Br. J. clin. Pharmac. (1991), 32, 489-493

The influence of co-administered organic acids on the kinetics and dynamics of frusemide De K. SOMMERS, E. C. MEYER & J. MONCRIEFF Department of Pharmacology, University of Pretoria, P.O. Box 2034, Pretoria 0001, South Africa

1 This study examines the effects of pretreatment with probenecid with and without pyrazinamide on the elimination kinetics and diuretic action of frusemide. 2 Six normal male volunteers received 40 mg frusemide i.v. on three occasions; i.e. once on its own and twice after pretreatment with 2 g probenecid with and without 3 g pyrazinamide. Both these latter drugs were administered orally 3 h before frusemide administration thereby attempting optimal suppression of proximal tubular secretion. Urinary losses were replaced i.v. with isovolumetric amounts of normal saline while insensible losses were compensated for by taking tap water orally. 3 The mean cumulative urinary frusemide excretion was significantly and similarly decreased by pretreatment with probenecid (34.9%) and probenecid plus pyrazinamide (33.6%), but the mean total volume of diuresis and the mean cumulative urinary sodium excretion did not differ significantly between treatments over the 5 h period. 4 The diuretic efficiency of frusemide was significantly increased with probenecid pretreatment during the first 90 min period after frusemide administration. Furthermore, in the first 30 min after administration the percent sodium fractional excretion was higher after pretreatment with probenecid even though the mean frusemide excretion rate was more than three times with frusemide alone than with probenecidfrusemide (374.4 ,ug min-1 vs 119.1 ,ug min- 1). Pretreatment with probenecid results in a higher concentration on the peritubular or blood side of the tubules and these results lead us to question the unconditional acceptance of a luminal site of action for the loop diuretics. Alternatively, probenecid may act in some other way to increase the effects of frusemide.

Keywords

frusemide

probenecid

diuretic efficiency

Introduction

Glomerular filtration of frusemide is greatly limited by its substantial (i.e. 95%) binding to plasma proteins (Cutler et al., 1974). Being a weak organic acid (pKa 3.9), it is primarily excreted by renal tubular secretion (Deetjen, 1966). The amount of frusemide in the tubular fluid rather than plasma concentration is the main determinant of frusemide diuresis (Rose et al., 1976) and, therefore, agents which interfere with its transport into the tubular lumen should antagonize its diuretic action. However, probenecid pretreatment in man increases the overall response to frusemide (Brater, 1978), in contrast to animal studies in which probenecid decreased response by inhibiting proximal renal tubular secretion of fruse-

mide to its active site (Hook & Williamson, 1965; Senft, 1965). In the studies of Brater (1978) and Honari et al. (1977) on humans the doses of probenecid were much smaller than those used in the animal studies. Other explanations could be either inter-species differences in the acid transport system (Holland & Williamson, 1979) or multiple systems. In snakes, for instance, urate and p-aminohippurate are secreted by separate mechanisms while probenecid depresses the secretion of both substances (Dantzler, 1970). In man, both pyrazinamide and probenecid can suppress the tubular secretion of uric acid (Fanelli et al., 1977), while the latter, but not pyrazinamide, depresses the secretion of ampicillin

Correspondence: Dr De K. Sommers, Department of Pharmacology, University of Pretoria, P.O. Box 2034, Pretoria 0001, South Africa

489

490

De K. Sommers, E. C. Meyer & J. Moncrieff

(Sommers et al., 1987). The renal organic anion transport system in man, therefore includes both a pyrazinoateand a probenecid-sensitive secretory mechanism. We report here our measurements in man of the effects of pretreatment with probenecid with and without pyrazinamide on the elimination kinetics and diuretic action of frusemide. Methods

Six healthy ambulatory male volunteers, mean age 21.7 years and mean weight 76.3 kg were the subjects. None smoked or had cardiac, hepatic or renal disease. The protocol had been approved by the Ethical Committee of the University of Pretoria and the volunteers gave their written informed consent. A cross-over experimental design was followed allowing at least a 2 week interval between treatments. The volunteers were requested to refrain from adding raw salt to their food but no attempt was made to modify their diet. However, on each day before admission to the Institute at 06.00 h three standard meals low in sodium content were provided. Then followed an overnight fast and breakfast was provided only at 3 h after the intravenous administration of frusemide. Before this i.v. injection the volunteers emptied their bladders, taking a urine specimen, and drank 500 ml tap water. The six subjects completed a total of six different studies performed in random order. This paper reports the results of the three studies assessing the effects of probenecid and pyrazinamide on the kinetics of and response to frusemide. (A separate paper is planned for the other three studies which examine the effects of indomethacin, labetalol and enalapril on the response to i.v. frusemide). Each subject performed one study with intravenous administration of 40 mg alone and two studies with the same dose of frusemide after pretreatment with 2 g probenecid (Benemid®) on one occasion and 2 g probenecid plus 3 g pyrazinamide (Pyrazide®) on the other. Both these latter drugs were administered orally 3 h before frusemide administration thereby attempting optimal suppression of proximal tubular secretion. Both drugs reach peak concentrations in plasma after about 2 h and the half-lives of probenecid and pyrazinamide are, respectively, 8 ± 3 h and 10 h (Anonymous, 1989; Weiner & Mudge, 1985). The subjects remained in a recumbent position throughout the study and stood only to void at the end of each collection period. Urine samples were collected by spontaneous voiding at 0, 30, 60, 90, 120, 180, 240 and 300 min after frusemide administration. Blood specimens were collected at 0, 0.5, 1, 2 and 4 h. Urinary losses were replaced intravenously (via a cannula placed in a forearm vein) with isovolumetric amounts of normal saline solution over the time interval of the subsequent collection period. Insensible losses were replaced orally with 50 ml h-1 tap water.

Laboratory determinations Sodium, potassium and creatinine were measured in blood and urine with a Beckman Astra-S Automated

Stat-Routine Analyzer employing an ion-specific electrode. The determination of frusemide in urine was performed by high performance liquid chromatography using a 250 mm x 4.6 mm i.d. Spherisorb S5 ODS2 column and isocratic elution with acetonitrile and methanol in 0.05 M (pH 2.5) ammonium phosphate buffer (20:15:65) at 2 ml min'-. The column temperature was ambient at about 240 C. The sample was injected through a loop injector (50 j,l). Clonazepam (80 ,ug ml -) was used as the internal standard and u.v. detection was at 233 nm. The peak height ratio standards curve was linear over the range tested, 2 p,g ml- -200 ug ml-l of frusemide in urine.

Pharmacodynamic analysis As previous studies by Alvan et al. (1990), Brater (1983) and Hammarlund et al. (1985) have shown that the diuretic effect of frusemide is well described by conventional concentration-effect modelling according to the Hill equation or the so called sigmoid Emax model (Holford & Sheiner, 1981) dose-response curves relating urinary frusemide excretion rate to response expressed as diuresis were analyzed by computer fitting to a sigmoidshaped curve of the format: a-d Y= +d 1 + (X/c)b

where Y is response (diuresis); X is dose (urinary excretion rate of frusemide); a is the lower asymptote (i.e. response when dose = 0); b is a 'slope factor' that determines steepness of the curve; c is the dose representing half maximal response (d + a/2); and d is the upper asymptote (i.e. response when dose = 00). Mean data (Kaojarern et al., 1982) were fitted to this format using a modified ALLFIT computer programme (DeLean et al., 1978) developed in the department. Using the non-linear regression programme NLIN for estimation of parameters which were used to construct the relationships between diuretic effects and diuretic efficiency vs frusemide excretion rate, respectively, efficiency (Eff) was calculated from the Hill equation (Alvan et al., 1990). Eff =

Emax ma

C-

C5o%S + C The percent fractional sodium excretion was calculated by the formula:

Urinary sodium Serum sodium

x

Serum creatinine x 100 Urinary creatinine

The cumulative sodium and frusemide urinary excretions were calculated for each time interval. Statistical analysis was by means of Student's paired ttest for comparing mean values, P < 0.05 being regarded as significant throughout. Values are expressed as mean + s.d. 95% confidence intervals were calculated on cumulative urine volume and cumulative sodium excretion.

Influence of organic acids on frusemide Results The diuretic efficiency of frusemide (calculated from data in Table 1) was significantly increased with probenecid pretreatment during the first 90 min period after frusemide administration (Figure 1). The mean cumulative urinary frusemide excretion (Figure 2) was significantly decreased by co-administration of probenecid (34.9%) and probenecid plus pyrazinamide (33.6%). Figure 3 illustrates the frusemide excretion rate over time. The mean total volume of diuresis did not differ significantly between treatments (frusemide: 3235.8 ± 709.4 ml 5 h-'; frusemide + probenecid: 3865.8 ± 398.1 ml 5 h-1 and frusemide + probenecid + pyrazinamide: 3420.0 ± 211.1 ml 5 h-1). The mean difference between the combination frusemide + probenecid and frusemide alone was 630 ± 655.4 ml, the 95% confidence interval being (-57.9; 1317.9). Similarly the mean difference between combination frusemide + probenecid + pyrazinamide and frusemide was 698.2 ± 1255.3 ml, the 95% confidence interval being (-619.4; 2015.8). Although the mean cumulative urinary sodium excretion (Figure 4) did not differ significantly between Table 1 Estimated parameters for the diuresis/frusemide excretion rate dose-response curve. Eo is basal diuresis and Emax is maximum obtainable diuresis caused by the drug, s is a slope factor, C50% is the frusemide excretion rate that gives half of the maximum induced diuresis

Eo (ml min-1)

3.15 ± 25.77 ± 2.04 ± 53.70 ±

Emax (ml min-1) s

C5o% (p.g min-')

3.7 3.88 1.4 18.14

-4.59 ± 78.79 ± 0.84 ± 240.64 ±

treatments, the average excretion was generally higher after the combination regimens than after frusemide alone. The mean difference between the combination frusemide + probenecid and frusemide alone was 97.2 ± 136.6 mmol, the 95% confidence interval being (-46.2; 240.6). Similarly, the mean difference between the combination frusemide + probenecid + pyrazinamide and frusemide was 147.7 (±, 196.8) mmol with the 95% confidence interval (-58.9; 354.3). At 30 min the percent fractional sodium excretion (Table 2) was significantly more with frusemide plus probenecid with or without pyrazinamide than with frusemide alone (7.39 ± 3.65 or 7.34 ± 4.44 vs 4.79 ± 1.84). This is also true for the values at 240 and 300 min and at 120 min with the combination probenecid and pyrazinamide. At 60 min the fractional excretion was higher with frusemide alone.

Discussion The evidence for ascribing the natriuretic action of frusemide exclusively to its luminal (rather than plasma

>" CD, . E) co

-x ' Ea) 2EU)

Frusemide + probenecid

Frusemide

491

7Ua) CO0

'

1.18 12.79 0.08 73.62

0-30

0-60

0-90

0-120 0-180 0-240 0-300

Time (min) Figure 2 Mean (± s.d.) cumulative urinary frusemide excretion after frusemide only (c), with probenecid (n), and with probenecid and pyrazinamide (U), at all time intervals.

0.5r 300 240

~

0.4

180

_ -033. 120 90 60 120 0 c 30 0 .90 D 0.2

E cm -)

60

0.1

0.1 .240 300 Ul

0 -

0

*

._o

3 100

200 300 Frusemide (,ug min- )

U)a.)cJ J

4 [00

Figure 1 Observed volume diuretic efficiency vs frusemide excretion rate after 40 mg frusemide i.v. (@) and after frusemide with probenecid pretreatment (U). Corresponding data points have been labelled by similar minute-times. * P < 0.05 (i.e. at 30, 60 and 90 min after frusemide administration). The corresponding values (± s.d.) in ml p.g-l for frusemide/ frusemide + probenecid are 0.07(0.01)/0.24(0.04); 0.14(0.01)/0.26(0.05); 0.21(0.01)/0.34(0.09).

x

a)

~0 E CnL

LL1

90 120

180 Time (min)

240

Figure 3 The frusemide excretion rate in ,ug min-1 after frusemide only (c), with probenecid (0), and with probenecid and pyrazinamide (A) at all time intervals.

De K. Sommers, E. C. Meyer & J. Moncrieff

492

concentration) is primarily based on microperfusion studies on dissected tubules from the kidneys of rats (Deetjin, 1966) and rabbits (Burg et al., 1973) and on in vivo experiments with dogs (Hook & Williamson, 1965; Rose et al., 1976). The ability of probenecid to completely block the natriuretic and diuretic actions of frusemide in the dog has never been demonstrated in man (Chennavasin et al., 1979; Homeida et al., 1977; Honari et al., 1977). Further examples of inter-species differences are that probenecid blocks the diuresis induced by frusemide, but not by bumetanide, in the cat (Friedman & Roche-Ramel, 1977), and in mice probenecid appears to cause dose-dependent reductions in frusemide-induced diuresis (Sandstrom, 1986). In man probenecid decreases the renal clearance of frusemide by a third (present study) to a half (Chennavasin et al., 1979). In the present study the simultaneous administration of probenecid and pyrazinamide in an attempt to block both the probenecid- and pyrazinoatesensitive acid secretory transport processes did not further reduce the amount of frusemide reaching the tubular lumen. The possibility that these organic acids could decrease the protein binding of frusemide thus allowing more to be filtered at the glomerulus deserves consideration. It is also possible that the metabolites of frusemide, e.g. the glucuronide conjugate, could contribute to natriuresis as intravenous frusemide has less than twice the natriuretic response than an oral dose, 1000 E

E

800

CD >0 >0 .;

600

._

xO

7

JEx :

a)

400 200 0-30

0-60

0-90

0-120 0-180 0-240 0-300

Time (min) Figure 4 Mean (± s.d.) cumulative urinary sodium excretion after frusemide only (c), with probenecid (0), and with probenecid and pyrazinamide (E) at all time intervals.

despite almost three times as much drug reaching the urine (Kaojarern et al., 1982). A similar phenomenon occurs when probenecid is co-administered with frusemide in that probenecid is found to actually increase the overall natriuretic response (Kaojarern et al., 1982). These phenomena were, however, ascribed to a changed time course of delivery of frusemide into urine resulting in concentrations at the 'steep' portion of the doseresponse curve for longer periods of time. The dosing schedule of probenecid may be important. Homeida et al. (1977) reported a marked reduction (52%) in the volume of distribution of frusemide after a 3 day pretreatment with probenecid, while Chennavasin et al. (1979) and Honari et al. (1977) who started probenecid pretreatment about 8 h before frusemide administration found the frusemide volume of distribution unchanged. In the present study the presence of organic acids did not significantly change the mean cumulative urinary sodium excretion over 5 h although the mean cumulative urinary frusemide excretion was reduced by a third. In the first 30 min after frusemide administration the percent fractional sodium excretion was higher after pretreatment with probenecid even though the mean frusemide urinary excretion rate was more than three times with frusemide alone than with probenecid-frusemide (374.4 pg min-' vs 119.1 ,ug min-'). With probenecidfrusemide the diuretic efficiency was significantly higher than with frusemide alone during the first 90 min after frusemide administration. As probenecid pretreatment significantly increases serum frusemide concentration (Chennavasin et al., 1977), which would result in a higher concentration on the basal or blood side of the tubules, the unconditional acceptance of the luminal site of action of the loop diuretics may be questioned. In conclusion there are several possible explanations for the phenomena (Kaojarern et al., 1982) that pretreatment of subjects with probenecid causes a greater overall natriuresis without affecting the total amount of frusemide delivered into the urine and that oral doses of frusemide cause the same cumulative natriuretic effect as identical doses administered intravenously despite delivering less unchanged drug into the urine. A pharmacokinetic explanation (Kaojarern et al., 1982) for the phenomena is that the amounts of frusemide reaching the intraluminal site more persistently approach the

Table 2 Percentage fractional sodium excretion (FENa%) with frusemide alone and with probenecid with and without pyrazinamide pretreatment Time (min) 60 120

240

0

30

0.31 + 0.19

4.79 + 1.84

*12.88

±2.30

5.67 + 3.2

+ 0.79

probenecid

0.20 ± 0.14

*7.34 ± 4.44

9.75 + 2.11

6.73 ± 2.10

+ 2.05

Frusemide + probenecid+

0.21 ± 0.15

*7.39 ± 3.65

10.02 + 2.27

*9.53 ± 1.15

+ 1.26

Frusemide Frusemide +

pyrazinamide *

P < 0.05

1.56

*4.93 *5.17

300 1.41 00.91

*3.26 ± 1.02

*4.25 ± 1.01

Influence of organic acids on frusemide amount with maximal efficiency. The present findings, i.e. that both the initial percent fractional sodium excretion and the diuretic efficiency were significantly increased by probenecid pretreatment could imply that frusemide is active from the peritubular or blood side of

493

the nephron or, alternatively, that probenecid increases the effects of frusemide. A further pharmacodynamic perspective could hypothesize that the 'ceiling effect' in response to frusemide may manifest because the drug acts indirectly via endogenous agents, e.g. prostaglandins.

References Alvan, G., Helleday, L., Lindholm, A., Santz, E. & Villen, T. (1990). Diuretic effect and diuretic efficiency after intravenous dosage of frusemide. Br. J. clin. Pharmac., 29, 215-219. Anonymous (1989). Antimycobacterial agents. In Martindale, The Extra Pharmacopoeia, 29th edn., ed. Reynolds, J. E. F., pp. 546-579. London: The Pharmaceutical Press. Brater, D. C. (1978). Effects of probenecid on furosemide response. Clin. Pharmac. Ther., 24, 548-554. Brater, D. C. (1983). Pharmacodynamic considerations in the use of diuretics. Ann. Rev. Pharmac. Tox., 23, 45-62. Burg, M., Stoner, L., Cardinal, J. & Green, N. (1973). Frusemide effect on isolated perfused tubules. Am. J. Physiol., 225, 119-124. Chennavasin, P., Seiwell, R., Brater, D. C. & Laing, W. M. M. (1979). Pharmacodynamic analysis of the furosemide-probenecid interaction in man. Kidney Int., 16, 187-195. Cutler, R. E., Forrey, A. W., Christopher, T. G. & Kimpel, B. M. (1974). Pharmacokinetics of furosemide in normal subjects and functionally anephric patients. Clin. Pharmac. Ther., 15, 588-596. Dantzler, W. H. (1970). Comparison of renal tubular transport of urate and PAH in water snakes. Evidence for differences in mechanisms and sites of transport. Comp. Biochem. Physiol., 34, 609-623. Deetjen, P. (1966). Micropuncture studies on site and mode of diuretic action of furosemide. Ann. N. Y. Acad. Sci., 139, 408-415. DeLean, A., Munson, P. J. & Rodbard, D. (1978). Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological doseresponse curves. Am. J. Physiol., 235, E97-E102. Fanelli, G. M., Bohn, D. L. & Zacchei, A. G. (1977). Renal excretion of a saluretic-uricosuric agent (MK-196) and interaction with a urate-retaining drug, pyrazinoate, in the chimpanzee. J. Pharmac. exp. Ther., 200, 413-419. Friedman, P. A. & Roch-Ramel, F. (1977). Hemodynamic and natriuretic effects of bumetanide and furosemide in the cat. J. Pharmac. exp. Ther., 203, 82-91. Hammerlund, M. M., Odlind, B. & Paalzow, L. K. (1985). Acute tolerance to furosemide diuresis in humans. Pharmacokinetic-pharmacodynamic modeling. J. Pharmac. exp. Ther., 223, 447-453.

Holford, N. H. G. & Sheiner, L. B. (1981). Understanding the dose-effect relationship: clinical application of pharmacokinetic-pharmacodynamic models. Clin. Pharmacokin., 6, 429-453. Holland, S. D., & Williamson, H. E. (1979). Probenecid inhibition of bumetanide-induced natriuresis in the dog (40540). Proc. Soc. exp. Biol. Med., 161, 299-302. Homeida, M., Roberts, C. & Branch, R. A. (1977). Influence of probenecid and spironolactone of furosemide kinetics and dynamics in man. Clin. Pharmac. Ther., 22, 402-409. Honari, J., Blair, A.D. & Cutler, R. E. (1977). Effects of probenecid on furosemide kinetics and natriuresis in man. Clin. Pharmac. Ther., 22, 395-401. Hook, J. B. & Williamson, H. E. (1965). Influences of probenecid and alterations in acid-base balance on the saluretic activity of furosemide. J. Pharmac. exp. Ther., 149, 404408. Kaojarern, S., Day, B. & Brater, D. C. (1982). The time course of delivery of furosemide into urine: an independent determinant of overall response. Kidney Int., 22, 69-74. Rose, H. J., Pruitt, A. W., Dayton, P. G. & McNay, J. L. (1976). Relationship of urinary furosemide excretion rate to natriuretic effect in experimental azotemia. J. Pharmac. exp. Ther., 199, 490-497. Sandstrom, P. (1986). Probenecid potentiates the hyperglycaemic effect but reduces the diuretic effect of frusemide in mice. Br. J. clin. Pharmac., 89, 307-312. Senft, G. (1965). Membrantransport und Pharmaka. In Normale und pathologische Funktionen des Nierentubulus, eds Ulrich, K. S. & Hierholzer, K., pp. 68-81. Bern: Hans Huber. Sommers, De K., Van Wyk, M., Moncrieff, J., Schoeman, H. S. & De Villiers, L. S. (1987). Renal secretory mechanisms and uric acid. S. Afr. J. Sci., 83, 114-115. Weiner, I. M. & Mudge, G. H. (1985). Inhibition of tubular transport of organic compounds. In The pharmacological basis of therapeutics, 7th edn., eds Gilman, A. G., Goodman, L. S., Rall, T. W. & Murad, F., pp. 920-925. New York: Macmillan Publishing Company.

(Received 25 October 1990, accepted 21 May 1991)

The influence of co-administered organic acids on the kinetics and dynamics of frusemide.

1. This study examines the effects of pretreatment with probenecid with and without pyrazinamide on the elimination kinetics and diuretic action of fr...
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