Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 μg mL^?1. It took approximately 8h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the ‘buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off 12000–14000) in a water bath shaker kept at 37°C and a rate of 50 oscillations min^?1. Azosemide was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24h. The binding of azosemide to 4% HSA was constant (95.5 ± 0.142%) at azosemide concentrations ranging from 5 to 100 μg mL^?1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6% respectively. The binding was also dependent on incubation temperature; the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37°C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9, and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 UmL^?1), AAG (up to 0.16%), sodium azide (NaN₃, up to 5%), its metabolite, Ml (up to 10 μg mL^?1), and anticoagulants (EDTA and citrate).     

Pharmacokinetics and pharmacodynamics of azosemide after intravenous and oral administration to rats: Absorption from various GI segments

Azosemide, 5, 10, 20, and 30 mg/kg, was administered both intravenously and orally to determine the pharmacokinetics and pharmacodynamics of azosemide in rats (n=7–12). The absorption of azosemide from various segments of GI tract and the reasons for the appearance of multiple peaks in plasma concentrations of azosemide after oral administration were also investigated. After intravenous (iv) dose, the pharmacokinetic parameters of azosemide such ast _1/2, MRT, V_SS, CL, CL_R, and CL_NR were found to be dose-dependent in the dose ranges studied. The percentages of the iv dose excreted in 8-hr urine as azosemide, MI (a metabolite of azosemide), glucuronide of azosemide, and glucuronide of MI—expressed in terms of azosemide—were also dose-dependent in the dose ranges studied. The data above suggest saturable metabolism of azosemide in rats. The measurements taken after the iv administrations such as the 8 hr urine output, the total amount of sodium and chloride excreted in 8-hr urine per 100 g body weight, and diuretic, natriuretic, kaluretic, and chloruretic efficiencies were also shown to be dose-dependent. However, the total amount of potassium excreted in 8-hr urine per 100 g body weight was dose-independent. Similar dose-dependency was also observed following oral administration. Azosemide was absorbed from all regions of GI tract studied and approximately 93.5, 79.1, 86.1, and 71.5% of the doses (5, 10, 20, and 30 mg/kg, respectively) were absorbed between 1 and 24 hr after oral administration. The appearance of multiple peaks after oral administration is suspected to be due mainly to the gastric emptying pattern. The percentages of azosemide absorbed from the GI tract as unchanged azosemide for up to 24 hr after oral doses of 5, 10, 20, and 30 mg/kg were significantly different with doses (decreased with increasing doses), suggesting that the problem of azosemide precipitating in acidic gastric juices or dissolution may have at least partially influenced the absorption of azosemide after oral administration. This paper was supported in part by Non-Directed Research Fund, Korea Research Foundation, 01-F-0124, 1994. This paper is taken from a dissertation submitted by Sun H. Lee to the Graduate School, Seoul National University, in partial fulfillment of Doctor of Philosophy Degree requirements.     

Development and Disappearance of Wavy Ribs Caused by Azose-mide in the Mouse Fetus

ABSTRACT Treatment of gravid mice (day 13 of gestation) with azosemide induces wavy ribs in their fetuses. The present study examined the morphological sequence of azosemide-induced wavy ribs, from their appearance through disappearance, by means of cartilage-bone double stain, hematoxylin and eosin stain, von Kóssa's stain for calcium salts and alkaline phosphatase stain. Both endochondral and intramembranous ossifications of the ribs were inhibited in day 14 azosemide-treated fetuses. A curvature of the ribs occurred on day 15 of gestation, and excessive uncalcified osteoid was present on the outer surfaces of the bent region. In the region of curvature, endochondral and intramembranous ossification, which is remarkable in normal, was not observed. Calcification, which began on day 14 in untreated fetuses, started including the bent region on day 16 of gestation. However, ribs in azosemide-treated fetuses exhibited alkaline phosphatase activity like in the control, during days 14–16 of gestation. These observations suggest that the defective ossification in azosemide-treated fetuses is caused by inhibition of calcium salts deposition on the uncalcified osteoid. During the lactation period, the bend of ribs was gradually normalized, and the bend was disappeared on day 9 postpartum.     

阿佐塞米的合成

以2－氟－4－氯苯甲酸为起始原料，经氨磺酰化、酰胺化、氰化、与2－噻吩甲胺缩合，最后与叠氮化钠和氯化铵进行1，3－环加成合成阿佐塞米，总收率22％。     

Pharmacokinetic and pharmacodynamic changes of azosemide after intravenous and oral administration of azosemide to uranyl nitrate-induced acute renal failure rats

The pharmacokinetic and pharmacodynamic differences of azosemide were investigated after intravenous (IV) and oral administration of azosemide, 10 mg kg⁻¹, to the control and uranyl nitrate-induced acute renal failure (U-ARF) rats. After IV administration, the plasma concentrations of azosemide were significantly higher in the U-ARF rats and this resulted in a significant increase in AUC (2520 versus 3680 μg min mL⁻¹) and significant decrease in Cl (3.96 versus 2.72 mL min⁻¹ kg⁻¹) of azosemide. The significant decrease in Cl in the U-ARF rats was due to the significant decrease in Cl_r of azosemide (1.55 versus 0.00913 mL min⁻¹ kg⁻¹) due to the decrease in kidney function in the U-ARF rats. After IV administration, the urine output (38.5 versus 8.45 mL 100 g⁻¹ body weight) and urinary excretion of sodium (4.60 versus 0.420 mmol 100 g⁻¹ body weight) decreased significantly in the U-ARF rats. After oral administration, the AUC_{0–8 h} of azosemide decreased significantly (215 versus 135 μg min mL⁻¹) in the U-ARF rats possibly due to the decreased GI absorption of azosemide. After oral administration, the 24-h urine output decreased considerably (16.1 versus 11.2 mL 100 g⁻¹ body weight, p <0.098) and the 24-h urinary excretion of sodium (1.74 versus 0.777 mmol 100 g⁻¹ body weight) decreased significantly in the U-ARF rats. The IV and oral doses of azosemide needed to be modified in the acute renal failure patients if the present rat data could be extrapolated to humans. © 1998 John Wiley & Sons, Ltd.     

Effects of the rate and composition of fluid replacement on the pharmacokinetics and pharmacodynamics of intravenous azosemide

The effects of differences in the rate and composition of intravenous fluid replacement for urine loss on the pharmacokinetics and pharmacodynamics of azosemide were evaluated using rabbit as the animal model. Each rabbit received a 4 h constant intravenous infusion of 1 mg kg⁻¹ azosemide with 0% replacement (treatment I, n =4), 50% replacement (treatment II, n =5), and 100% replacement (treatment III, n =5) with lactated Ringer's solution, as well as with 100% replacement with 5% dextrose in water (D-5-W, treatment IV, n =5). Renal clearance and urinary excretion rate of the drug in treatment III were considerably higher than those in treatments I, II, and IV. In spite of the similarities in kinetic properties, diuretic and/or natriuretic effects of azosemide were markedly different among the four treatments. For example, the mean 8 h urine output values were 98·2, 178, 733, and 237 mL for treatments I–IV, respectively, and the corresponding values for sodium excretion were 11·1, 19·4, 76·4, and 14·2 mmol, and for chloride 13·4, 23·8, 78·9, and 17·1 mmol. Except for treatment III, diuresis and/or natriuresis were found to be time dependent, generally decreasing with time until reaching a low plateau during the later hours of infusion. The present findings also show that (i) no fluid replacement and 100% replacement with D-5-W both produce the same degree (not significantly different) of severe acute tolerance in natriuresis, indicating the insignificance of water compensation in tolerance development; (ii) in treatment II, where neutral sodium balance was achieved, the development of acute tolerance in diuresis can mainly be attributed to negative water balance under this special condition; and (iii) at steady state the hourly diuresis and natriuresis can differ up to about 6·87- and 5·21-fold between treatments. Some implications for the bioequivalence evaluation of dosage forms of azosemide are discussed. © 1997 John Wiley & Sons, Ltd.     

EFFECT OF PHENOBARBITAL, 3-METHYLCHOLANTHRENE,AND CHLORAMPHENICOL PRETREATMENT ON THE PHARMACOKINETICS AND PHARMACODYNAMICS OF AZOSEMIDE IN RATS

The effects of pretreatment with the enzyme inducers phenobarbital (PB) and 3-methylcholanthrene (3-MC) and the enzyme inhibitor chloramphenicol (CM) on the pharmacokinetic and pharmacodynamic parameters of azosemide were examined after intravenous (IV) administration of azosemide, 10 mg kg⁻¹, to rats. The nonrenal clearance (1·63 versus 3·30 mL min⁻¹ kg⁻¹) of azosemide increased significantly in 3-MC pretreated rats. This suggested that the nonrenal metabolism of azosemide increased by pretreatment with 3-MC. This relationship was supported by the significant decrease in 24 h urinary excretion of unchanged azosemide in 3-MC pretreated rats (54·1 versus 41·1% of IV dose). This relationship was also supported at least in part by the results of a liver homogenate study; the amount of azosemide remaining per gram of liver decreased significantly (48·2 versus 43·0 μg) and the amount of M1 formed increased significantly (4.88 versus 6.66 μg when expressed in terms of azosemide) in 3-MC pretreated rats after 30 min incubation of 50 μg azosemide in 9000 g supernatant fractions of liver homogenates. The content of hepatic cytochrome P-450 (0·751 versus 1·57 nmol/mg protein) and the weight of liver (3.53 versus 4·20% of body weight) increased significantly in 3-MC pretreated rats, suggesting that the metabolizing enzyme(s) for azosemide seemed to be induced by pretreatment with 3-MC. The 8 h urine output (29·2 versus 18·1 mL) and 8 h urinary excretion of sodium (4·02 versus 2·39 mmol) and chloride (4·01 versus 2·73 mmol) per 100 g body weight decreased significantly in 3-MC pretreated rats. However, the diuretic, natriuretic, kaluretic, and chloruretic efficiencies were not significantly different between the control and 3-MC pretreated rats. The pharmacokinetic and pharmacodynamic parameters of azosemide were not significantly different between the control and PB pretreated rats, and similar results were also obtained from the control and CM pretreated rats. The above data indicate that the metabolizing enzyme(s) for azosemide seem(s) to be neither induced by PB pretreatment nor inhibited by CM pretreatment. However, the content of hepatic cytochrome P-450 and the weight of liver increased significantly in PB pretreated rats, while the values were not significantly different between the control and CM pretreated rats. © 1997 John Wiley & Sons, Ltd.     

THE EFFECT OF INTRAVENOUS INFUSION TIME ON THE PHARMACOKINETICS AND PHARMACODYNAMICS OF THE SAME TOTAL DOSE OF AZOSEMIDE IN RABBITS

The pharmacokinetics and pharmacodynamics of azosemide were evaluated after intravenous (IV) administration of the same total dose of azosemide, 1 mg kg⁻¹, in different infusion times, 1 min (treatment I) and 4 h (treatment II) to rabbits (n =5, each). The loss of water and electrolytes in urine induced by azosemide was immediately replaced with infusion of equal volume of lactated Ringer's solution. Some pharmacokinetic parameters of azosemide were different between treatments I and II. For example, the mean value of terminal half-life (70·5 versus 107 min), total body clearance (5·88 versus 8·32 mL min⁻¹ kg⁻¹), renal clearance (3·45 versus 6·51 mL min⁻¹ kg⁻¹), and mean residence time (18·5 versus 31·7 min) increased significantly in treatment II. The 8 h urine output (236 versus 733 mL) and 8 h urinary excretion of sodium (29·2 versus 76·4 mmol) and chloride (27·5 versus 78·9 mmol) increased significantly in treatment II although the total amount of 8 h urinary excretion of unchanged azosemide increased by only 15% in treatment II. This could be due to the fact that the urinary excretion rates of azosemide in treatment II remained for a longer period of time close to the maximally efficient urinary excretion rates of azosemide for both urine output and urinary excretion rates of sodium than in treatment I. Plasma concentrations of azosemide and hourly urine output and hourly urinary excretion of azosemide, sodium, potassium, and chloride during the apparent steady state (between 2 and 4 h) in treatment II were fairly constant. © 1997 by John Wiley & Sons, Ltd.     

Teratogenicity of Azosemide, a Loop Diuretic, in Rats, Mice and Rabbits

Teratogenic effects of azosemide, a loop diuretic, were investigated in rats, mice and rabbits. Azosemide was given orally to pregnant rats, mice and rabbits during organogenesis. The pregnant animals were killed at term and their fetuses were examined for external, visceral and skeletal abnormalities. In rats, azosemide at 10–30 mg/kg/day did not affect intrauterine growth, resorptions and rates of external and visceral malformations. Treatment with 90 mg/kg/day resulted in a significant increase in skeletal abnormalities such as wavy ribs, bent scapula and bent humerus. However, the skeletal abnormalities observed in term fetuses could not be found in adult offspring, indicating that they were temporary. In mice, 1250 mg/kg/day of azosemide caused maternal death, abortion, and retarded maternal and fetal weight. Treatment with 200–500 mg/kg/day did not induce fetal mortalities, external and visceral malformations. Skeletal abnormalities increased in dose-dependent fashion. The type of abnormalities was identical to that encountered in rat fetuses. Furosemide as a positive control also produced similar types of skeletal abnormalities in mouse fetuses. In rabbits, azosemide did not have embryolethal or teratogenic effects even at the highest dose (6 mg/kg/ day), which caused maternal death. Treatment for a different 3-day period and then a different day during organogenesis in rats and mice showed that the sensitive period was days 15–17 of gestation with a peak on day 16 in rats, and days 12–15 with a peak on day 13 in mice.     

Pharmacokinetics and pharmacodynamics of azosemide

Azosemide is used in the treatment of oedematous states and hypertension. The exact mechanism of action is not fully understood, but it mainly acts on both the medullary and cortical segments of the thick ascending limb of the loop of Henle. Delayed tolerance was demonstrated in humans by homeostatic mechanisms (principally an increase in aldosterone secretion and perhaps also an increase in the reabsorption of solute in the proximal tubule). After oral administration to healthy humans in the fasting state, the plasma concentration of azosemide reached its peak at 3-4 h with an absorption lag time of approximately 1 h and a terminal half-life of 2-3 h. The estimated extent of absolute oral bioavailability in humans was approximately 20.4%. After oral administration of the same dose of azosemide and furosemide, the diuretic effect was similar between the two drugs, but after intravenous administration, the effect of azosemide was 5.5-8 times greater than that in furosemide. This could be due to the considerable first-pass effect of azosemide. The protein binding to 4% human serum albumin was greater than 95% at azosemide concentrations ranging from 10 to 100 microg/ml using an equilibrium dialysis technique. The poor affinity of human tissues to azosemide was supported by the relatively small value of the apparent post-pseudodistribution volume of distribution (Vdbeta), 0.262 l/kg. Eleven metabolites (including degraded products) of azosemide including M1, glucuronide conjugates of both M1 and azosemide, thiophenemethanol, thiophencarboxylic acid and its glycine conjugate were obtained in rats. Only azosemide and its glucuronide were detected in humans. In humans, total body clearance, renal clearance and terminal half-life of azosemide were 112 ml/min, 41.6 ml/min and 2.03 h, respectively. Azosemide is actively secreted in the renal proximal tubule possibly via nonspecific organic acid secretory pathway in humans. Thus, the amount of azosemide that reaches its site of action could be significantly modified by changes in the capacity of this transport system. This capacity, in turn, could be predictably changed in disease states, resulting in decreased delivery of the diuretic to the transport site, as well as in the presence of other organic acids such as nonsteroidal anti-inflammatory drugs which could compete for active transport of azosemide. The urinary excretion rate of azosemide could be correlated well to its diuretic effects since the receptors are located in the loop of Henle. The diuretic effects of azosemide were dependent on the rate and composition of fluid replacement in rabbits; therefore, this factor should be considered in the evaluation of bioequivalence assessment.