Propranolol glucuronide cumulation during long-term propranolol therapy: A proposed storage mechanism for propranolol The comparative disposition of propranolol glucuronide (PC) and propranolol was determined in 35 patients with h}pertension or coronary artery disease during initiation of propranolol therapy, during steady-state conditions, and after discontinuation of propranolol (dose range, 40 to 960 mg daily, every 6 hr). The 2.3jold PC cumulation in plasma was identical to propranolol cumulation. PC plasma levels were about 4 times as high as propranolol levels OI'er the whole dose range. Unexpectedly slow terminal elimination rate of propranolol (t 1/2 approximately 16 to 24 hr)
0/1
discontinuation of propranolol appeared to be related to equally
slow PC elimination. PC and propranolol could be detected in plasma and urine up to 3 to 5 days ajier propranolol discontinuation. The PC formed in man was deconjugated to propranolol in the dog ajier intravenous administration, suggesting that PC may serve as a storage pool for propranolol. Observations consistent with systemic and ellleric deconjugation of PC, including ellTero/7!:'Paric recirculation, may, at least ill part, explain the obsen'ed propranolol ClImulation as well as the slow elimination of propranolol aper its discontinuatio/l. PC renal clearance (29 to 70 /Ill/min) a/ld PC plasma levels were highly dependent on glomerular{iltration rate, suggesting that PC may cumulate abnormally in patie/lts with severe renal disease.
Thomas Walle, Ph.D., Edward C. Conradi, M.D., U. Kristina Walle, R.Ph., Timothy C. Fagan, M.D., and Thomas E. Gaffney, M.D. Charleston, S. C. Departments uf Pharmacology and Medicine, Medical University of South Carolina
In a recent publication we described presystemic propranolol glucuronidation in normal subjects. 2o Propranolol glucuronide (PG) plasma levels exceeded propranolol levels by about 7 times after oral doses, whereas PG and proSupported by National Institute of General Medical Sciences grants GM 20387 and RR 0 I070. Presented in part at the Federation of American Societies for Experimental Biology annual meeting in Anabeim, Calif., April, 1976 (abstracted in Fed Proc 35:665, 1976). Received for publication April 6, 1979. Accepted for publication June 20, 1979. Reprint requests to: Thomas Walle, Ph.D., Department of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29403.
686
pranolol levels were of the same order after intravenous propranolol. In preliminary studies, high PG plasma concentrations relative to propranolol were also observed in patients during long-term oral therapy. 19 This suggested PG cumulation after repeated propranolol administration, an observation that stands in contrast to the disposition generally accepted for a drug glucuronide. PG cumulation might, at least in part, explain the slow and avid propranolol cumulation during long-term therapy. 17. 26 Our study describes PG disposition relative to that of propranolol during initiation of propranolol therapy, at steady state, and up to 5
0009-9236179/120686+10$01.0010 © 1979 The C. V. Mosby Co.
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days after propranolol discontinuation in patients with hypertension or coronary artery disease. PG was also isolated from human urine and its kinetics, including deconjugation to propranolol, were studied after intravenous administration in the dog. The biliary excretion of PG and of propranolol was also studied in the dog. Methods
Studies in man. Thirty-five patients, aged 18 to 63 yr, with mild to moderately severe hypertension or coronary artery disease were hospitalized in a general clinical research center or at the Veterans Administration Hospital for this study. Prior to the study they had an admission history, physical examination, chest film, electrocardiogram, complete blood count, urinalysis, and selected blood chemistry determinations. A rigorous characterization of these patients with respect to sex, age, weight, race, and the concomitant use of other drugs has been reported. 17 None had liver disease. Creatinine clearance was 40 mllmin in two patients, whereas in all other patients studied it was normal with respect to age (80 to 145 mllmin). Five patients had not received propranolol before hospitalization, whereas 30 patients had been on propranolol for weeks to several years at the time of admission. PC cumulation in plasma. The cumulation of PO in plasma from initiation of propranolol therapy, 10 to 40 mg every 6 hr, to steady state was determined in 5 patients as described for propranolol. 1 7 Peak PO plasma levels were measured at 2 hr after the first dose, given in the morning in a fasting state, and then 2 hr after the ninth and thirteenth doses. PC disposition during long-term therapy. Propranolol was administered in divided doses every 6 hr at 15 different dose levels (dose range, 40 to 960 mg daily). Thirteen of the patients were studied at 2 or more of these dose levels, making the total number of steady-state observations 64. To determine the time to reach the peak PO plasma levels within a dosage interval, PO levels were measured in 7 patients on long -term therapy, 10 to 80 mg every 6 hr, on day 4 of the hospitalization at 0.5, 1, 1.5, 2, 2.5, 3, 4, and 6 hr after the morning dose. The
Propranolol glucuronide cumulation
687
peak plasma PO level was reached at 2.3 ± 0.5 hr (mean ± SD), at much the same time as that found for propranolol. I i Due to a flat plasma concentration-time curve between 1.5 and 2.5 hr, a 2-hr sample could be regarded as highly representative of the peak PO level. Steady-state plasma PO levels are expressed as the mean of two determinations, on days 3 and 4 of the hospitalization as described for propranolol. l i Peak concentrations, measured in all patients, are represented by the mean of the concentrations at 2.0 hr after the morning dose on these days. Trough concentrations were measured in 19 patients at 6.0 hr after the same doses. The daily urinary PO excretion in 24 patients is similarly reported as mean excretion during days 4 and 5 of the hospitalization. PC and propranolol disappearance after discontinuation of propranolol. In 3 hypertensive patients who had no evidence of coronary artery disease and who had been on long-term propranolol therapy, 80 to 160 mg! day, for several weeks, propranolol was abruptly discontinued. Starting 2 hr after the last dose, 9 to 10 blood samples were collected over a 3- to 5-day period. Serial l2-hr urine collections were also made. Sample col/ecting and handling techniques. All blood samples were obtained with the patient at rest by separate venipuncture or through an indwelling butterfly cannula for serial samples. Blood and urine samples were collected, handled and stored as described previously. 17 Isolation of PC from urine. Aliquots of urine (400 m!) from patients on long-term propranolol treatment were added to an XAD-2 column, 25 x 200 mm. After washing the column with 400 ml water, PG was eluted with methanol and collected in lO-ml fractions. Seventy-five percent of the PG, determined by enzymatic hydrolysis and measurement of propranolol, appeared in the 50- to 120-ml fractions. After evaporation under vacuum, the residue was dissolved in 5 ml water and added to a second XAD-2 column and treated as above. Collection of the 60- to 90-ml fractions gave a 50% yield of partially purified PO. After evaporation under vacuum, the residue was dissolved in pH 10 carbonate buffer to a concentration of 10 mg/ml and extracted twice with ethyl acetate to remove traces of propranolol. The pH was adjusted to 7.4
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Clin. Pharmacal. Ther. December 1979
Table I. Cumulation of plasma PG in 5 subjects after initiation of propranolol therapy to steady state
Subject
1 2 3 4 5
X±
Propranolol dose (mg)* 10 10
20 20 40
PG in plasma (ng/ml) Dose It
28 40 60 70 120
I
SD
Dose
75 81 135 140 280
9t
J
Dose I3t
86 73 127 165 260
Cumulation factor*
2.9 1.9 2.2 2.2 2.3 2.3 ± 0.4
'Given every 6 hr. tGiven in a fasting state 2 hr after each morning dose. :j:Mean concentration after doses 9 and 13/concentration after dose l.
before the solution was administered. There was no trace of propranolol in this solution «0.01% of the PG). Studies in the dog. Intravenous administration of PG. Single doses, 0.5 to 1.0 mg/kg, of the partially purified PG were injected as a rapid bolus into the femoral vein of 4 conscious dogs (8 to 12 kg). Starting at 0.5 hr after PG administration, blood samples were collected from the jugular vein through an indwelling catheter over a 16- to 32-hr period. Biliary PG. Three dogs (12 to 16 kg) were given oral doses of propranolol every 6 hr (10 mg/kg/day). The animals were killed 4 hr after the last dose on day 7. Gall bladder bile, venous blood, and bladder urine were collected. Analytical methodology. Plasma, urine, and bile levels of propranolol were determined by gas chromatography- mass spectrometry using hexadeuterium-labeled propranolol as the internal standard. 21, 23 Plasma, urine, and bile levels of PG were obtained as the difference in propranolol levels between enzyme-hydrolyzed and nonhydrolyzed samples. 20 The sensitivity limit was about 0.4 ng/ml for PG and 0.1 ng/ml for propranolol.23 Results
Studies in man. PG cumulation in plasma. Peak plasma PG levels determined in 5 patients after the first, ninth, and thirteenth doses of propranolol are shown in Table I. Steady-state levels appear to
be reached after the ninth dose in all patients, i.e., after 2 days of propranolol therapy. There was 2.3-fold cumulation of PG. PG levels after the ninth and thirteenth doses are all within the range of PG levels after weeks to several years on propranolol (Fig. 1). Disposition of PG in plasma during longterm therapy. The peak (2-hr) plasma PG levels compared with those of propranolol (dose range 40 to 960 mg daily) in the 35 patients studied are shown in Fig. 1. The inset shows the result in a single patient who was studied at 6 dose levels, 40 to 320 mg daily. The PG levels, as reported earlier for propranolol,17 were nonlinearly related to dose at doses up to 160 mg daily and linearly related to dose at higher doses. The linear portions of the curves, characterized by the equations in Fig. 1, were extrapolated to zero levels at a dose of about 100 mg daily. The PG levels were 4.2 times as high as the propranolol levels over the whole dose range studied. In the 2 patients who had low creatinine clearance (40 mll min), the PG levels were markedly elevated (Fig. 1) at each of the dose levels. Trough (6-hr) PG levels determined in 19 of the patients were 51 ± 11 (SD) % of peak (2-hr) levels or much the same as the trough/peak level relationship for proprano101. 17 The mean plasma PG levels and propranolol levels during a 6-hr dosage interval were 72% of peak levels, calculated from the area under the plasma concentration-time curve (ng . hr/ml) divided by the dosage interval (6 hr). The between-patient variation in plasma PG
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Propranolol glucuronide cumulation
Table II. Renal clearance of PG and creatinine clearance in 5 patients on long term treatment with 640 mg propranolol per day
4000
Subject I
2 3 4 5
Creatinine clearance (mllmin)
PG renal clearance * (mllmin)
141 123 116 80 40
67.0 72.9 74.6 42.8 29.0
-' ""-
:E:
= ""
~'
..-
3000
0
• •
..- ,,"
PG
100 200 300
~
i= cc
10
5
1
c:r
2:
cc
=
DAYS AFTER PROPRANOLOL DISCONT1NUATION Fig. 3. Plasma concentrations and urinary excretion of PG (e) and propranolol (0) in 3 hypertensive patients after withdrawal of long-term propranolol therapy. Numbers refer to individual patients.
Studies in the dog. Intravenous PC kinetics. The plasma decay curves, tVz, and volume of distribution (Vd) following intravenous PG administered to 4 dogs are shown in Fig. 4. The PG level declined monoexponentially during the first 12 hr with a tV2 of 1.5 to 2.2 hr. A further slow phase of PG elimination with an estimated tl/2 of7.5 to 16 hr was observed in 3 of the animals. The mean plasma PG clearance (dose/area under the plasma concentration-time curve) was 58 mil
min. The mean volume of distribution in all animals (dose/plasma concentration at time zero or calculated from its plasma clearance! 0.693 --w2) was 1.0 l/kg.
Deconjugalion of PC to propranolol. Propranolol appeared in the plasma of each animal after intravenous PG (Fig. 4). The maximum propranolol plasma level (3.5 to 9.2 ng/ml) was reached at 1 to 8 hr. The propranololleve]s then declined monoexponentially with t16 ranging
692
Walle et al.
Clin. Pharmacal. Ther. December 1979
0,5 MG/KG:
1.0 MG/KG:
---------
---------
#2
#1
#3
500 T~
....J
:.::
"'-" :z
""
500
1.5
100 50
100 50
10 5
10 5
1 ,5
,5
:z
2
t-
«
t""
:z
w
u
:z
T~ 12
•
0
u
""
:.:: VJ
« ....J Cl..
1
T~
.1
2.0
0
T" 9.0
T" 11.5
T" 7.0
0
0
5 1015
0 5 10 15
0 5 10 15 20 25
,1
o5
10 15 20 25 30
HOURS AFTER INTRAVENOUS PG ADMINISTRATION Fig. 4. PG (e) and propranolol (0) plasma levels in 4 dogs after intravenous PG doses (0.5 to 1.0 mg/kg). The tl/z is expressed in hours (hr) and Vd in liters per kilogram (II kg).
Table III. PG and propranolol concentrations in 3 dogs (mean ± SD) treated with propranolol. 10 mg/kg/day,for 7 days
PG (J-Lg/ml) Propranolol (J-Lg/ml)
Bile
Plasma
Urine
106 ± II ND
0.30 ± 0.05 0.053 ± 0.019
14.0 ± 3.1 0.43 ± 0.15
NO = not detectable.
from 2.0 to 11.5 hr. The PG injected contained no measurable propranolol, emphasizing that the early appearance of propranolol in plasma was the result of deconjugation to propranolol. Propranolol levels I to 2 hr after the intravenous PG were significantly higher than 0.5 hr after the dose in all animals. The area under the plasma concentration-time curve ratio propranolol/PG was 0.033 ± 0.022 (mean ± SD; n = 4) with the curves extrapolated to infinite time. There was a PG/ propranolol plasma level ratio of about 4 beyond 12 hr in 3 of the 4 animals, much the same as the PG/propranolol ratio in man during long-term propranolol therapy and persisting for 3 to 5 days after propranolol withdrawal. Biliary PG. PG and propranolol levels in bile, plasma, and urine of 3 dogs undergoing
long-term treatment with oral propranolol are shown in Table III. The ability of the biliary excretory system to concentrate PG is reflected in the high bile/plasma level ratio of 353. Note that the PG/propranolol plasma level ratio of 5.7 in the dog is much the same as that observed in man (4.2). No propranolol was detected in the bile. Discussion
Reports that propranolol cumulates more than twofold in plasma of patients treated at 6-hr intervals with oral doses of propranolo]3' 7, 17 and that steady-state concentrations are not reached until after 2 to 3 days of treatment 17 have been difficult to reconcile with a single oral dose t 1/2 of only 3 to 4 hr. 7, 15, 20 Although the propranolol tV2 is increased to 4 to 6 hr after
Volume 26 Number 6
long-term therapy,3, 7, 26 this does not explain its avid and slow cumulation, Observations that satisfactory blood pressure control can be maintained with propranolol throughout dosage intervals of 12 1, 12, 24 or even 24 hr25 and that effects of propranolol can persist for 3 days after drug withdrawaF are also difficult to reconcile with the reported short plasma propranolol tV2, Whereas the duration of the antihypertensive and other actions of propranolol would suggest a longer dosage interval than is generally used, the kinetic basis for such therapy is lacking, The unexpected finding that PO cumulates more than twofold after long-term propranolol dosing with steady-state plasma concentrations 4 times as high as those of propranolol suggested that propranolol cumulation, at least in part, may depend on PO cumulation, The observation that propranolol does not cumulate after repeated intravenous doses,7 a route of administration that generates very little PO, 20 supports such a hypothesis, The finding of a change in the propranolol tVz from 4 to 6 hr to approximately 16 to 24 hr, starting at 12 to 24 hr after cessation of longterm propranolol therapy, is of particular importance, This change in propranolol kinetics, which has not been previously observed, appeared to be related to a parallel decrease in PO elimination, Whereas with previously available methodology no residual propranolol or propranolol metabolites were detected at 24 hr after withdrawal,4, H, II both PO and propranolol were detected in plasma and urine 3 to 5 days after withdrawal in the present study with PO concentrations exceeding propranolol concentrations by about fourfold throughout the drugwithdrawal period. Although plasma levels at 36 to 72 hr are quite low for both PO (2 to 6 ng/ml) and propranolol (0.1 to 2 ng/ml), they may still be of biological significance. Persistent biological activity has been observed at 36 11 , 25 and even at 72 hr2 after withdrawal of long-term propranolol. The unexpected cumulation and slow disappearance of PO may be explained by the distribution of PO into a deep storage site. The very high concentrations of PO in the bile of the dog after long-term propranolol suggest enterohe-
Propranolol glucuronide cumulation
693
patic recirculation. The irregular shape of the terminal elimination phase of both PO and propranolol in man could be explained by such a mechanism. Enteric deconjugation of PO and subsequent reabsorption of propranolol are also suggested by the reports that only a small fraction of radioactive doses of propranolol are recovered in human feces. 14 Through such mechanisms, deconjugation of PO to propranolol could serve to replenish an active pool of propranolol including such sites as have recently been described in postganglionic sympathetic neurons. 5 The slow and avid cumulation of propranolol and its slow elimination may, however, also be explained by the distribution of propranolol into a high-affinity binding site independent of PO. The slow release of such a pool of propranolol should, by normal metabolic clearance, result in a parallel slow disappearance of PO. Direct support for the deconjugation of PO to propranolol was obtained by intravenous administration of human PO to the dog. The rapid appearance of propranolol in plasma as early as 0.5 hr after the PO dose is the result of systemic deconjugation which could take place in the blood as well as in other tissues. ,B-Olucuronidase activity is detectable in almost all tissues including blood. 16 The very slow decline of propranolol in 3 of the dogs given intravenous PO (tV2, 7.5 to 11.5 hr) indicates continuing deconjugation of PO with the deconjugation rate being much slower than the known elimination rate of propranolol (tllz, 0.5 to 2 hr)6, 9, 10, 22 in this species. This slow deconjugation to propranolol coincides with a marked increase in the PO tV2 from 2 to 12 hr, which is similar to observations in man. This change in PO kinetics may be due to enterohepatic recirculation of PO including enteric deconjugation to propranolol. Although the amount of propranolol formed after intravenous PO appears small relative to the amount of PO administered (A ue proprano10l/AUe PO = 0.033), it represents about 10% of PO if corrected for the threefold difference in volume of distribution between propranolol 9 and PO. Furthermore, since the deconjugated propranolol is eliminated mainly by metabolism, the measured propranolol only represents
694
Walle et al.
a fraction of total deconjugated PO. Systemic PO deconjugation may also be quantitatively more important in disease conditions associated with elevated .a-glucuronidase activity. 16 Although deconjugation of PO to yield propranolol has been demonstrated in the dog, mechanisms and sites of deconjugation require further studies including enterohepatic recirculation of PO and propranolol. It also remains to be determined whether these processes occur in man. The high plasma PO concentrations observed in man can in part be explained by the smaller volume of distribution of PO (It/kg in the dog) than of propranolol. It is clear, however, that PO occupies a larger pool of distribution than the vascular space. It is not unlikely that PO, retaining the highly lipophilic naphthalene ring system, could penetrate into cell membranes and concentrate in tissues. Potential plasma binding or renal tubular reabsorption could explain the relatively low renal clearance of PO. PO renal clearance of only 51 mllmin is less than half the normal glomerular filtration rate in healthy individuals. The dependence of plasma PO levels on renal clearance was reflected in the elevated levels in the few patients studied who had subnormal glomerular filtration rates, suggesting that PO may cumulate more readily in patients with severe renal disease. This has been shown to be the case with the glucuronic acid conjugate of oxazepam. 13 Abnormal PO cumulation in association with PO deconjugation may lead to elevated propranolol plasma levels in patients with chronic renal disease. A recent study in the dog,22 demonstrating 4 times higher plasma concentrations of the t- isomer of PO than of the d - isomer after single oral doses of d,t-propranolol, suggests that our findings of PO cumulation, slow disappearance, and deconjugation to propranolol may apply mainly to the .a-blocking I-isomer. Although the size of the deep pool identified for propranolol in man is small and therefore can explain only in part total propranolol cumulation in the body, the detection of a deep storage site introduces a concept that may be useful in our gradually developing understanding of the disposition and mechanism of action of propranolol. The saturation of a metabolic pro-
Clin. Pharmacal. Ther. December 1979
cess other than glucuronidation is probably of greater significance for the total body accumulation of propranolol. This saturable process very likely involves naphthalene ring oxidation.17, IH Finally, the importance of highly sensitive and specific analytical methodology for the identification of deep storage sites for drugs in man cannot be overemphasized. We thank John Brubaker for valuable assistance in the experiments performed in the dog.
References I. Berglund G, Andersson 0, Hansson L, Olander R: Propranolol given twice daily in hypertension. Acta Med Scand 194:513-515, 1973. 2. Brundin T, Edhag 0, Lundman T: Effects remaining after withdrawal of long-term betareceptor blockade. Reduced heart rate and altered haemodynamic response to acute propranolol administration. Br Heart J 38: 1065-1072, 1976. 3. Chidsey CA, Morselli P, Bianchetti G, Morganti A, Leonetti G, Zanchetti A: Studies of the absorption and removal of propranolol in hypertensive patients during therapy. Circulation 52:313-318, 1975. 4. Coltart DJ, Cayen MN, Stinson EB, Davies RO, Harrison DC: Determination of the safe period for withdrawal of propranolol therapy. Circulation 47, 48(suppl. IV):IV-7, 1973. 5. Daniell HB, Walle T, Gaffney TE, Webb JG: Stimulation induced release of propranolol and norepinephrine from adrenergic neurons. J Pharmacol Exp Ther 208:354-359, 1979. 6. Evans GH, Nies AS, Shand DG: The disposition of propranolol. III. Decreased half-life and volume of distribution as a result of plasma binding in man, monkey, dog and rat. J Pharmacol Exp Ther 186:114-122,1973. 7. Evans GH, Shand DG: Disposition of propranolol. V. Drug accumulation and steady-state concentrations during chronic oral administration in man. CUN PHARMACOL THER 14:487-493, 1973. 8. Faulkner SL, Hopkins JT, Boerth RC, Young JL, Jellett LB, Nies AS, Bender HW, ~hand DG: Time required for complete recovery from chronic propranolol therapy. N Engl J Med 289:607-609, 1973. 9. George CF, Orme ML 'E, Buranapong P, Macerlean D, Breckenridge AM, Dollery CT: Contribution of the liver to overall elimination of propranolol. J Pharmacokinetic Biopharm 4: I7 -27, 1976. 10. Hayes A, Cooper RG: Studies on the absorption, distribution and excretion of propranolol in rat,
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11.
12.
13.
14.
15.
16.
17.
18.
dog and monkey. J Pharmacol Exp Ther 176: 302-3 11, 1971. Leaman DM, Levenson L W, Shiroff RA, Babb JD, Dejoseph RL, Hayes AH, Zelis R: Persistence of biological activity after disappearance of propranolol from the serum. J Thorac Cardiovasc Surg 72:67-72, 1976. MacLeod S, Hamet P, Larochelle P, Nadeau J, Ogilvie R, Rangno R, Ruedy J, Sellers E, Ti T: Antihypertensive efficacy of b.i.d. propranolol. Ann R Coli Physicians Surg Can II:34, 1978. Odar-CederiOf I, Vessman J, Alvan G, Sj6qvist F: Oxazepam disposition in uremic patients. Acta Pharmacol Toxicol 40(suppl. 1):52-62, 1977. Paterson JW, Conolly ME, Dollery CT, Hayes A, Cooper RG: The phannacodynamics and metabolism of propranolol in man. Pharmacol Clin 2: 127-133, 1970. Shand DG, Nuckolls EM, Oates JA: Plasma propranolol levels in adults with observations in four children. CUN PHARMAcaL THER II: I 12120, 1970. Wakabayashi M: f3-Glucuronidases in metabolic hydrolysis, in Fishman WH, editor: Metabolic conjugation and metabolic hydrolysis. New York, 1970, Academic Press, Inc., vol. II. Walle T, Conradi EC, Walle UK, Fagan TC, Gaffney TE: The predictable relationship between plasma levels and dose during chronic propranolol therapy. CUN PHARMAcaL THER 24:668-677, 1978. Walle T, Conradi EC, Walle UK, Fagan TC, Gaffney TE: 4-Hydroxy-propranolol and its glucuronide in man after single and chronic doses of propranolol. CUN PHARMAcaL THER. (In press.)
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19. Walle T, Conradi EC, Walle UK, Gaffney TE: Steady-state plasma concentrations and urinary excretion of propranolol-O-glucuronide and propranolol in patients during chronic oral propranolol therapy. Fed Proc 35:665, 1976. 20. Walle T, Fagan TC, Conradi EC, Walle UK, Gaffney TE: Presystemic and systemic glucuronidation of propranolol. CUN PHARMAcaL THER 26:167-172, 1979. 21. Walle T, Morrison J, Walle K, Conradi E: Simultaneous determination of propranolol and 4-hydroxypropranolol in plasma by mass fragmentography. JChromatogr 114:351-359,1975. 22. Walle T, Walle UK: Stereoselective oral bioavailability of (±)-propranolol in the dog. A GC-MS study using a stable isotope technique. Res Commun Chern Pathol Pharmacol 23:453464, 1979. 23. Walle T, Walle UK, Bridges DR, Conradi EC, Gaffney TE: Quantitative GC-MS of proprano101, a key to an improved understanding of the relationship between plasma concentrations and dose. Clin Chern 24:991, 1978. 24. Wilkinson PR, Dixon N, Hunter KR: Twicedaily propranolol treatment for hypertension. J Int Med Res 2:220-223, 1974. 25. Wilson M, Morgan G, Morgan T: The effect on blood pressure of f3-adrenoreceptor-blocking drugs given once daily. Clin Sci Mol Med 51:5275-5285, 1976. 26. Wood AJJ, Carr K, Vestal RE, Belcher S, Wilkinson GR, Shand DG: Direct measurement of propranolol bioavailability during accumulation to steady-state. Br J Clin Phannacol 6:345-350, 1978.
0/1
discontinuation of propranolol appeared to be related to equally
slow PC elimination. PC and propranolol could be detected in plasma and urine up to 3 to 5 days ajier propranolol discontinuation. The PC formed in man was deconjugated to propranolol in the dog ajier intravenous administration, suggesting that PC may serve as a storage pool for propranolol. Observations consistent with systemic and ellleric deconjugation of PC, including ellTero/7!:'Paric recirculation, may, at least ill part, explain the obsen'ed propranolol ClImulation as well as the slow elimination of propranolol aper its discontinuatio/l. PC renal clearance (29 to 70 /Ill/min) a/ld PC plasma levels were highly dependent on glomerular{iltration rate, suggesting that PC may cumulate abnormally in patie/lts with severe renal disease.
Thomas Walle, Ph.D., Edward C. Conradi, M.D., U. Kristina Walle, R.Ph., Timothy C. Fagan, M.D., and Thomas E. Gaffney, M.D. Charleston, S. C. Departments uf Pharmacology and Medicine, Medical University of South Carolina
In a recent publication we described presystemic propranolol glucuronidation in normal subjects. 2o Propranolol glucuronide (PG) plasma levels exceeded propranolol levels by about 7 times after oral doses, whereas PG and proSupported by National Institute of General Medical Sciences grants GM 20387 and RR 0 I070. Presented in part at the Federation of American Societies for Experimental Biology annual meeting in Anabeim, Calif., April, 1976 (abstracted in Fed Proc 35:665, 1976). Received for publication April 6, 1979. Accepted for publication June 20, 1979. Reprint requests to: Thomas Walle, Ph.D., Department of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29403.
686
pranolol levels were of the same order after intravenous propranolol. In preliminary studies, high PG plasma concentrations relative to propranolol were also observed in patients during long-term oral therapy. 19 This suggested PG cumulation after repeated propranolol administration, an observation that stands in contrast to the disposition generally accepted for a drug glucuronide. PG cumulation might, at least in part, explain the slow and avid propranolol cumulation during long-term therapy. 17. 26 Our study describes PG disposition relative to that of propranolol during initiation of propranolol therapy, at steady state, and up to 5
0009-9236179/120686+10$01.0010 © 1979 The C. V. Mosby Co.
Volume 26 Number 6
days after propranolol discontinuation in patients with hypertension or coronary artery disease. PG was also isolated from human urine and its kinetics, including deconjugation to propranolol, were studied after intravenous administration in the dog. The biliary excretion of PG and of propranolol was also studied in the dog. Methods
Studies in man. Thirty-five patients, aged 18 to 63 yr, with mild to moderately severe hypertension or coronary artery disease were hospitalized in a general clinical research center or at the Veterans Administration Hospital for this study. Prior to the study they had an admission history, physical examination, chest film, electrocardiogram, complete blood count, urinalysis, and selected blood chemistry determinations. A rigorous characterization of these patients with respect to sex, age, weight, race, and the concomitant use of other drugs has been reported. 17 None had liver disease. Creatinine clearance was 40 mllmin in two patients, whereas in all other patients studied it was normal with respect to age (80 to 145 mllmin). Five patients had not received propranolol before hospitalization, whereas 30 patients had been on propranolol for weeks to several years at the time of admission. PC cumulation in plasma. The cumulation of PO in plasma from initiation of propranolol therapy, 10 to 40 mg every 6 hr, to steady state was determined in 5 patients as described for propranolol. 1 7 Peak PO plasma levels were measured at 2 hr after the first dose, given in the morning in a fasting state, and then 2 hr after the ninth and thirteenth doses. PC disposition during long-term therapy. Propranolol was administered in divided doses every 6 hr at 15 different dose levels (dose range, 40 to 960 mg daily). Thirteen of the patients were studied at 2 or more of these dose levels, making the total number of steady-state observations 64. To determine the time to reach the peak PO plasma levels within a dosage interval, PO levels were measured in 7 patients on long -term therapy, 10 to 80 mg every 6 hr, on day 4 of the hospitalization at 0.5, 1, 1.5, 2, 2.5, 3, 4, and 6 hr after the morning dose. The
Propranolol glucuronide cumulation
687
peak plasma PO level was reached at 2.3 ± 0.5 hr (mean ± SD), at much the same time as that found for propranolol. I i Due to a flat plasma concentration-time curve between 1.5 and 2.5 hr, a 2-hr sample could be regarded as highly representative of the peak PO level. Steady-state plasma PO levels are expressed as the mean of two determinations, on days 3 and 4 of the hospitalization as described for propranolol. l i Peak concentrations, measured in all patients, are represented by the mean of the concentrations at 2.0 hr after the morning dose on these days. Trough concentrations were measured in 19 patients at 6.0 hr after the same doses. The daily urinary PO excretion in 24 patients is similarly reported as mean excretion during days 4 and 5 of the hospitalization. PC and propranolol disappearance after discontinuation of propranolol. In 3 hypertensive patients who had no evidence of coronary artery disease and who had been on long-term propranolol therapy, 80 to 160 mg! day, for several weeks, propranolol was abruptly discontinued. Starting 2 hr after the last dose, 9 to 10 blood samples were collected over a 3- to 5-day period. Serial l2-hr urine collections were also made. Sample col/ecting and handling techniques. All blood samples were obtained with the patient at rest by separate venipuncture or through an indwelling butterfly cannula for serial samples. Blood and urine samples were collected, handled and stored as described previously. 17 Isolation of PC from urine. Aliquots of urine (400 m!) from patients on long-term propranolol treatment were added to an XAD-2 column, 25 x 200 mm. After washing the column with 400 ml water, PG was eluted with methanol and collected in lO-ml fractions. Seventy-five percent of the PG, determined by enzymatic hydrolysis and measurement of propranolol, appeared in the 50- to 120-ml fractions. After evaporation under vacuum, the residue was dissolved in 5 ml water and added to a second XAD-2 column and treated as above. Collection of the 60- to 90-ml fractions gave a 50% yield of partially purified PO. After evaporation under vacuum, the residue was dissolved in pH 10 carbonate buffer to a concentration of 10 mg/ml and extracted twice with ethyl acetate to remove traces of propranolol. The pH was adjusted to 7.4
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Clin. Pharmacal. Ther. December 1979
Table I. Cumulation of plasma PG in 5 subjects after initiation of propranolol therapy to steady state
Subject
1 2 3 4 5
X±
Propranolol dose (mg)* 10 10
20 20 40
PG in plasma (ng/ml) Dose It
28 40 60 70 120
I
SD
Dose
75 81 135 140 280
9t
J
Dose I3t
86 73 127 165 260
Cumulation factor*
2.9 1.9 2.2 2.2 2.3 2.3 ± 0.4
'Given every 6 hr. tGiven in a fasting state 2 hr after each morning dose. :j:Mean concentration after doses 9 and 13/concentration after dose l.
before the solution was administered. There was no trace of propranolol in this solution «0.01% of the PG). Studies in the dog. Intravenous administration of PG. Single doses, 0.5 to 1.0 mg/kg, of the partially purified PG were injected as a rapid bolus into the femoral vein of 4 conscious dogs (8 to 12 kg). Starting at 0.5 hr after PG administration, blood samples were collected from the jugular vein through an indwelling catheter over a 16- to 32-hr period. Biliary PG. Three dogs (12 to 16 kg) were given oral doses of propranolol every 6 hr (10 mg/kg/day). The animals were killed 4 hr after the last dose on day 7. Gall bladder bile, venous blood, and bladder urine were collected. Analytical methodology. Plasma, urine, and bile levels of propranolol were determined by gas chromatography- mass spectrometry using hexadeuterium-labeled propranolol as the internal standard. 21, 23 Plasma, urine, and bile levels of PG were obtained as the difference in propranolol levels between enzyme-hydrolyzed and nonhydrolyzed samples. 20 The sensitivity limit was about 0.4 ng/ml for PG and 0.1 ng/ml for propranolol.23 Results
Studies in man. PG cumulation in plasma. Peak plasma PG levels determined in 5 patients after the first, ninth, and thirteenth doses of propranolol are shown in Table I. Steady-state levels appear to
be reached after the ninth dose in all patients, i.e., after 2 days of propranolol therapy. There was 2.3-fold cumulation of PG. PG levels after the ninth and thirteenth doses are all within the range of PG levels after weeks to several years on propranolol (Fig. 1). Disposition of PG in plasma during longterm therapy. The peak (2-hr) plasma PG levels compared with those of propranolol (dose range 40 to 960 mg daily) in the 35 patients studied are shown in Fig. 1. The inset shows the result in a single patient who was studied at 6 dose levels, 40 to 320 mg daily. The PG levels, as reported earlier for propranolol,17 were nonlinearly related to dose at doses up to 160 mg daily and linearly related to dose at higher doses. The linear portions of the curves, characterized by the equations in Fig. 1, were extrapolated to zero levels at a dose of about 100 mg daily. The PG levels were 4.2 times as high as the propranolol levels over the whole dose range studied. In the 2 patients who had low creatinine clearance (40 mll min), the PG levels were markedly elevated (Fig. 1) at each of the dose levels. Trough (6-hr) PG levels determined in 19 of the patients were 51 ± 11 (SD) % of peak (2-hr) levels or much the same as the trough/peak level relationship for proprano101. 17 The mean plasma PG levels and propranolol levels during a 6-hr dosage interval were 72% of peak levels, calculated from the area under the plasma concentration-time curve (ng . hr/ml) divided by the dosage interval (6 hr). The between-patient variation in plasma PG
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Propranolol glucuronide cumulation
Table II. Renal clearance of PG and creatinine clearance in 5 patients on long term treatment with 640 mg propranolol per day
4000
Subject I
2 3 4 5
Creatinine clearance (mllmin)
PG renal clearance * (mllmin)
141 123 116 80 40
67.0 72.9 74.6 42.8 29.0
-' ""-
:E:
= ""
~'
..-
3000
0
• •
..- ,,"
PG
100 200 300
~
i= cc
10
5
1
c:r
2:
cc
=
DAYS AFTER PROPRANOLOL DISCONT1NUATION Fig. 3. Plasma concentrations and urinary excretion of PG (e) and propranolol (0) in 3 hypertensive patients after withdrawal of long-term propranolol therapy. Numbers refer to individual patients.
Studies in the dog. Intravenous PC kinetics. The plasma decay curves, tVz, and volume of distribution (Vd) following intravenous PG administered to 4 dogs are shown in Fig. 4. The PG level declined monoexponentially during the first 12 hr with a tV2 of 1.5 to 2.2 hr. A further slow phase of PG elimination with an estimated tl/2 of7.5 to 16 hr was observed in 3 of the animals. The mean plasma PG clearance (dose/area under the plasma concentration-time curve) was 58 mil
min. The mean volume of distribution in all animals (dose/plasma concentration at time zero or calculated from its plasma clearance! 0.693 --w2) was 1.0 l/kg.
Deconjugalion of PC to propranolol. Propranolol appeared in the plasma of each animal after intravenous PG (Fig. 4). The maximum propranolol plasma level (3.5 to 9.2 ng/ml) was reached at 1 to 8 hr. The propranololleve]s then declined monoexponentially with t16 ranging
692
Walle et al.
Clin. Pharmacal. Ther. December 1979
0,5 MG/KG:
1.0 MG/KG:
---------
---------
#2
#1
#3
500 T~
....J
:.::
"'-" :z
""
500
1.5
100 50
100 50
10 5
10 5
1 ,5
,5
:z
2
t-
«
t""
:z
w
u
:z
T~ 12
•
0
u
""
:.:: VJ
« ....J Cl..
1
T~
.1
2.0
0
T" 9.0
T" 11.5
T" 7.0
0
0
5 1015
0 5 10 15
0 5 10 15 20 25
,1
o5
10 15 20 25 30
HOURS AFTER INTRAVENOUS PG ADMINISTRATION Fig. 4. PG (e) and propranolol (0) plasma levels in 4 dogs after intravenous PG doses (0.5 to 1.0 mg/kg). The tl/z is expressed in hours (hr) and Vd in liters per kilogram (II kg).
Table III. PG and propranolol concentrations in 3 dogs (mean ± SD) treated with propranolol. 10 mg/kg/day,for 7 days
PG (J-Lg/ml) Propranolol (J-Lg/ml)
Bile
Plasma
Urine
106 ± II ND
0.30 ± 0.05 0.053 ± 0.019
14.0 ± 3.1 0.43 ± 0.15
NO = not detectable.
from 2.0 to 11.5 hr. The PG injected contained no measurable propranolol, emphasizing that the early appearance of propranolol in plasma was the result of deconjugation to propranolol. Propranolol levels I to 2 hr after the intravenous PG were significantly higher than 0.5 hr after the dose in all animals. The area under the plasma concentration-time curve ratio propranolol/PG was 0.033 ± 0.022 (mean ± SD; n = 4) with the curves extrapolated to infinite time. There was a PG/ propranolol plasma level ratio of about 4 beyond 12 hr in 3 of the 4 animals, much the same as the PG/propranolol ratio in man during long-term propranolol therapy and persisting for 3 to 5 days after propranolol withdrawal. Biliary PG. PG and propranolol levels in bile, plasma, and urine of 3 dogs undergoing
long-term treatment with oral propranolol are shown in Table III. The ability of the biliary excretory system to concentrate PG is reflected in the high bile/plasma level ratio of 353. Note that the PG/propranolol plasma level ratio of 5.7 in the dog is much the same as that observed in man (4.2). No propranolol was detected in the bile. Discussion
Reports that propranolol cumulates more than twofold in plasma of patients treated at 6-hr intervals with oral doses of propranolo]3' 7, 17 and that steady-state concentrations are not reached until after 2 to 3 days of treatment 17 have been difficult to reconcile with a single oral dose t 1/2 of only 3 to 4 hr. 7, 15, 20 Although the propranolol tV2 is increased to 4 to 6 hr after
Volume 26 Number 6
long-term therapy,3, 7, 26 this does not explain its avid and slow cumulation, Observations that satisfactory blood pressure control can be maintained with propranolol throughout dosage intervals of 12 1, 12, 24 or even 24 hr25 and that effects of propranolol can persist for 3 days after drug withdrawaF are also difficult to reconcile with the reported short plasma propranolol tV2, Whereas the duration of the antihypertensive and other actions of propranolol would suggest a longer dosage interval than is generally used, the kinetic basis for such therapy is lacking, The unexpected finding that PO cumulates more than twofold after long-term propranolol dosing with steady-state plasma concentrations 4 times as high as those of propranolol suggested that propranolol cumulation, at least in part, may depend on PO cumulation, The observation that propranolol does not cumulate after repeated intravenous doses,7 a route of administration that generates very little PO, 20 supports such a hypothesis, The finding of a change in the propranolol tVz from 4 to 6 hr to approximately 16 to 24 hr, starting at 12 to 24 hr after cessation of longterm propranolol therapy, is of particular importance, This change in propranolol kinetics, which has not been previously observed, appeared to be related to a parallel decrease in PO elimination, Whereas with previously available methodology no residual propranolol or propranolol metabolites were detected at 24 hr after withdrawal,4, H, II both PO and propranolol were detected in plasma and urine 3 to 5 days after withdrawal in the present study with PO concentrations exceeding propranolol concentrations by about fourfold throughout the drugwithdrawal period. Although plasma levels at 36 to 72 hr are quite low for both PO (2 to 6 ng/ml) and propranolol (0.1 to 2 ng/ml), they may still be of biological significance. Persistent biological activity has been observed at 36 11 , 25 and even at 72 hr2 after withdrawal of long-term propranolol. The unexpected cumulation and slow disappearance of PO may be explained by the distribution of PO into a deep storage site. The very high concentrations of PO in the bile of the dog after long-term propranolol suggest enterohe-
Propranolol glucuronide cumulation
693
patic recirculation. The irregular shape of the terminal elimination phase of both PO and propranolol in man could be explained by such a mechanism. Enteric deconjugation of PO and subsequent reabsorption of propranolol are also suggested by the reports that only a small fraction of radioactive doses of propranolol are recovered in human feces. 14 Through such mechanisms, deconjugation of PO to propranolol could serve to replenish an active pool of propranolol including such sites as have recently been described in postganglionic sympathetic neurons. 5 The slow and avid cumulation of propranolol and its slow elimination may, however, also be explained by the distribution of propranolol into a high-affinity binding site independent of PO. The slow release of such a pool of propranolol should, by normal metabolic clearance, result in a parallel slow disappearance of PO. Direct support for the deconjugation of PO to propranolol was obtained by intravenous administration of human PO to the dog. The rapid appearance of propranolol in plasma as early as 0.5 hr after the PO dose is the result of systemic deconjugation which could take place in the blood as well as in other tissues. ,B-Olucuronidase activity is detectable in almost all tissues including blood. 16 The very slow decline of propranolol in 3 of the dogs given intravenous PO (tV2, 7.5 to 11.5 hr) indicates continuing deconjugation of PO with the deconjugation rate being much slower than the known elimination rate of propranolol (tllz, 0.5 to 2 hr)6, 9, 10, 22 in this species. This slow deconjugation to propranolol coincides with a marked increase in the PO tV2 from 2 to 12 hr, which is similar to observations in man. This change in PO kinetics may be due to enterohepatic recirculation of PO including enteric deconjugation to propranolol. Although the amount of propranolol formed after intravenous PO appears small relative to the amount of PO administered (A ue proprano10l/AUe PO = 0.033), it represents about 10% of PO if corrected for the threefold difference in volume of distribution between propranolol 9 and PO. Furthermore, since the deconjugated propranolol is eliminated mainly by metabolism, the measured propranolol only represents
694
Walle et al.
a fraction of total deconjugated PO. Systemic PO deconjugation may also be quantitatively more important in disease conditions associated with elevated .a-glucuronidase activity. 16 Although deconjugation of PO to yield propranolol has been demonstrated in the dog, mechanisms and sites of deconjugation require further studies including enterohepatic recirculation of PO and propranolol. It also remains to be determined whether these processes occur in man. The high plasma PO concentrations observed in man can in part be explained by the smaller volume of distribution of PO (It/kg in the dog) than of propranolol. It is clear, however, that PO occupies a larger pool of distribution than the vascular space. It is not unlikely that PO, retaining the highly lipophilic naphthalene ring system, could penetrate into cell membranes and concentrate in tissues. Potential plasma binding or renal tubular reabsorption could explain the relatively low renal clearance of PO. PO renal clearance of only 51 mllmin is less than half the normal glomerular filtration rate in healthy individuals. The dependence of plasma PO levels on renal clearance was reflected in the elevated levels in the few patients studied who had subnormal glomerular filtration rates, suggesting that PO may cumulate more readily in patients with severe renal disease. This has been shown to be the case with the glucuronic acid conjugate of oxazepam. 13 Abnormal PO cumulation in association with PO deconjugation may lead to elevated propranolol plasma levels in patients with chronic renal disease. A recent study in the dog,22 demonstrating 4 times higher plasma concentrations of the t- isomer of PO than of the d - isomer after single oral doses of d,t-propranolol, suggests that our findings of PO cumulation, slow disappearance, and deconjugation to propranolol may apply mainly to the .a-blocking I-isomer. Although the size of the deep pool identified for propranolol in man is small and therefore can explain only in part total propranolol cumulation in the body, the detection of a deep storage site introduces a concept that may be useful in our gradually developing understanding of the disposition and mechanism of action of propranolol. The saturation of a metabolic pro-
Clin. Pharmacal. Ther. December 1979
cess other than glucuronidation is probably of greater significance for the total body accumulation of propranolol. This saturable process very likely involves naphthalene ring oxidation.17, IH Finally, the importance of highly sensitive and specific analytical methodology for the identification of deep storage sites for drugs in man cannot be overemphasized. We thank John Brubaker for valuable assistance in the experiments performed in the dog.
References I. Berglund G, Andersson 0, Hansson L, Olander R: Propranolol given twice daily in hypertension. Acta Med Scand 194:513-515, 1973. 2. Brundin T, Edhag 0, Lundman T: Effects remaining after withdrawal of long-term betareceptor blockade. Reduced heart rate and altered haemodynamic response to acute propranolol administration. Br Heart J 38: 1065-1072, 1976. 3. Chidsey CA, Morselli P, Bianchetti G, Morganti A, Leonetti G, Zanchetti A: Studies of the absorption and removal of propranolol in hypertensive patients during therapy. Circulation 52:313-318, 1975. 4. Coltart DJ, Cayen MN, Stinson EB, Davies RO, Harrison DC: Determination of the safe period for withdrawal of propranolol therapy. Circulation 47, 48(suppl. IV):IV-7, 1973. 5. Daniell HB, Walle T, Gaffney TE, Webb JG: Stimulation induced release of propranolol and norepinephrine from adrenergic neurons. J Pharmacol Exp Ther 208:354-359, 1979. 6. Evans GH, Nies AS, Shand DG: The disposition of propranolol. III. Decreased half-life and volume of distribution as a result of plasma binding in man, monkey, dog and rat. J Pharmacol Exp Ther 186:114-122,1973. 7. Evans GH, Shand DG: Disposition of propranolol. V. Drug accumulation and steady-state concentrations during chronic oral administration in man. CUN PHARMACOL THER 14:487-493, 1973. 8. Faulkner SL, Hopkins JT, Boerth RC, Young JL, Jellett LB, Nies AS, Bender HW, ~hand DG: Time required for complete recovery from chronic propranolol therapy. N Engl J Med 289:607-609, 1973. 9. George CF, Orme ML 'E, Buranapong P, Macerlean D, Breckenridge AM, Dollery CT: Contribution of the liver to overall elimination of propranolol. J Pharmacokinetic Biopharm 4: I7 -27, 1976. 10. Hayes A, Cooper RG: Studies on the absorption, distribution and excretion of propranolol in rat,
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11.
12.
13.
14.
15.
16.
17.
18.
dog and monkey. J Pharmacol Exp Ther 176: 302-3 11, 1971. Leaman DM, Levenson L W, Shiroff RA, Babb JD, Dejoseph RL, Hayes AH, Zelis R: Persistence of biological activity after disappearance of propranolol from the serum. J Thorac Cardiovasc Surg 72:67-72, 1976. MacLeod S, Hamet P, Larochelle P, Nadeau J, Ogilvie R, Rangno R, Ruedy J, Sellers E, Ti T: Antihypertensive efficacy of b.i.d. propranolol. Ann R Coli Physicians Surg Can II:34, 1978. Odar-CederiOf I, Vessman J, Alvan G, Sj6qvist F: Oxazepam disposition in uremic patients. Acta Pharmacol Toxicol 40(suppl. 1):52-62, 1977. Paterson JW, Conolly ME, Dollery CT, Hayes A, Cooper RG: The phannacodynamics and metabolism of propranolol in man. Pharmacol Clin 2: 127-133, 1970. Shand DG, Nuckolls EM, Oates JA: Plasma propranolol levels in adults with observations in four children. CUN PHARMAcaL THER II: I 12120, 1970. Wakabayashi M: f3-Glucuronidases in metabolic hydrolysis, in Fishman WH, editor: Metabolic conjugation and metabolic hydrolysis. New York, 1970, Academic Press, Inc., vol. II. Walle T, Conradi EC, Walle UK, Fagan TC, Gaffney TE: The predictable relationship between plasma levels and dose during chronic propranolol therapy. CUN PHARMAcaL THER 24:668-677, 1978. Walle T, Conradi EC, Walle UK, Fagan TC, Gaffney TE: 4-Hydroxy-propranolol and its glucuronide in man after single and chronic doses of propranolol. CUN PHARMAcaL THER. (In press.)
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19. Walle T, Conradi EC, Walle UK, Gaffney TE: Steady-state plasma concentrations and urinary excretion of propranolol-O-glucuronide and propranolol in patients during chronic oral propranolol therapy. Fed Proc 35:665, 1976. 20. Walle T, Fagan TC, Conradi EC, Walle UK, Gaffney TE: Presystemic and systemic glucuronidation of propranolol. CUN PHARMAcaL THER 26:167-172, 1979. 21. Walle T, Morrison J, Walle K, Conradi E: Simultaneous determination of propranolol and 4-hydroxypropranolol in plasma by mass fragmentography. JChromatogr 114:351-359,1975. 22. Walle T, Walle UK: Stereoselective oral bioavailability of (±)-propranolol in the dog. A GC-MS study using a stable isotope technique. Res Commun Chern Pathol Pharmacol 23:453464, 1979. 23. Walle T, Walle UK, Bridges DR, Conradi EC, Gaffney TE: Quantitative GC-MS of proprano101, a key to an improved understanding of the relationship between plasma concentrations and dose. Clin Chern 24:991, 1978. 24. Wilkinson PR, Dixon N, Hunter KR: Twicedaily propranolol treatment for hypertension. J Int Med Res 2:220-223, 1974. 25. Wilson M, Morgan G, Morgan T: The effect on blood pressure of f3-adrenoreceptor-blocking drugs given once daily. Clin Sci Mol Med 51:5275-5285, 1976. 26. Wood AJJ, Carr K, Vestal RE, Belcher S, Wilkinson GR, Shand DG: Direct measurement of propranolol bioavailability during accumulation to steady-state. Br J Clin Phannacol 6:345-350, 1978.
