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本文研究了氨基酸对大鼠肝脏中美托洛尔代谢的抑制作用，探讨了食物对药物代谢影响的可能机制。美托洛尔是一种β阻断剂，它在人体内的首过效应高，意味着在药物进入全身循环之前，它在肝脏中已被大量代谢。这种代谢过程涉及多种药物代谢酶。研究中使用了与美托洛尔结构相似但组织结合性较低的药物普萘洛尔作为参考，以确定在有无氨基酸存在的情况下，美托洛尔及其两个代谢物的Vmax和KM值。研究设计了六组实验，每组包含来自24只雄性Sprague-Dawley大鼠的4个肝脏。这些肝脏在单一通路模式下用含有不同浓度的美托洛尔（3.74, 4.49, 5.61, 7.48, 18.7, 44.9 μM）的氧合缓冲液中灌注130分钟，流速为每分钟每克肝3毫升。从第50分钟到第90分钟，缓冲液中加入了平衡氨基酸混合物。每5分钟取样一次，使用高效液相色谱法（HPLC）分析美托洛尔及其两个代谢物，分别是α-羟基美托洛尔和O-去甲基美托洛尔。研究通过非线性曲线拟合确定了在氨基酸存在或不存在的条件下的稳态药物浓度，进而确定了Vmax和表观KM值。结果表明，氨基酸可逆地降低了美托洛尔及其两个代谢物的Vmax值约50%，而对表观KM值没有显著影响。因此，在低美托洛尔入流浓度时，药物的可用性显著增加（超过90%）。此外，氨基酸还增加了氧气的消耗，直到流出缓冲液几乎耗尽。关于可能影响Vmax的机制，研究假设氨基酸可能直接抑制了代谢酶，或者是辅因子（如NADPH或氧气）的限制。氨基酸介导的肝脏窦状隙中心区域的氧气消耗可能导致药物代谢酶的抑制，并与氨基酸共灌注期间流出缓冲液中氧气消耗和Vmax降低相一致。研究推测，这些机制中的一个或多个可能贡献于人类观察到的食物与高首过代谢药物之间的相互作用。该研究提供了关于氨基酸如何影响药物代谢酶活性的新见解，并且对于了解食物成分如何影响药物在人体内的代谢具有重要意义。这些发现可能有助于优化药物治疗方案，减少食物和药物相互作用带来的不良影响。

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INHIBITION OF METOPROLOL METABOLISM BY AMINO ACIDS IN PERFUSED

RAT LIVERS

Insights into the Food Effect?

BO WANG AND HUGH A. SEMPLE

College of Pharmacy and Nutrition, University of Saskatchewan

(Received June 18, 1996; accepted December 10, 1996)

ABSTRACT:

A mixture of amino acids inhibits propranolol metabolism in per-

fused rat livers. To obtain mechanistic information about the inter-

action, a related but less tissue-bound drug, metoprolol, was used

to determine V

max

and K

for parent drug and two metabolites in

the presence and absence of amino acids. Six groups of 4 livers

from 24 male Sprague-Dawley rats were perfused in the single-

pass mode at 3 ml/min/g liver for 130 min with oxygenated buffer

containing 3.74, 4.49, 5.61, 7.48, 18.7, or 44.9

M metoprolol. From

50 to 90 min, a balanced amino acid mixture was included in the

buffer. Samples of liver effluent taken every 5 min were analyzed by

HPLC for metoprolol and two metabolites,

-hydroxymetoprolol

and O-demethylmetoprolol. Steady-state concentrations of drug

determined before, during, and after amino acids were used to

determine V

max

and apparent K

values by nonlinear curve-fitting

under each condition. Amino acids reversibly reduced the V

max

values of metoprolol and both metabolites by ;50% without sig-

nificantly affecting apparent K

values. As a result, large increases

in availability occurred, especially at low metoprolol inlet concen-

trations (>90%). Amino acids also increased oxygen consumption

until the effluent buffer was almost depleted. Possible mechanisms

influencing V

max

include direct inhibition of metabolic enzymes by

amino acids or cosubstrate (NADPH or oxygen) limitation. Amino

acid-mediated pericentral oxygen depletion in the hepatic sinu-

soids could result in inhibition of drug-metabolizing enzymes, and

is consistent with a reduction of V

max

and oxygen depletion in the

effluent buffer during amino acid coinfusion. We postulate that one

or more of these mechanisms could contribute to the interaction

between food and high first-pass drugs observed in humans.

Rac-metoprolol is one of several drugs known to exhibit increased

oral availability when coadministered with a high-protein meal, in-

cluding propranolol, propafenone, labetolol, zuclopenthixol, and

dixyrazine (1). Although this food interaction was first observed two

decades ago, its mechanism remains to be fully elucidated, despite

intensive study (1). The mechanism is likely to be complex, with

contributions from more than one of the physiological responses to

food combining to cause a net increase in AUC

oral

Because drugs

that show the “food effect” are almost completely absorbed from the

gastrointestinal tract, it is generally agreed that the interaction is due

to a reduction in first-pass metabolism (1). Studies to date on pro-

pranolol have concentrated on the liver, although recent studies in

rabbits suggest the possible contribution of intestinal metabolism (2).

A reduction in hepatic first-pass metabolism could occur through

changes to hepatic blood flow, plasma protein binding, or metabolic

activity. The hypothesis that the increase in AUC

oral

of propranolol

could be caused by a transient increase in hepatic blood flow (3) was

questioned, because flow changes could not account for the magnitude

of the increase (4, 5). It is also unlikely that food causes an increase

in the unbound fraction of propranolol in plasma (6). Experiments in

humans to test for transient changes in metabolic activity have shown

that food causes inhibition of presystemic primary conjugation of

propranolol (7), although data relating to the more important phase I

pathways are inconclusive (8, 9). Nevertheless, simulations have

indicated that propranolol availability would be most sensitive to

changes in apparent V

max

and somewhat sensitive to changes in

apparent K

, both of which contribute to intrinsic clearance (10).

To explore further whether nutrients could inhibit propranolol

metabolism, amino acids were coinfused into rat livers perfused with

buffer containing propranolol (11). Global inhibition of metabolism

was observed, indicating that at concentrations achieved after a high

protein meal, amino acids could inhibit all of the pathways of pro-

pranolol metabolism and that the degree of metabolic inhibition was

related to the concentration of amino acids in the buffer. Hepatic

tissue binding prevented further exploration of the mechanism using

propranolol as a model drug. Pilot studies showed, however, that

metoprolol is much less extensively tissue bound and its metabolism

was inhibited by amino acids, making it an appropriate model drug for

mechanistic studies involving the measurement of Michaelis-Menten

parameters. The metabolism of metoprolol is depicted in fig. 1. We

describe herein experiments that confirm our pilot observations in

perfused rat livers and that focus on amino acid effects on the apparent

max

and K

of metoprolol metabolism. As part of the liver viability

assessment, effluent buffer O

content was measured.

Abbreviations used are: AUC, area under the plasma concentration-time

curve; O

, oxygen; OH, hydroxy; C

, concentration at steady-state; t

, time to

steady-state; C

, inlet concentration; C

out

, outlet concentration; Q, buffer flow

rate; E, organ extraction ratio; CL, organ clearance; F, organ availability;

velocity of metabolism; C

out,m

, outlet concentration of metabolite; [S], substrate

concentration; MW, molecular weight; CYP, cytochrome P450.

This study was supported by the Medical Research Council of Canada, De-

velopment Grant DG-405 and by the Heart and Stroke Foundation of Saskatchewan.

This work has been presented in part as an abstract [ISSX Proc. 8, 311 (1995)].

Send reprint requests to: Dr. Hugh A. Semple, College of Pharmacy and

Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon,

Saskatchewan, Canada S7N 5C9.

0090-9556/97/2503-0287–295$02.00/0

RUG METABOLISM AND DISPOSITION Vol. 25, No. 3

287

at NSTL on June 16, 2012dmd.aspetjournals.orgDownloaded from

Materials and Methods

Chemicals. (6)-Metoprolol tartrate and nadolol (internal standard) were

purchased from Sigma Chemical Co. (St. Louis, MO). H119/66 p-OH benzoate

(

-hydroxymetoprolol), H105/22 p-OH benzoate (O-demethylmetoprolol),

and H117/04 HCl (metoprolol acid) were gifts from Astra Ha¨ssle (Mo¨lndal,

Sweden). Solvents were HPLC grade (BDH, Toronto, Canada). All other

chemicals used were of analytical grade.

Animals. Livers used in the study were from 24 male Sprague-Dawley rats

(198–250 g, Charles River, St. Constant, Quebec, Canada) maintained on

standard laboratory chow and water ad libitum in accordance with the guide-

lines of the Canadian Council on Animal Care. Animals were randomly

assigned to six groups of four in a parallel design, one group for each of six

concentrations of metoprolol in the perfusion medium (3.74, 4.49, 5.61, 7.48,

18.7, and 44.9

M). Perfusions were performed sequentially according to the

random assignments.

Surgical Procedure. After anesthetizing the rat with methoxyflurane

(Pitman-Moore, Mississauga, Ontario, Canada), the liver was isolated and

perfused in the single-pass mode with oxygenated Krebs bicarbonate buffer in

a temperature- and humidity-controlled cabinet as previously described (12,

13). During the surgical procedure, there was no prolonged period of anoxia

because perfusion was started immediately after cannulation of the portal vein.

The entire surgical procedure was conducted rapidly (,20 min) to minimize

the chance of an unsuccessful preparation. Liver viability was assessed by: 1)

measurement of the O

tension in the liver effluent using a biological O

monitor (YSI model 5300; YSI, Inc., Yellow Springs, OH) equipped with a

micro-O

probe attached to a low-flow cell; 2) the physical appearance of the

liver; and 3) the maintenance of metoprolol steady-state concentrations.

Liver Perfusions. The study was designed both to evaluate the apparent

Michaelis-Menten parameters and to measure the effect of a balanced mixture

of amino acids on apparent V

max

and K

values for metoprolol and its

metabolites. Metoprolol in Krebs bicarbonate buffer was infused at 3 ml/min/g

liver into different livers at various concentrations over the range of saturation

of metabolism. Because the liver outlet steady-state concentration reflected the

velocity of metabolism or formation of each metabolite, plots of velocity vs.

concentration were constructed from which the apparent V

max

and K

for

metoprolol and each metabolic pathway were calculated. To assess the effect

of amino acids, the metoprolol and/or metabolite steady-state concentrations in

the effluent were perturbed by adding a balanced mixture of amino acids to the

buffer. Based on the measurement of portal venous concentration of phenyl-

alanine (250

M) in dogs after a high-protein meal (14), a balanced mixture of

amino acids in the perfusion buffer-medium was added as a dilution of

Aminosyn* II 10% Amino Acids Injection (Abbott Laboratories, Montreal,

Canada). Final concentrations of constituent amino acids in the inlet buffer

were as previously described (11). Velocity vs. concentration plots were again

constructed, and the apparent V

max

and K

values in the presence of amino

acids were calculated for comparison with the preamino acid values. Once

steady-state liver outlet concentrations had been measured in the presence of

amino acids, buffer without amino acids was perfused again to verify the

reversibility of the amino acid interaction.

Time 0 was defined by the initiation of perfusion with Krebs bicarbonate

buffer containing metoprolol at 1 of the 6 inlet concentrations. A blank

perfusion buffer sample was collected before time 0; after time 0, perfusate

samples were collected over 20 sec at 1, 2.5, 5, 7.5, 10 min, and then every 5

min until 130 min. After the initial 50-min control phase, during which

metoprolol and metabolite concentrations in the effluent buffer had been at

steady state for ;30 min, the perfusion medium was switched to Krebs buffer

containing amino acids in addition to metoprolol. The metoprolol and amino

acids perfusion was continued for 40 min, then the perfusion medium was

returned to the original Krebs buffer containing only metoprolol for 40 min.

During the perfusion, the temperature and O

tension in the effluent were

recorded when samples were taken. Samples of liver effluent were analyzed for

metoprolol and metabolites immediately following each perfusion experiment.

Analytical Method. After addition of the internal standard (50

l of a 6.46

M nadolol solution in water) to 1.0 ml of the perfusate sample, 50

lofthe

mixed sample was directly injected onto the HPLC system consisting of a

Waters model 510 Pump, a Shimadzu model SIL-9A Autoinjector, a NovaPak

8 3 100 mm, 4-

m particle size column, an ABI Spectroflow 980

Programmable Fluorescence detector (excitation wavelength 224 nm; no emis-

sion filter), and a Shimadzu model C-R3A Chromatopac Integrator. The

mobile phase consisted of water:acetonitrile:triethylamine (91:9:0.3, v/v/v),

adjusted to pH 3.0 with orthophosphoric acid and pumped at 3.0 ml/min. Run

time was 22 min. The assay was validated according to generally accepted

criteria (15).

Data Analysis. When the single-pass perfusion system was at steady-state,

and the distribution/binding processes of the drug in the liver preparation were

essentially complete, the rate of drug loss across the liver equaled the rate of

metoprolol elimination (i.e. the rate of metabolism). C

and t

were defined as

previously described (12). The hepatic tissue binding of metoprolol was calculated

from the effluent steady-state metoprolol data as previously described (12).

Hepatic disposition of drug was described by standard steady-state mass

balance equations for drug elimination by an organ (16). Parameters calculated

from C

, C

out

, and Q included E, CL (ml/min) and F. The

(nmol/min/g liver)

FIG.1.Metabolism of metoprolol.

288

WANG AND SEMPLE

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