Original articles Naphthoxylactic acid after single and long-term doses of propranolol The disposition of naphthoxylactic acid (NLA) and propranolol was determined after single intravenous and oral doses of propranolol in 5 subjects and during long-term therapy in 8 hypertensive patients. The area under the plasma concentration/time curve (AUC) of NLA exceeded that of propranolol twofold after an intravenous 4-mg dose of propranolol but tenfold after single oral 20-mg and 80-mg doses, indicating a high degree of presystemic formation of NLA. The time to peak concentration (1.5 to 2 hr) and the plasma half-life of NLA (3.1 to 4.2 hr) were in the same range as for propranolol. A two- to threefold accumulation of NLA in plasma was indicated after chronic propranolol therapy. Whereas plasma concentrations and urinary excretion of NLA increased linearly with dose on long-term propranolol dosage, there was a nonlinear relationship between propranolol plasma levels and dose. This resulted in a markedly reduced NLA /propranolol plasma level ratio with increasing doses (from 10 to 28 at a daily 80-mg propranolol dose to about 3 at a daily 960-mg dose). The urinary excretion of NLA accounted for about 14% of the propranolol dose over the dose range studied. The finding that the renal clearance of NLA (18 to 54 mllmin) and plasma levels were dependent on glomerular filtration rate (GFR) indicates that renal elimination is the main determinant of total body clearance of NLA and that it may accumulate strikingly in patients 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 of Pharmacology and Medicine, Medical University of South Carolina
Naphthoxylactic acid (NLA) has been reported to be a propranolol metabolite in man. 1, 4. 14 Semiquantitative measurements suggested that NLA may account for 20% of propranolol metabolism. 4 The main route of formation of NLA depends on N -dealkylation of propranolol to N -desisopropylpropranolol folSupported by National Institute of General Medical Sciences grants GM 20287 and RR 0 1070, Received for publication April 24, 1979, Accepted for publication May 7, 1979, Reprint requests to: Thomas Walle, Ph,D" Department of Pharo macology, Medical University of South Carolina, 171 Ashley Ave" Charleston, SC 29403,
lowed by deamination and oxidation of an intermediate aldehyde. 3 , 13, 14 An alternate route involves direct deamination of propranolol to this aldehyde. 15 Involvement of both mitochondrial and plasma monoamine oxidase in the formation of NLA has been indicated. 13 We recently described a gas chromatographic assay of NLA in plasma. 2 Preliminary observations using this assay demonstrated NLA levels in plasma about 10 times that of propranolol in patients on long-term propranolol therapy. This was recently observed by others. 5 Our study focused on a comparison of the disposition of NLA and propranolol after single
0009-9236/79/110548+07$00.70/0 © 1979 The C. y, Mosby Co,
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intravenous and oral doses of propranolol in nonnal subjects as well as during chronic therapy over a wide dosage range in hypertensive patients. NLA was detennined by a molecularly specific gas chromatography-mass spectrometry technique using deuterium-labeled NLA as the internal standard. Methods
All subjects were hospitalized in a general clinical research center. In each, an admission history, physical examination, chest film, electrocardiogram, complete blood count, urinalysis, and selected blood chemistry detenninations were perfonned. Single doses of propranolol. Five healthy male subjects, 22 to 30 yr of age, weighing 62 to 83 kg, were given single oral 20-mg and 80-mg doses of propranolol (lnderal) on 2 different occasions. The doses were administered in the morning after overnight fasting. Blood samples were collected from a forearm vein 0.5, 1, 1.5,2,3,4,6, 10, and 16 hr after the dose. Single intravenous doses, 0.05 mg/kg (3.7 ± 0.6 mg), were given to 3 of these subjects at a rate of 1 mg/min on a separate occasion. Blood samples were collected from the opposite arm 0.25,0.5, 1,2,4,6, and 8 hr after the dose. Long-term doses of propranolol. Eight patients, 26 to 51 yr of age, weighing 61 to 103 kg, with mild to moderately severe hypertension were also studied. On admission all were on long-tenn propranolol therapy, 80 to 480 mg daily (every 6 hr). Four of the patients were also on trichlonnethiazide, and 4 on hydralazine. None of the patients had liver disease. Two patients had creatinine clearances of 40 mllmin, whereas creatinine clearance in the other patients was nonnal with respect to age (80 to 145 mllmin). After 3 days of hospitalization, a blood sample was collected 2 hr after a morning dose following an overnight fast. The daily dose was then increased and a blood sample was again collected at 2 hr after the morning dose following 3 days at this dose level. The total number of steady-state doses studied in each patient ranged from 2 to 6. The highest dose level was 960 mg/ day. In 5 of the patients a blood sample was also collected 6 hr after the
Naphthoxylactic acid after propranolol
highest dose. Twenty-four-hour urine collections were also made in 5 of the patients on the third day at each dose level. Sample collecting and handling techniques. All blood samples were collected with the subject at rest by separate venipuncture or via an indwelling Butterfly cannula for serial samples. Blood and urine samples were collected, handled, and stored as described previously. 10 Analytical methodology. NLA was extracted from plasma and urine and derivatized as previously described. 2 Separation and detection were made by gas chromatography-mass spectrometry focusing on the molecular ion of NLA (m/e, 442)2 and that of a deuterium-labeled eH2-ring) internal standard (m/e, 444). The internal standard was prepared from NLA by deuterium/hydrogen exchange as described for propranolol. 17 The method was rapid, specific, and sensitive down to a concentration of I ng/ml. Propranolol was measured by gas chromatography-mass spectrometry using hexadeuterium-Iabeled internal standard as described previously. 16. 18 Calculations. The area under the plasma concentration/time curves (AUCs) for NLA and propranolol from 0 to 16 hr was determined according to the trapezoidal rule. For the intravenous dose the plasma concentration/time curves were extrapolated from 8 to 16 hr. Plasma half-lifes (tV2S) were calculated by regression analysis. Results
Plasma NLA and propranolol in normal subjects after single doses of propranolol. The plasma concentrationltime curves for NLA and propranolol after a single 0.05-mg/kg (about 4-mg) intravenous dose of propranolol in 3 subjects and 20- and 80-mg oral doses in 5 subjects are shown in Fig. 1. The time to reach the peak concentration of NLA (1.5 to 2 hr) was the same after the intravenous and the 2 oral doses. The tV2 for NLA (3.1 to 4.2 hr) was virtually identical to the tV2 for propranolol (3.8 to 4.2 hr). The NLA concentrations were considerably higher than the propranolol concentrations, especially after oral doses (Fig. I and Table I). While the AUC for propranolol was very much the same after the 4-mg intravenous dose (58
Walle et al.
Clin. Pharmacol. Ther. November 1979
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....J C> ....J C>
Y=O .1l0X-1. 8 20
O+-----r----.-----r----,----. 1000 800 600 o 200 400
PROPRANOLOL DOSE, MG/DAY Fig. 3. The relationship between the NLAI proprano-
lol plasma level ratio and propranolol dose in 8 hypertensive patients during chronic propranolol therapy. Symbols are the same as in Fig. 2.
tionship between NLA excretion and daily propranolol dose over the whole dose range, which was extrapolated through the origin. A mean of II % (w /w) of the propranolol dose appeared as NLA in urine which on a molar basis corresponds to 14%. The renal clearance of NLA was compared with the creatinine clearance in 5 of the patients (Fig. 5). The renal clearance of NLA (18 to 54 mil min) correlated well (correlation coefficient, 0.90; p < 0.05) with the creatinine clearance in these patients (40 to 145 mllmin). The patient with the lowest NLA renal clearance was one of the patients with the markedly elevated plasma NLA levels (Fig. 2, A). Treatment of both plasma and urine samples with Glusulase* 12 did not even minimally increase NLA levels. No NLA was detected in the bile of dogs chronically treated with proprano101,8 not even after enzymatic hydrolysis. ',a-Glucuronidase + sulfatase. Endo Labs.
PROPRANOLOL DOSE, MG/DAY Fig. 4. Daily urinary NLA excretion in 5 of the hy-
pertensive patientsl dose during chronic propranolol therapy. Symbols are the same as in Fig. 2. Discussion
NLA plasma levels, five- to sixfold higher after a 20-mg single oral dose of propranolol than after a 4-mg intravenous dose (both doses giving the same plasma propranolol levels), demonstrate deamination and subsequent oxidation of propranolol to NLA as an important metabolic pathway involved in the presystemic removal of oral doses of propranolol. Other studies have shown that the formation of propranolol glucuronide 12 and 4-hydroxypropranolol and its glucuronide 9 are also involved in the presystemic metabolism of propranolol. The total plasma concentrations of these 4 metabolites together with the estimated concentrations of a large number of minor metabolites 6 , 11, 14 exceed the plasma concentrations of propranolol by about 30 times after 20-to 80-mg oral doses of propranolol. This is very similar to the total radioactivity /proprano101 plasma level ratio of 25 to 35 reported after radioactive 40-mg oral doses. 4 These observations suggest that the single oral dose of propranolol, at least as it appears in plasma, may be accounted for by known metabolites and that NLA is by far the major metabolite. A comparison of NLA plasma levels after the
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80-mg single oral dose of propranolol with the concentrations observed after chronic doses of 80 mg given every 6 hr indicates that NLA accumulates in plasma two- to threefold as a result of repeated propranolol administration which resembles the cumulation characteristics of propranolol 10 and propranolol glucuronide. 8 The nonlinear relationship between plasma levels of propranolol and dose as previously reported lO and also observed in this study does not involve saturation of the metabolic pathways leading to NLA formation, since NLA levels in both plasma and urine appear to increase linearly with propranolol dose over the whole therapeutic dose range. The nonlinear propranolol disposition during long-term therapy has to be explained by saturation of another elimination process, which could involve naphthalene ring oxidation. 9 The elimination of NLA appears to depend on excretion by the kidneys. The fairly low renal clearance of NLA (18 to 54 mUmin) is, therefore, probably the main determinant of the high plasma levels. This is supported by findings of NLA plasma concentrations in the 2 patients with subnormal GFR 2 to 3 times that in patients with normal GFR, although plasma levels of propranolol were not affected. The low renal clearance of NLA could be due to plasma binding or renal tubular reabsorption of NLA, or both. The fact that a low GFR did not affect the plasma concentrations of propranolol is consistent with the observations that the total body clearance of propranolol is entirely due to metabolism. 10 Since propranolol and most of its metabolites are, to a very large extent, conjugated at the ,B-hydroxyl group 12, 14 it is surprising that not even a trace of NLA is conjugated with either glucuronic acid or sulfate. This may be due to strong intermolecular hydrogen bonding between the hydroxyl and carboxyl groups. Although the presence of naphthoxyacetic acid,5, 14 hydroxynaphthoxylactic acid,6 and methoxyhydroxynaphthoxylactic acid ll in man indicate further oxidation of NLA, this only occurs to a minor extent. Whereas the total concentration of known propranolol metabolites in plasma appears to account for single oral doses of propranolol, the
Naphthoxylactic acid after propranolol
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