s. For a stimulus interval of 3.3 s the ST forces exceeded the T1 forces by 7.6% (95% CI: 4.4-10.8%); no significant differences were found between the ST and T1 forces for stimulus intervals of 10 and 30 s. CONCLUSIONS: The ST or T1 force during stimulation with a stimulus interval of 10 s or more during a stable 50% neuromuscular block in the tibialis anterior muscle of the cat is not affected by the preceding stimulus. In addition, ST and T1 forces do not differ when employing a stimulus interval of 10 s or more under these circumstances. Our results thus indicate that the known differences between ST and T1 forces after a bolus injection of a muscle relaxant can not be explained by differences in acetylcholine release when the stimulus interval exceeds 10 s.

Pharmacokinetics and disposition of pipecuronium bromide in the cat.

The pharmacokinetics and hepatic disposition of pipecuronium have been investigated in cats with normal and absent renal function. A combined fluorimetric and chromatographic technique was used to determine the concentrations of pipecuronium and its metabolites in the samples. Following intravenous injection of 150 micrograms kg-1, pipecuronium disappeared from the plasma bi-exponentially with half-lives of 9.8 +/- 5.4, 77.7 +/- 9.7 min and 7.2 +/- 5.0, 100, 6 +/- 23.7 min; the Vd was 362.3 +/- 74.9 ml kg-1 and 123.7 +/- 14.6 ml kg-1 and the clearance was 5.0 +/- 0.9 ml min-1 kg-1 and 1.0 +/- 0.1 ml min-1 in the animals with and without renal function, respectively. In the animals with normal kidney function 53%, 12% and 8% of the administered dose of pipecuronium were recovered within 8 h in the urine, bile and liver, respectively. In 'nephrectomized' cats the lack of renal elimination was to a great extent compensated for by increased hepato-biliary elimination. Absence of renal function significantly altered the pharmacokinetic parameters and prolonged the time-course of the neuromuscular blocking effects of pipecuronium. Neither temporary hepatic exclusion nor intraportal administration of pipecuronium indicated any significant role of the liver in handling pipecuronium. Renal excretion seems to be the predominant route of elimination. No metabolites were found in this study.

Hemodynamic effects of high dosages of verapamil and the lack of protection by 4-aminopyridine in the rabbit.

4-Aminopyridine (1 mg/kg) restored a 35% fall in arterial blood pressure in rabbits evoked by a verapamil infusion (0.5 mg/kg/min) more effectively than calcium-levulate (approx. 13 mg/kg). However, the lethal dose of verapamil for rabbits (mean: 7.1 mg/kg given by intravenous infusion in 14.3 min) was not affected by 4-aminopyridine pretreatment.

Direct action of 4-aminopyridine on the contractility of a fast-contracting muscle in the cat.

1. The effects of 4-aminopyridine on the contractility of the fast-contracting tibialis anterior and the slow-contracting soleus muscles of cats under chloralose anaesthesia have been studied. 2. 4-Aminopyridine, in doses of 0.5 mg/kg and above, produced a slowly developing increase in the twitch tension of directly stimulated chronically denervated and of indirectly stimulated innervated tibialis anterior muscles, but had little or no effect on twitches of soleus muscles. The effect of innervated tibialis anterior muscles was more pronounced than that on chronically denervated muscles, but it was nevertheless concluded that the whole effect on innervated muscles was the result of a direct action on the muscle fibres. The simultaneously occurring facilitatory action on neuromuscular transmission, which is manifested in the anti-curare action of 4-aminopyridine, had a faster time-course and occurred in both the tibialis anterior and the soleus muscles. 3. 4-Aminopyridine antagonized dantrolene sodium on on the tibialis anterior muscle but not on the soleus muscle. The antagonism could be described as physiological antagonism since it simply reflected the opposing actions on contractility of the two drugs. 4. 4-Aminopyridine was without effect on maximal tetanic tension of either the tibialis anterior or the soleus muscle. 5. It seems clear from the literature that a species difference exists with regard to the ability of 4-aminopyridine to increase muscle contractility. The results described in this paper show that muscle differences within the same species also exist.

Mechanisms underlying the prolonged duration of action of muscle relaxants caused by extrahepatic cholestasis.

The neuromuscular blocking effects of Org 6368 and Org NC 45 were studied in rats with experimental cholestasis and in controls. The effect of Org NC 45 during infusion of taurocholate was investigated. In cholestatic rats we observed a three-fold increase of the duration of action of Org 6368 and Org NC 45. The same was observed for Org NC 45 with taurocholate. In the rat phrenic nerve-hemidiaphragm preparation taurocholate potentiated the neuromuscular blockade of Org 6368, pancuronium and gallamine, but not Org NC 45. Increased bile salt concentrations caused strong inhibition of hepatic uptake and biliary excretion of Org 6368 and tubocurarine in isolated perfused livers. Taurocholate and glycocholate were more potent than cholate and chenodeoxycholate. Cholestatic livers exhibited a clearance of Org 6368 which was 50% of control. We conclude that the prolonged duration of action of certain muscle relaxants because of cholestasis results from both inhibition of hepatic uptake by the accumulated bile salts and a general deterioration of liver transport function.

Disposition of vecuronium bromide in the cat.

The disposition of vecuronium bromide has been investigated in six normal cats (group I) and in six cats with ligated renal pedicles (group II). A combined fluorimetric and chromatographic technique was used to determine the concentrations of vecuronium and its metabolites in biological material. After i.v. injection of 0.6 mg kg-1, vecuronium disappeared rapidly from the plasma of the normal cat. Concentrations decreased bi-exponentially with half-lives of 4.6 and 31 min, respectively. The steady state volume of distribution was 0.23 litre kg-1 and the clearance 11 ml min-1 kg-1. Seventy percent of an i.v. dose of vecuronium (or its metabolites) was recovered: 15% in the urine, 40% in the bile and 15% in the liver. Only 3.8% of this consisted of the 3-hydroxy metabolite. There were no significant differences in pharmacokinetic data or in the amounts of vecuronium and its metabolites recovered in cats with ligated renal pedicles. The 15% of vecuronium normally excreted by the kidney was compensated for by increased hepatic and biliary concentration of vecuronium.

The pulmonary first-pass uptake of five nondepolarizing muscle relaxants in the pig.

BACKGROUND: It is not known whether the lungs influence the early pharmacokinetics of muscle relaxants and, if they do, whether differences in pulmonary uptake contribute to the differences in potency and/or onset time among muscle relaxants. Because the lungs are uniquely positioned, receive the entire cardiac output, have a large capillary surface area, and can temporarily store various basic drugs, the authors determined whether substantial pulmonary first-pass uptake of muscle relaxants occurs. METHODS: In 14 pigs, rocuronium, vecuronium, Org 9487, Org 7617, or d-tubocurarine were administered simultaneously with indocyanin green within 1 s into the right ventricle, and then arterial blood was sampled every 1.2 s (in the first min). The tibialis muscle response was registered mechanomyographically. RESULTS: The maximum block was 93% (68-100% [median and range]). Onset times ranged from 83 s (78-86 s) for rocuronium to 182 s (172-192 s) for d-tubocurarine. Fraction-versus-time outflow curves showed that the peak of muscle relaxants and indocyanin green occurred almost simultaneously. Pulmonary first-pass retention was negligible. The retention of muscle relaxants at 95% passage of indocyanin green was -9% (-31 to 18%). The difference in the mean transit time between muscle relaxant and indocyanin green was 1.0 (0.8 to 1.4), 0.2 (-0.8 to 0.3), 0.3 (0.2 to 0.4), 0.5 (0.2 to 1.3), and -2.2 s for rocuronium, vecuronium, Org 9487, Org 7617, and d-tubocurarine, respectively. CONCLUSIONS: There is no substantial pulmonary first-pass uptake of rocuronium, vecuronium, Org 9487, Org 7617, or d-tubocurarine in pigs. Therefore, differences in pulmonary first-pass uptake do not contribute to the differences in potency and/or onset time among muscle relaxants.