Introduction

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Diet is a well recognized variable affecting drug disposition in humans (Walter-Sack and Klotz, 1996; Wilkinson, 1997). In some cases, diet has been found to affect intestinal or hepatic drug metabolism. For example, grapefruit juice increased plasma concentrations of the calcium channel blocker felodipine by decreasing intestinal cytochrome P-450 (CYP) 3A4 (Lown et al., 1997; Schmiedlin-Ren et al., 1997). Another more recently recognized factor in drug disposition is the function of drug transporters such as the MDR1 gene product P-glycoprotein (Schinkel et al., 1995; Wacher et al., 1995; Kim et al., 1998), which is expressed in liver and intestine, as well as other sites. Therefore, modification of drug transporter function by dietary constituents may also play a role in diet-induced changes in drug disposition.

The antiarrhythmic drug quinidine is a substrate of both CYP3A4 (Guengerich et al., 1986) and P-glycoprotein (Terao et al., 1996; Fromm et al., 1998). Recently, we identified dietary salt as a modulator of the prehepatic disposition of p.o. quinidine, a CYP3A substrate (Darbar et al., 1997). We found that when subjects ingested a high-salt diet (400 mEq/day), the oral bioavailability of quinidine was 69 ± 12% compared with 76 ± 17% with a low-salt diet (10 mEq/day). Furthermore, there was a much greater reduction (~3-fold) in the area under the concentration-time curves (AUCs) in the first hour after drug administration, and there was no effect on terminal elimination half-life or plasma concentrations after i.v. administration of quinidine. Taken together, these data suggested that dietary salt modulates a prehepatic (likely intestinal) component of quinidine disposition.

The studies in which we initially identified the effect of dietary salt on quinidine disposition were designed to manipulate dietary salt to indirectly modulate sympathetic function, as shown by Fraser et al. (1981). Thus, one possible mechanism underlying the effect of salt on drug disposition may relate to altered sympathetic modulation of gut function. In a further test of the link between dietary salt and sympathetic function, we now report the effect of -adrenoceptor blockade with nadolol on salt-related changes in quinidine disposition.

It is also not known whether the effect of dietary salt is due to local (i.e., intestinal) salt-induced alterations in CYP3A4 and/or P-glycoprotein function or to a primary systemic effect, subsequently modifying the prehepatic component of quinidine disposition. We therefore distinguished between these competing hypotheses by studying the disposition of quinidine after i.v. rather than p.o. salt loading. If the previously observed alteration in prehepatic disposition of quinidine by dietary salt was reproduced by i.v. salt loading, a systemic effect of sodium chloride could be inferred. On the other hand, if it were not, we would deduce that the primary effect of sodium chloride on quinidine disposition is at the local level.

	Materials and Methods

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Subjects

Healthy volunteers of either sex (age range, 18-40 years) were recruited into both studies. No clinically significant abnormalities were found by history, physical examination, or routine laboratory tests, including complete blood count, biochemistry, and ECG. No subjects took long-term medications, and all refrained from ingesting caffeine, grapefruit juice, and alcohol for the duration of the study. The protocols were approved by the Vanderbilt Institutional Review Board, and all subjects gave written informed consent before the study.

Protocols

The diets used in these studies were low salt (10 mEq/day), prepared by the metabolic kitchen of the Vanderbilt Clinical Research Center. The diets were supervised by a registered dietician. Each food item was weighed to ensure correct salt content of the diet.

Nadolol Study. Quinidine disposition was determined on two occasions, once after 1 week on the low-salt diet (10 mEq/day) and once after 1 week on a high-salt diet (400 mEq/day), as achieved with supplementary salt tablets. The order of administration of the two diets was randomized, and the dietary treatments were separated by at least 2 weeks. Subjects received nadolol (20-40 mg once daily) for the entire two dietary treatments. The blocker nadolol was chosen because it lacks first-pass hepatic metabolism, so its effect is less likely to be modulated by dietary salt than would be an agent such as propranolol. All of the subjects had participated in the earlier investigation, and their data from that study therefore were used for comparison of the effect of dietary salt in the absence of -adrenoceptor blockade.

Intravenous Quinidine. This was conducted in two parts: once after 9 days on a high-salt diet (400 mEq/day) and once after 9 days on a low-salt diet (10 mEq/day). The high-salt intake was accomplished by administering the low-salt diet and i.v. sodium chloride supplementation daily, as described below. The order of the sodium chloride loading and sodium chloride restriction arms was randomized, and both parts were separated by at least 1 week.

Quinidine Assay

Blood samples were centrifuged immediately, and the separated plasma was stored at 4°C and protected from light until analysis (within 2 days). Plasma quinidine concentrations were measured by the Vanderbilt Clinical Laboratory Service by immunoassay (TD_x/TD_xFL_x; Abbott Laboratories, Chicago, IL, or Emit 2000 immunoassay; Behring Diagnostics Inc., Cupertino, CA). The assays are designed to measure total plasma quinidine. A contaminant of all administered quinidine preparations, dihydroquinidine, which has pharmacological effects similar to those of quinidine, also was measured (Thompson et al., 1987). The assay results combine quinidine and dihydroquinidine values, but the level of dihydroquinidine is reported to be less than 10% of the total quinidine level in serum (Kates et al., 1978). Controls were run with each batch of samples; the assays conformed to the ranges stipulated by the manufacturers. The sensitivity of the assays is stated to be 0.2 µg/ml; coefficients of variation for human serum containing known concentrations of quinidine (1.5, 3.0, and 6.0 µg/ml) are typically less than 5%.

Pharmacokinetic Evaluation

The AUCs of i.v. quinidine (AUC_i.v.) and p.o. quinidine (AUC_p.o.) were calculated by the trapezoidal rule with extrapolation of the terminal phase to infinity. Systemic serum clearance (CL_S) was calculated as CL_S = dose_i.v./AUC_i.v., and apparent oral clearance (CL_O) was calculated as CL_O = dose_p.o./AUC_p.o.. Bioavailability (F) was calculated by F = (AUC_p.o.·dose_i.v.)/(AUC_i.v.·dose_p.o.). Volume of distribution (V_C) was calculated as V_C = dose_i.v./C(0), with C(0) being the extrapolated quinidine plasma concentration at 0 h after i.v. administration. The terminal elimination half-life (t_1/2) was estimated by linear regression of the log-transformed plasma concentration-time curve after i.v. administration.

Statistical Analysis

All data are presented as mean ± 1 S.D. Paired comparisons were analyzed by Student's t test. Multiple comparisons (Table 1) were analyzed by ANOVA with subsequent Student-Newman-Keuls tests if the null hypothesis of equal means could be rejected. A value of p < .05 was required for statistical significance.

	Results

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Nadolol Study. Eight subjects (six men and two women; age range, 23-39; mean age, 31.6 ± 5.5 years; Table 1), all of whom had participated in our earlier study (Darbar et al., 1997), were included. There was no significant difference in the weight of the subjects while on the two diets (68 ± 12 kg for low salt versus 73 ± 15 kg for high salt), and no subjects reported altered bowel habits. The reduction in peak exercise HR was 24.3 ± 2.9%, which was achieved with 20 mg/day nadolol in six subjects and 40 mg/day in the remaining two subjects (Table 1). Twenty-four-hour urinary sodium excretions showed compliance with the diets (34 ± 22 mEq/24 h for low salt versus 454 ± 52 mEq/24 h for high salt; p < .05).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Plasma concentration-time profiles of quinidine after p.o. administration with and without nadolol on a low-salt diet (top) and high-salt diet (bottom). *p < .05, quinidine versus quinidine + nadolol.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Quinidine clearance after p.o. administration with (+) and without () nadolol on the low- and high-salt diets.

Intravenous Quinidine. Body weight and systolic and diastolic blood pressures during the low- and high-salt loading phases were not significantly different from the respective baseline parameters (Table 3). HR during salt loading (day 8) was significantly lower than that before both diets were started and during the low-salt diet (day 8, Table 3). Urinary sodium excretions showed compliance with the diets (21 ± 25 mEq/24 h for low salt versus 349 ± 31 mEq/24 h for i.v. salt; p < .001; Table 3).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Mean plasma quinidine concentration-time curves in eight healthy volunteers during low-salt diet or low-salt diet and i.v. salt loading (top) after i.v. administration of quinidine gluconate and (bottom) after p.o. administration of quinidine gluconate.

	Discussion

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In this and our earlier studies, dietary salt had no effect on the t_1/2 of quinidine (regardless of route of drug administration). Thus, we inferred that the previously observed decrease in plasma quinidine concentrations and bioavailability during a high-salt diet with p.o. drug likely reflected an effect on intestinal drug disposition rather than on hepatic and renal elimination processes. A simple decrease in the extent of quinidine absorption with the high-salt diet seems unlikely, given that in our previous study the recoveries of quinidine and its major metabolite were similar after p.o. high- and low-salt diets (Darbar et al., 1997). If absorption was decreased on a high-salt diet, then decreased recovery would be expected.

In the present study, we report that -blockade did not alter the disposition of quinidine when the drug was administered to subjects receiving a high-salt diet. However, -blockade inhibited the effect of the low-salt diet to increase plasma concentrations shortly after an p.o. dose: clearance with the low-salt diet plus -blockade was equivalent to that on the high-salt diet. This result strongly supports our working hypothesis that sympathetic activation, a recognized result of salt restriction (Fraser et al., 1981), mediates the change in quinidine disposition during the low-salt diet. Our other major finding is that the pharmacokinetics of p.o. quinidine were not significantly altered when salt loading was accomplished by the i.v., rather than the p.o., route. This result indicates that although systemic sympathetic activation contributes to the effect of dietary salt, another important element must be directly at the level of the intestine. It should be noted that there was a (nonsignificant) trend to smaller AUCs after p.o. administration of quinidine during i.v. salt loading in comparison to the low-salt diet in this study, indicating that i.v. sodium chloride might also have minor effect on disposition of quinidine. However, this effect is small compared with the effect after p.o. administration of the same amount of sodium chloride. The power calculation indicates that it is highly unlikely that the present results can be attributed to a Type II error.

Molecular Determinants of Quinidine Disposition. Quinidine is a substrate of both CYP3A4 and P-glycoprotein (Guengerich et al., 1986; Terao et al., 1996; Fromm et al., 1998). Because both proteins are expressed in the intestinal mucosa (Thiebaut et al., 1987; Watkins et al., 1987; Kolars et al., 1992; Lown et al., 1994; Watkins, 1997), we hypothesize that modification in the function of one or both of these proteins is the cause for our pharmacokinetic observations. P-glycoprotein, the product of the MDR1 gene, functions as an energy-dependent drug efflux pump (Higgins, 1995). In cancer cells, up-regulation of P-glycoprotein results in the efflux of antineoplastic drugs and, hence, resistance to drug therapy, the multidrug resistance phenomenon (Pinedo and Giaccone, 1995). Importantly, functional P-glycoprotein is expressed not only tumor cells but also in many cell types that express CYP3A, including liver, kidney, and the intestine (Gatmaitan and Arias, 1993; Kivistö et al., 1995; Levêque and Jehl, 1995; Wacher et al., 1995). Although initial studies of P-glycoprotein function focused on anticancer drugs, it was recognized that a number of other drugs that are known to be metabolized by common isoforms of CYPs, such as CYP3A4, are also known either to be transported or to inhibit the function of P-glycoprotein. Examples include quinidine, verapamil, and cyclosporin (Levêque and Jehl, 1995; Wacher et al., 1995). Therefore, -adrenergic activation by a low-salt diet may be one component of a signaling pathway whereby the activities involved in intestinal drug disposition such as CYP3A or P-glycoprotein are suppressed, resulting in increased drug availability.

Role of Sympathetic Modulation in Drug Disposition. The studies in which we first identified the effect of dietary salt on quinidine disposition were not designed to study disposition but rather to assess the extent to which dietary salt modulated sympathetic function (Fraser et al., 1981). The unexpectedly higher plasma quinidine concentrations on the low-salt diet raised the possibility that one mechanism underlying the effect of dietary salt on drug disposition may relate to altered sympathetic modulation of gut function. Furthermore, the extent to which the sympathetic nervous system is activated may also be important because we have found that subjects with urinary sodium values of >40 mEq while on a low-salt diet had AUCs similar to those when the same subjects were on a high-salt diet after p.o. verapamil (Darbar et al., 1998). Activation of the sympathetic nervous system may therefore be a major component of the salt sensitivity of drug disposition that this study has demonstrated. Suppression of this system by nadolol attenuates the effect of a low-salt diet and increases quinidine clearance.

Clinical Importance. We have found that the effect of dietary salt is not limited to quinidine but that dietary salt also modulates the disposition of p.o. verapamil, which is also a substrate of CYP3A4 and P-glycoprotein in humans (Darbar et al., 1998). The effect we now describe may be important in determination of drug bioavailability in conditions associated with altered salt balance such as congestive heart failure and hypertension. For example, it is known that after the administration of equivalent doses, plasma quinidine concentrations are generally higher in patients with congestive heart failure than in control groups (Bellet et al., 1971; Kessler et al., 1974; Ueda and Dzindzio, 1978; Gillis et al., 1995). Although a number of explanations have been proposed, there remain some doubts regarding the precise underlying mechanism or mechanisms (Bellet et al., 1971; Ueda and Dzindzio, 1978). Our data raise the strong possibility that an alteration in salt balance in patients with congestive heart failure may be responsible. The effect we observed suggests that the coadministration of a -adrenoceptor blocker may reduce plasma quinidine concentrations in these patients. In addition, because salt intake is highly variable among individuals and among different ethnic groups (Wilkinson, 1997), differences in salt intake are likely to be one confounding factor for observed variable pharmacokinetics of quinidine and other drugs.