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Effects of Chronic Ethanol Ingestion on Pharmacokinetics of Procainamide in Rats
Effects of Chronic Ethanol Ingestion on Pharmacokinetics of Procainamide in Rats DILIPJ. GOLE**
AND
JANARDAN B. NAGWEKAR*'
Received June 27, 1989, from the "Department of yha!maceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State 'Present address: Mediventures, Inc., Research and Development Accepted for publication April 13, 1990. Universit Detroit, MI 48202. Center, &5 Phoenix Drive, Ann Arbor, MI 48108.
Abstract 0 Blood level studies were carried out in rats to determine the effects of chronic ethanol ingestion on the distribution pharmacokinetic parameters and tissue steady-state partition coefficients of procainamide. The ethanol-treated rats received 4g/kg of ethanol daily for 28 days in Treatment A and 4 g/kg of ethanol for an initial 7 days, followed by 8 gikg of ethanol for the subsequent 21 days in Treatment 8; the control rats received isocaloric sucrose in the respective groups. As determined from two-compartment analysis of the blood level data, both ethanol treatments significantly decreased the distribution clearance (CL,; k,?Vd,) and the apparent first-order rate constant for drug transfer from the centralCompartment to the tissue compartment (k,*) of procainamide without affecting the total body clearance of drug (CL) or the apparent volumes of distribution of drug in the body at steady state (Vd,,) and at pseudo-equilibrium ( Vdp).Additionally, the apparent volume of distribution of the drug in the central compartment (Vd,) was 57-62% greater due to both ethanol treatments. Furthermore, the steady-state partition coefficients of the drug were found to be significantly lower in heart and kidneys and greater in fat of the ethanol-treatedrats (Treatment B) as compared with those in the control rats. Possible mechanisms are proposedto account for these various effects in light of the known effects of chronic ethanol ingestion on the chemical composition of cell membranes of tissues and organs.
In recent years, chronic ingestion of ethanol has been shown to increase the concentration of cholesterol, to decrease the synthesis of protein, a n d to alter the fatty acid composition of phospholipids in the membranes of a variety of tissues (heart, liver, muscles) of animals, including rats.1-4 Since cholesterol, phospholipid, a n d protein constituents of membranes act as barriers for the transport of drugs through biological membranes and since one of the major mechanisms responsible for the localization of drugs in tissues is their reversible binding to cellular membrane components, the purpose of t h i s study w a s t o determine the effects of chronic ethanol ingestion on the distribution pharmacokinetic parameters a n d t h e steady-state partition coefficients of procainamide, an antiarrhythmic drug, i n rats. Although m a n y studies reported in the literature have demonstrated the effects of chronic ethanol o n drug metabolism due to the induction of microsomal drug metabolizing enzymes, this study appears t o be the first to investigate t h e effects of chronic ethanol exposure on the kinetics of d r u g distribution in the body a n d on the steady-state partition coefficients of d r u g in various tissues. We considered procainamide to be suitable for the study because its metabolism, which occurs primarily by non-microsomal enzymes, w a s not expected to be affected by chronic ethanol treatment, thereby m a k i n g i t possible to isolate the effect of chronic ethanol treatment on the distribution pharmacokinetic parameters of t h e drug. Furthermore, since the pK, of this basic drug is 9.2, it was expected t o remain almost entirely i n the cationic form at the physiological pH of 7.4. 232 I Journal of Pharmaceutical Sciences Vol. 80,No. 3, March 1991
Experimental Section Chemicals-Procainamide HCl (mp 167 "C) was purchased from Sigma Chemical Company (St. Louis, MO) and N-acetylprocainamide HC1 (mp 185 "C) was purchased from Aldrich Chemical Company (Milwaukee, WI).Ethanol and sucrose were of U.S.P. grade and the reagents used in the analytical procedures were of analytical grade. Animals and Ethanol Treatment-Sprague-Dawley male rats with initial body weights of 200-220 g were used in the study. The rats were treated in the following manner Ethanol Treatment A-The rats in this group received 4 g/kg of ethanol orally per day for 28 days. Ethanol was administered as a 20% (vlv) solution in water. The corresponding control group received 5 g/kg of sucrose orally per day for 28 days. Sucrose was administered as a 20% (w/v) solution in water. Ethanol Treatment B-The rats in this group received 4 g/kg of ethanol orally per day for the initial 7 days and 8 g/kg of ethanol for the subsequent 21 days. Ethanol was administered as 20% (v/v) and 40% (v/v) solutions in water, respectively. The 8-g/kg ethanol dose was administered in two equally divided doses, -2 h apart, to avoid gastric discomfort. The control rats received 5 g/kg of sucrose orally per day for the initial 7 days and 10 g/kg of sucrose for the subsequent 21 days. Sucrose was administered as 20% (wlv) and 40% (w/v) solutions in water, respectively. The 10-g/kg sucrose dose was administered in two equally divided doses, -2 h apart. The volume of ethanol or sucrose solution administered to each rat was 25 mL/kg. The sucrose dose received by the control rats was isocaloric with the ethanol dose received by the ethanol-treated rats, thereby maintaining similar caloric content in both groups. The oral administration of sucrose or ethanol solution was accomplished in -15 s using a syringe equipped with round tip oral needle. The body weights of rats were recorded daily before sucrose or ethanol administration. After sucrose or ethanol administration, each rat was given five to six Purina Chow pellets and free access to water. Each pellet weighed -3 g and contained 22.5% protein, 4.5% fat, and a negligible amount of cholesterol (220 ppm), and provided 12 Kcal. The rats were housed in individual cages. Procainamide Administration-For the iv administration of procainamide HCl (hereafter referred to as procainamide), either as a single dose or by continuous infusion, the external jugular vein of each rat was surgically cannulated on the 28th day of the treatment -4 h prior to the administration of the last dose of ethanol or sucrose. Food was withheld from the rat for 12 h prior to drug administration. Intravenous Single-Dose Studies-On the 29th day, a 20-mg dose of procainamide, contained in 1mL of normal saline and adjusted to pH 7.4, was administered to each rat via the jugular vein cannula. A blank blood sample (0.3 mL) was obtained from the rat immediately prior to the administration of the drug. Blood Level Pharmacokinetics (Treatment A Rats)-The pharmacokinetics of procainamide was studied in the ethanol-treated rats (n = 5) and the corresponding control rats (11 = 5). Following the iv administration of the dose of the drug, and by taking necessary precaution, uncontaminated blood samples (0.3 mL each) were withdrawn from the rat at 3, 6 , 10, 15, 25,40, 50, 60, and 80 min, using 1-mL heparinized syringes. The blood samples were stored at - 10 "C until they were analyzed for procainamide. Blood Level Pharmacokinetics (Treatment B Rats)-Following the iv administration of the single dose of procainamide to the ethanoltreated rats (n = 24) and the corresponding control rats (n = 24) the
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0022-3549/91/0300-0232$07.00/0 0 1997, American Pharmaceutical Association
rats were sacrificed a t predetermined times; only one blood sample was obtained per rat. The rats were lightly anesthetized with ether prior to their decapitation. Three rats were sacrificed at each given time (3,6, 10, 15,25, 40,60, and 80 min). The blood of each rat was collected separately in the heparinized beakers. The 0.3-mL blood samples of individual rats were stored a t -10 "C until they were analyzed for procainamide. (This approach of obtaining only one blood sample per rat was adopted because we were interested in collecting various tissues of the rats for another purpose which is not a part of this report. It also allowed comparison of the drug pharmacokinetics in the control rats of Treatments A and B, as discussed later.) Urinary Excretion Study (Treatment E Rats)-The overall urinary excretion studies of procainamide were carried out in a separate group of ethanol-treated rats (n = 5) and the corresponding control rats (n = 5). The effect of ethanol treatment on the extent of procainamide metabolism was determined by measuring the total recovery of the intact drug and its major metabolite, N-acetylprocainamide. Following the iv administration of a single dose of the drug, the rats were transferred to individual urine-collection cages. Urine samples were carefully collected over a period of 28 h and the urine volumes were recorded. All urine samples were stored a t - 10 "C until they were analyzed for procainamide and N-acetylprocainamide. Although no food or water was given to the rats during the initial 7-h period of urine collection, two pellets ofrat chow and access to water were allowed during the 7-28-h period. Intravenous Infusion Studies (Treatment E Rats)-Tissue Partition Coefficients-The steady-state partition coefficients of procainamide in various tissues were determined in ethanol-treated rats (n = 5) and the corresponding control rats (n = 5) after iv infusion of the drug. On the 29th day, after withdrawing 0.3 mL of blank blood from the rat, a freshly prepared pH 7.4 solution of procainamide (20 mg/mL) was infused into each rat at a rate of 0.4 mg/min; the average infusion time was 407 ? 47 min. These infusion conditions provided the steady-state concentration of the drug as determined from the preliminary studies. Immediately prior to decapitation, the rat was lightly anesthetized with ether, the infusion was discontinued, and the residual drug solution remaining in the cannula was promptly withdrawn in <5 s with a 1-mL tuberculin syringe. Immediately after decapitation, blood was collected in a heparinized beaker and heart, liver, kidneys, and portions of thigh muscle and fat were excised. The excised tissues were promptly rinsed with ice-cold saline and blotted dry. Each tissue was minced and -500 mg of fat and 200300 mg of each of the other tissues were transferred to pre-weighed 17 x 60-mm sample vials and weighed accurately. The accurately measured 0.3 mL of blood and plasma from a given rat were transferred to sample vials. The plasma was obtained after centrifuging part of the blood a t 3000 rpm for 15 min. The blood, plasma, and tissue samples were stored a t - 10 "C until they were analyzed for procainamide. Blood and Plasma Binding-The binding of procainamide to whole blood or plasma was studied using the equilibrium dialysis method. The blood and plasma samples (containing procainamide) used in the binding studies were obtained from the decapitated ethanol-treated rats (n = 4) and the corresponding control rats (n = 4) employed in the partition coefficient studies described above. Plasma was diluted with an equal volume of isotonic Sorensen's pH 7.4 buffer before using it in the binding studies; this was done to provide the realistic concentration of plasma proteins present in blood. The 1-mL dialysis cells were set up for equilibrium dialysis study a t 37 "C as described elsewhere.5 One side of the dialysis cell contained 1 mL of pH 7.4 Sorensen's phosphate buffer and the other side contained 1 mL of blood or plasma (containing procainamide) from a single rat. The equilibrium was achieved in 12 h, as deterwere noted mined from a preliminary study. Volume shifts of 4% during dialysis. As determined separately, N-acetylprocainamide, the metabolite of procainamide, did not hydrolyze under the equilibrium dialysis conditions employed in this study. The fractions of free drug in blood (f,) or in plasma of the control and ethanol-treated rats were calculated according to the following equation:
100 - % Bound fB =
100
Extraction Procedure for Blood, Tissue, and Urine Samples-To each blood or tissue sample (except fat), 0.4 mL of 1M NaOH solution was added and the samples were vortexed for 10 s. Then, 2 mL of
1-octanol was added to each sample, the blood samples were vortexed for -1 min, and the tissue samples were homogenized for 2 min using a model I" 10135 Brinkman homogenizer. The samples were then centrifuged to separate the octanol phase. An accurately measured 1.4-mL aliquot of supernatant octanol phase from a given sample was transferred to a test tube and was extracted with 0.2 mL of 0.1 M HCI by vortexing for 1 min; a clear acidic aqueous phase was obtained by centrifuging the sample for 15 min. The aqueous phase samples were then subjected to the HPLC analysis of procainamide. To each fat sample, 2 mL of 0.1 M HCI (containing 22% NaCI) was added and the samples were homogenized for 2 min and then centrifuged to separate the aqueous phase. An accurately measured 1.4-mL volume of the aqueous phase from each sample was transferred to test tubes. To each tube was then added 0.1 mL of 4 M NaOH and the whole mixture was extracted with 2 mL of octanol. The drug was extracted from the octanol phase by employing the procedure described previously for the blood samples. The procedure used for the extraction of procainamide and N-acetylprocainamide from the urine samples was essentially the same as that described for the blood samples. High-Performance Liquid Chromatography Analysis-A Perkin-Elmer Series 10 liquid chromatograph, equipped with LC 85 variable wavelength detector and model R 100 microprocessor chart recorder was used for the quantitative determination of procainamide and its major metabolite, N-acetylprocainamide. A Perkin Elmer HS-C,, reversed-phase column was used. The mobile phase consisted of 20% (v/v) methyl alcohol, 5 8 (v/v) acetonitrile, and 75% (vlv) phosphate buffer; the phosphate buffer solution contained 0.1 M sodium dihydrogen phosphate and 0.1 M disodium hydrogen phosphate. The flow rate of mobile phase was 1.5 mumin. The chromatograms of procainamide and N-acetylprocainamide were obtained a t a wavelength of 280 nm. The volume of the aqueous extract of each blood and tissue sample injected into the C,, reversed-phase column was 15 fiL. The retention time for procainamide was 2 min and 10 sand that forN-acetylprocainamide was 3 min and 30 s. The concentrations of procainamide in blood, tissue, and urine samples were calculated with reference to the corresponding standard curves of peak height versus amount of procainamide; the standard solutions of the drug were appropriately spiked with rat blank blood, urine, or a given tissue. The concentrations of procainamide in these standard solutions ranged from 2.5 to 160 pg/mL. The standard curves of procainamide obtained for the blood, urine, and tissue samples were identical. The standard curves were also similarly prepared for N-acetylprocainamide. Treatment of Pharmacokinetic Data-The blood concentration (C) versus time data of procainamide, which were normalized on the basis of an 80-mg iv dose per kilogram of body weight of rats, were fitted to a polyexponential equation:
(2) where A, and A, are the coefficients and constants, respectively. Curve fitting was initially performed with the ESTRIP computer program and was refined by the PCNONLIN7 computer program. Statistical Treatment of Data-An unpaired t test was used to determine significant differences (p < 0.05) between the data obtained in the ethanol-treated rats and the corresponding control rats.
Results Selection of Ethanol Dose(s1 and Duration of Treatment and Their Effects on Body W e i g h t d i n c e pretreatment of animals (including rats) with 2.5 to 14 glkg of ethanol per day for 8 to 35 days has been shown3.&10 to alter the membrane composition of tissues, the ethanol pretreatment schedules described in Treatments A and B were utilized because of the objective of the study. The reason for using the mixed doses of ethanol in Treatment B, instead of an ethanol schedule of 8 g/kg P r day for 28 consecutive days, was that when the latter treatment schedule was used, >50% ofthe rats became ill, lost considerable body weight, and eventually died. Journal of Pharmaceutical Sciences I 233 Vol. 80, No. 3, March 1997
The body weight versus time data obtained for the Treatment B ethanol-treated rats (n = 29) and their corresponding control rats (n = 29) used in the blood level kinetic study and urinary excretion study are presented in Figure 1. Figure 1 indicates that while the body weights of the control rats increased with time during the entire treatment period of 28 days, the body weights of the ethanol-treated rats increased initially with time up to 8 days, decreased with time during the 8-12-day period, and then increased with time during the 12-28 day period.The loss in body weights occurred soon after the treatment with the higher dose ofethanol (8glkglday) was initiated and this effect lasted for the next 4 days, mainly due to the reduced consumption of food by these rats. It was noted that although the body weights of the ethanol-treated rats were lower (by 5 to 7%) than those of the control rats during the 12- to 28-day period, the rate of gain in the body weight of the ethanol-treated rats (2.6 ? 0.7) was similar to that of the control rats (2.8 ? 0.3), apparently indicating normal physiological behavior of the ethanol-treated rats. The body weights of the ethanol-treated rats (Treatment A) were similar to those of their respective control rats during the entire treatment period. Procainamide Pharmacokinetics (Treatment A)Computer analysis of the blood level data (Figure 2) indicated that the disposition of procainamide in all five control rats and in four out of five ethanol-treated rats was best described by a two-compartment open model, with drug elimination occurring from the central compartment. Procainamide disposition in untreated rats has been described by a two-compartment open model by several investigators.11-13 The distribution of most drugs from blood into the highly perfused tissues, such as liver and kidneys, is known to be quite rapid and that into the moderately to poorly perfused tissues, such as muscles and fat, is rathef slow. Therefore, in a two-compartment open model, it is considered that the liver and kidneys are part of the central compartment and muscles and fat are part of the peripheral tissue compartment. As mentioned earlier, we also studied the concentration-time profiles of procainamide in the individual tissues of the ethanol-treated and control rats of Treatment B for another report. In that study, we observed that the drug uptake curve noted in the muscles and fat was absent in the kidneys, heart, and liver, indicating that the distribution of procainamide from blood to the liver, kidneys, and heart was indeed rapid and that to the muscles and fat was slow (unpublished results). Therefore, liver, kidneys, and heart are included in the central compartment, while muscles and fat are included in the peripheral tissue compartment. The elimination of procainamide is known to occur due to its metabolism and urinary excretion. The metabolism of procainamide occurs14 in the rat liver due to N-acetylation,
A
5 W
s
I-
U <
-
200[.'
0
" ' . ' " 5
'
10
.
'
'
"
"
l
'
'
l
15
20
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25
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Figure l-hnparative body weight versus time profiles of Treatment B rats. Key: ( 0 )ethanol-treated; (0)Control. 234 I Journal of Pharmaceutical Sciences Vol. 80. No. 3, March 1997
TIUE BIN1
.A
m
I
d
Table CAverage Values (* SD)of the Two-Compartment Open Model Pharmacoklnetic Parameters Determined for Procainamlde In Control and Ethanol-Treated Rats
k12,min-‘
hl,min-‘
k,,, min-’ A’. min-’ A,, min-’
Vd,, mUkg Vd, mUkg Vd,, mUkg CL, mUmin/kg CL,,, mL/min/kg
Control
Control (n = 5)
Treatment A (n = 4)a
0.2108 f: 0.1069 0.1494 f 0.0349 0.0667 f: 0.0288 0.4030f 0.1422 0.0239f 0.003 787.9f 274 1767 f 251 1967 t 247 46.4f 1.2 144.3 f 25.6
0.0655f: 0.0581 0.0758f:0.0254’ 0.0472f 0.0195 0.1661 f 0.0975b 0.0224f 0.004 1237 f 322 2073 f 109 2583 * 798 58.5 k 24 67.7f 44.6’
Parameter
(n
’
=
24)
0.2412f 0.0972 0.1486f 0.0223 0.0549f 0.0139 0.4255f: 0.1262 0.0192f:0.0022 787.9f 190 2064 ? 657 2253 2 829 43.2f 15 190.0f 18.2
Treatment B (n = 24) 0.0873f 0.0703’ 0.1446f 0.08 0.0357f 0.0079’ 0.24662 0.1509’ 0.02092 0.0039 1280 f 239’ 2048 f 207 2186 f 753 45.7f 13 111.7f 18.7’
stated in the text, one of the five rats displayed one-compartmentopen model pharmacokinetics for procainamide, with Vd = 2615 mL, 0.0217 min-’, and CL = 56.8 mUmin/kg. ’Significantly different from the corresponding control (p < 0.05).
a As
A2
=
ponentially (Figure 2) according to the one-compartment open model equation (C = C,e-’*‘), where all terms have the usual meaningsl.17 The apparent volume of distribution (Vd),the elimination rate constant (A,), and the CL of the drug were calculated using the PCNONLIN program. The values of these parameters are listed in Table I. Procainamide Pharmacokinetics (Treatment Bt-Since the blood samples from the ethanol-treated rats (Treatment B) and the corresponding control rats were collected by sacrificing three rats at each of the eight time periods, the blood level data of rats of a given group (n = 24) were pooled for the pharmacokinetic analysis and are presented in Figure 3. The computer analysis indicated that the disposition of procainamide in both groups of rats was best described by a two-compartment open model. The values of various pharmacokinetics parameters are listed in Table I. The standard deviations of k12, k,,, A,, A,, and k,, were provided by the
100
10
I
I
I
I
20
40
60
80
TIME [MINI Flgure +Comparative
concentration-time biexponential profiles of procainamide obtained in blood of the control (0)and ethanol-treated(W) rats of Treatment B after the administrationof an 80-mg iv dose to each rat. The solid lines are the computer predicted profiles and the symbols are average of three concentrations (with standard errors) of procainamide obtained in three rats sacrificed at a given time.
PCNONLIN Program, while those of Vd,, Vd,,, Vd,, CL, and CLd were calculated as described by others.ls.19 The distribution pharmacokinetic parameters A,, k,,, Vd,, and CL, observed in the ethanol-treated rats (Treatment B) were significantly different from those observed in the corresponding control rats. The elimination rate constant (klo)of the drug was also found to be significantly affected by the ethanol treatment. Overall Urinary Excretion of Procainamide (Treatment B k T h e overall urinary excretion data of procainamide were measured only in terms of the intact procainamide and its major metabolite (N-acetylprocainamide). It was observed that the amount of the intact drug excreted in the urine of the control rats (9.0 ? 0.5 mg) was not significantly different from that of the Treatment B ethanol-treated rats (9.2 ? 0.5 mg). Similarly, the amount of N-acetylprocainamide recovered in the urine of the ethanol-treated rats (3.8 2 0.4 mg) was not significantly different from that of the control rats (4.8 ? 1.1 mg). The total urinary recovery of procainamide and N-acetylprocainamide observed in this study was comparable to that reported by others.11.20 Partition Coefficients of Procainamide in Tissues (Treatment B k T h e apparent partition coefficient of procainamide in a given tissue (R,)was calculated by dividing the steadystate drug concentration in a given tissue CC), by the steadystate drug concentration in pooled blood (CB),according to the following equation:17
The contribution of the residual blood drug concentration to the total tissue drug concentration was considered to be negligible. The mean partition coefficients of the drug determined in heart (RH), liver ( R J ,kidneys (RK), thigh muscle (RTM),and fat (RF) are listed in Table 11. It was noted that the apparent partition coefficients of the drug in ethanol-treated rats (Treatment B) were significantly different from those in control rats only for heart, kidneys, and fat; in ethanol-treated rats, the R H and R K values were lower by 24 and 13%, respectively, and the RF value was higher by 38%. The normalized average steady-state concentrations of procainamide in blood (72.0 -C 13.6 pg/mL) and plasma (70.4 2 12.4 pg/mL) of the control rats were not significantly different from the normalized average concentrations of procainamide in blood (72.2 IT 13.3 pg/mL) and plasma (71.0 5 18.5 pg/mL) of the ethanol-treated rats (Treatment B). Furthermore, the drug concentration in blood was not significantly different from that in plasma either in control rats or the ethanol-treated rats. Journal of Pharmaceutical Sciences I 235 Vol. SO,No. 3, March 1991
Table ICAverage Steadystate Partitlon Coefficlents of Procainamlde Determined In Various Tlssues of Control and Ethanol-Treated Rats (Treatment B)
Tissue Partition Coefficient (4)
Rats Ethanol-Treated
Control
Heart (I?,,) Liver (RL) Kidney (RK)
2.472 0.27" 3.172 0.81" 6.36 t 0.94" 3.09 2 0.39" 0.13t 0.02'
Thigh muscle (&)
Fat ( 4 )
1.872 0.34" 3.35 2 1.22a 5.53 2 0.52' 2.75 2 0.72" 0.18? 0.02'
P
<0.05 NS
<0.05 NS
C0.05
" Standard deviation based on n = 7. Standard deviation based on n
3.
=
Binding of Procainamide to Whole Blood and Plasma (Treatment B)-The extent of binding of procainamide to blood (7.3 +. 2.7%) or plasma (8.5 2 7.8%) of the ethanoltreated rats (Treatment B) was not found to be significantly different from that for blood (7.9 +. 5.3%)or plasma (8.0 7.8%)of the control rats. The extent of binding of procainamide to whole blood was similar to that for plasma in both the control and ethanol-treated rats. The average of f B values determined for the ethanol-treated and control rats are listed in Table 111. Fractions of Free Procainamide in Tissues-The fractions of free procainamide in various tissues ( f T ) of the control and ethanol-treated rats (Treatment B) were calculated according to the equation:17
*
(4) The f B and RT values were known, as shown in Tables I1 and 111. The fH and fK values of the drug in ethanol-treated rats were found to be significantly greater than those in control rats by 32 and 21%, respectively.
Discussion It should be noted that, although the approach we used for rat blood sampling in pharmacokinetic studies related to Treatment A (blood samples were taken serially from each rat) was different from that we used in those studies related to Treatment B (only one blood sample per rat was obtained), the magnitudes of the pharmacokinetic parameters of procainamide determined in the control rats of Treatment A were similar to the control rats of Treatment B (Table I). Therefore, it can be inferred that the removal of 3.0 to 3.5 mL of blood from the individual rats of Treatment A had no effect on the pharmacokinetics of the drug and that the effects observed in the ethanol-treated rats of Treatments A and B were primarily due to chronic ethanol exposure. Effects on Procainamide Pharmacokinetics-Body Clearance and Metabolism-The ethanol treatments employed in Table Ill-Average Fractions of Free Procalnamlde in Blood (f,) and Various Tlssues (fT) of the Control and Ethanol-Treated Rats (Treatment B)
Tissue ~~~~~
~~~
Blood (&) Heart (f,) Liver (fL) Kidney (f,) Thigh muscle ( f m ) a
n
~~
P
~
0.922 0.05' 0.38 t 0.04' 0.31 t 0.07' 0.14t 0.02' 0.30 t 0.04b
Standard deviation based on n
=
Rats Ethanol-Treated
Control
= 4.
0.93t 0.03" 0.502 O . l l b 0.30 5 0.11 ' 0.17t 0.02' 0.362 0.12'
NS
<0.05 NS <0.05 NS
Standard deviation based on
7.
236 I Journal of Pharmaceutical Sciences Vol. 80, No. 3, March 7997
this study did not affect the CL (k,,Vd,) of procainamide (Table I), indicating that the irreversible removal of the drug from the central compartment was not affected. However, the k,, value of the drug was significantly lower in the ethanoltreated (Treatment B) rats than in the control rats (Table I). The decrease in k,, cannot be attributed to the direct effect of the ethanol Treatment B on the hepatic or renal elimination functions of the rats because the extent of urinary excretion of the intact drug and its major metabolite, N-acetylprocainamide, observed in the ethanol-treated and control rats were similar. However, in view of the fact that the ethanol treatment had no effect on CL of the drug, the decrease in k,,, which is a dependent variable,'l is most probably the consequence of the significant increase in Vd, of the drug caused by the ethanol treatment. Distribution Parameters-Table I shows that the elimination-independent distribution pharmacokinetic parameters k,,, A,, Vd, and, more importantly, the distribution clearance (k12 Vd,) were significantly affected by the ethanol Treatment B. The same distribution pharmacokinetic parameters, except Vd,, were also significantly affected by ethanol Treatment A. Although the Vd, of the drug in the ethanol-treated rats (Treatment A) was not significantly different from that observed in the corresponding control rats, the average Vd, value of the drug in these ethanol-treated rats was -57% greater than that in the control rats. This percentage increase in Vd, was quite comparable to the 62% increase in Vd, observed in ethanol-treated rats (Treatment B). The fact that one of the five ethanol-treated rats (Treatment A) exhibited one-compartment characteristics for procainamide appeared to be indicative of the effect of ethanol (Treatment A) on the distribution kinetics of procainamide. Thus, this study demonstrated that both ethanol treatments significantly decreased the distribution clearance (intercompartmental clearance) of procainamide. Since the tissue transport or uptake rate of drugs of molecular weights similar to that of procainamide is limited not only by their transport across the tissue cell membranes but also by the tissue perfusion rates,22 the decrease in procainamide distribution clearance observed in this study might be due to the alteration in blood flow andlor permeability of the tissues. Moravi et al.23 demonstrated that rats treated with 11.512.5 glkg of ethanol per day in a liquid diet for 4 weeks experienced a decrease in cardiac output as well as in blood flows of myocardium, kidneys, skin, and carcass by -2630%. Ituriaga e t al.24observed that the rats, which were fed ethanol in a liquid diet in doses similar to those used in our study, experienced a 28% decrease in hepatic blood flow. Although we did not measure the tissue blood flows, we cannot eliminate the possibility that the ethanol treatments in our study might have also lowered the cardiac output and the blood flows in various tissues, especially in the tissues (muscles) of the peripheral compartment, and thereby contributed to the reduction of the distribution clearance of procainamide in the ethanol-treatedrats. However, the increase in Vd, of the drug that we observed in the ethanol-treated rats cannot be explained by a reduction in tissue blood flows.Therefore, it seems that the permeability characteristics of tissue cell membranes were substantially affected by the chronic ethanol treatment. It is recognized that the large size of the apparent volume of distribution of the central compartment of procainamide is clearly indicative of the fact that it exceeds both the blood volume and extracellular space and that it includes not only the highly perfused tissues, but also part of the moderately perfused peripheral tissue which is in rapid equilibrium due to its close proximity to the capillary network of the tissue. Since chronic ethanol ingestion affects the concentration of cholesterol and phospholipid-associated fatty acids in the cell
membranes of a variety of tissues,1-' it is conceivablethat the ethanol treatment might have rendered additional parts of the tissues of the peripheral compartment (which largely consists of muscles) more readily accessible or permeable to the drug, thereby bringing it into rapid equilibrium with the central compartment, making it kinetically a part of the central compartment, and consequently yielding a higher value of Vd, in the chronically ethanol-treated rats. This mechanism appears to be consistent with our findings that Vd, and Vd, of procainamide (Table I), as well as the fractions of free procainamide in blood and muscles, remained unaffected by the ethanol treatments. It is noted in Table I that the body clearance and the total apparent volume of distribution of procainamide in the only ethanol-treated rat (Treatment A) which displayed the onecompartment characteristics for procainamide were similar to those in other ethanol-treated and control rats. It appears that the ethanol treatment in this rat rendered a large part of the tissues of the peripheral compartment readily permeable to the drug and brought it into rapid equilibrium with the central compartment, thereby rendering the tissue compartment kinetically indistinguishable. Although we did not measure the blood levels of ethanol when the pharmacokinetics of procainamide were studied, we assumed that the concentration of ethanol in blood was negligible at the time procainamide was administered. This assumption was considered reasonable because, at the time of procainamide administration, 24 h had already elapsed since the last dose of ethanol was administered either in Treatment A or Treatment B and, as demonstrated by others2627 in rats, the blood ethanol concentration becomes negligible even after 8 h following the last dose of ethanol in chronic ethanol treatments similar to those employed in our study. Therefore, the effects of chronic ethanol on the distribution pharmacokinetics of procainamide noted in the present study are attributed to the effects of ethanol on the composition and permeability characteristics of tissue cell membranes. Effect on Partition Coefficients-The fact that the R, and R, values of procainamide observed in the ethanol-treated rats were significantly lower than those in the control rats was rationalized by considering the alternative mathematical definition of tissue partition coefficient (R,) of drug which is expressed as follows:17 R T = fB/fT
where fT is a fraction of free procainamide in a given tissue. The usual assumption made in the derivation of this equation is that, a t steady state, the concentration of free drug in blood is the same as that in tissue fluids. This equation indicates that any alteration that may occur in the binding of drug to a tissue and/or blood would alter the partition coefficient of drug in that tissue. Since the f B values of procainamide observed in the ethanol-treated and control rats were similar, the lower RH and RK values of the drug noted for the ethanol-treated rats can be attributed to the increase in f H and f K values of the drug in their hearts and kidneys, respectively (Table 111); the increase in fH and f K may have resulted from the decreased binding of procainamide to membranes and intracellular constituents of these tissues. One of the major mechanisms known to be responsible for the localization of drugs in tissues is their reversible binding to the cellular membrane components and the intracellular constituents. The binding of the cationic forms of basic drugs, including procainamide, to phospholipids and contractile proteins (myosin and actin) of the heart and skeletal muscle have been demonstrated28.29 in vitro. Therefore, it seems reasonable to attribute the decrease in partition coefficientsof
procainamide in heart and kidneys of the ethanol-treated rats to the possible decrease in binding of the drug to proteins and phospholipids of these tissues. A decrease in the binding of procainamide to cell membranes and intracellular components might have resulted from the decreased protein synthesis,30 the modified acyl chains of cellular phospholipids,*,31-33 and/or the alterations in the protein conformations,33 all of which are brought about by chronic ethanol ingestion. Such ultrastructural changes in various organs have been shown to occur without causing hypertrophy of the organs. As noted by other workers3636 for ethanol treatment similar to that employed in our study, we also observed that the weights of hearts, livers, and kidneys of the ethanoltreated rats were very similar to those of the control rats. We also considered the possibility of the effect of chronic ethanol on the intracellular and/or extracellular pH of tissues which might affect the partition coefficients of procainamide in tissues. To our knowledge, liver is the only tissue that has been investigated37-3*and shown to have its intracellular pH lowered by -0.2 pH units due to a higher NADH:NAD+ratio resulting from a constant oxidation of ethanol. Since kidney and heart are not known to be the sites of metabolism for ethanol, chronic ethanol exposure would seem highly unlikely to alter the intracellular or extracellular pH of these tissues and affect the partition coefficientsof procainamide. It is interesting to note that the partition coefficient of procainamide was not affected in the liver of ethanol-treated rats, despite the possibility that the liver intracellular pH of these rats was affected due to chronic ethanol exposure. Furthermore, even if the tissue intracellular or extracellular pH were slightly altered by ethanol exposure, it is doubtful if it would influence the tissue partition coefficientof procainamide (pK, 9.2) which exists entirely in the cationic form at a physiological pH and would certainly remain in that form at a lower pH. While the ethanol treatment decreased RH and RK, the same ethanol treatment significantly increased RF (Table 11). The RF values of procainamide in both the control and ethanol-treated rats were much less than unity, indicating that the solubility of the drug in fat is small. In view of the report that chronic ethanol ingestion for a period as few as 4 weeks in doses similar to those used in our study lowered the triglyceride content of adipocytes due to decreased lipogenesis,39 it is conceivable that a decreased lipid content in adipocytes of the ethanol-treated rats might have resulted in increasing the solubility of procainamide in fat and consequently increasing its RF. Significance-One of the striking pharmacokinetic findings of this study which might be pharmacologically important is that the significant changes that may occur due to chronic ethanol exposure in the concentration profiles and partition coefficientsof drugs in individual tissues containing the sites of action may not necessarily manifest in exhibiting significant differences in their blood or plasma concentration profiles and body clearance values. This may result because the amounts of drugs involved in tissues containing the sites of action (i.e., heart and kidney) are usually too small (as compared with the total amount of drug present in the body) to influence their overall blood levels or clinically useful pharmacokinetic parameters, namely, body clearance and area under the blood level curves. In view of the results of this study, it becomes apparent that taking notice of subtle changes that might occur in the distribution pharmacokinetic parameters of drugs due to chronic ethanol ingestion might be useful in discovering potential changes that occur in drug concentration a t the site of action and in drug binding to receptors present in small organs. It has been recognized that chronic ethanol exposure affects the myocardial membrane and consequently impairs membrane-mediated functions in the heart.40.41 Therefore, since Journal of Pharmaceutical Sciences I 237 Vol. 80, No. 3, March 1991
procainamide has its site of action i n the membranes of myocardial fibers,42 a decrease in the binding ofprocainamide to myocardial tissue inferred in this s t u d y could be of clinical significance.
References and Notes 1. Chin, J. H.; Goldstein, D. B.; Parsons, L. M. Alcoholism: Clin. Exp. Res. 1979.3, 47-49. 2. LaDroitte, P. H., Lamboeuf, Y.; Blanquat, G. D. Biochem. Pharmmol. 1984,33, 615-624. 3. Littleton, J . M.; John, G. R.; Grieve, S. J . Alcoholism: Clin. Exp. Res. 1979, 3, 50-56. 4. Reitz, R. C.; Helsabeck, E.; Mason, D. B.; Lipids 1973,8, 80-84. 5. Amin, Y. M.; Nagwekar, J. B. J . Pharm. Sci. 1975, 64, 18041812. 6. Brown, R. D.; Manno, J. E. J . Pharm. Sci. 1978,67, 1687-1691. 7. Weiner, D.; Metzler, C. PCNONLZN, Statistical Consultants, Inc., 1984. 8. Rubin, E., Rottenberg, H. Fed. P m . 1982,41, 2465-2471. 9. Chin, J. H.; Goldstein, D. B. Science 1977, 196, 684-685. 10. Littleton, J. M.; John, G. R. J. Pharm. Phnrmacol. 1977, 29, 579-580. 11. Schneck, D. W.; Grove, K.; Dewitt, F. 0.;Shiroff, R. A.; Hayes, A. H. J . Pharmacol. Exp. Ther. 1976,204, 219-225. 12. Basseches, P. J.; Digregoria, G. J. J . Pharm. Sci. 1982, 72, 1256-1259. 13. Pang, K. S.; Huang, J. C.; Finkle, C.; Kong, P.; Cherry, W. F., Fayz, S. Drug Metab. Dispos. 1982,12, 314-322. 14. Uetrecht, J . P.;Woosley, R. L.; Freeman, R. W.; Sweetman, B. J.; O a k s , J . A. Drug Metab. Dispos. 1981,9, 183-187. 15. Schneck, D. W., S rouse, J. S.; Hays, A. H.; Shiroff, R. A. J . Phnrmacol. Exp. 1978,204, 212-218. 16. Weber, W. W.; Glowinski, I. B. In Enzymatic Basis ofDetoxication, Vol. 11; Jakoby, W. B., Ed.; Academic: New York, 1980; Chapter 9. p 174. 17. Gibaldi, M.; Perrier, D. Pharmacokinetics; Marcel Dekker: New York, NY, 1982. 18. Deming, W. E. Statistical Adjustment ofData; Dover: New York, NY. - . - , 1939. -- -- . 19. Szymanski, D. J.; Nagwekar, J . B. J . Phnrm. Sci. 1982, 71, 275-281. 20. Cerna, J.; Zidek, 2.;Janku, I. J . Phnrm. Phnrmacol. 1970, 31, 71 1-712. 21. Gibaldi, M.; Koup, J . R. Eur. J . Clin. Pharmacol. 1981, 20,
Tier.
238 / Journal of Pharmaceutical Sciences Vol. 80, No. 3, March 1991
299-305. 22. Gerlowski, L. E.; Jain, R. K. Microvasi:ularRes. 1986,31,228305. 23. Moravi, v.; Ungvary, G.; Varga, K.; Albert, ,K.; Foll , G. Acta Physiologrca Academiae ScientLarum Hungarmze 197{ 54,369379. 24. I t u r i p , , H . ; Bunout, D., Petermann, M.; Ugarte, G.; Israel, Y. Alco olism Clin. Exp. Res. 1981, 5, 221-224. 25. Khanna, J. M.; Israel, Y.; Kalant, H.; Mayer, J . M. Biochem. Pharmacol. 1982,31,3140-3141. 26. Jones, A. W.; Alling, C.; Becker, W.; Anngard, E. Pharmacol. Biochem. Behav. 1983,19, 115-119. 27. York, J. L. Pharmacol. Biochem. Beh.av. 1983, 19, 687-691. 28. Fichtl, B.; Bondy, B.; K u n , H. J . Phurmacol. Exp. Ther. 1980, 215.248-253. 29. K u n , H.; Fichtl, B. Drug Metab. Rers. 1983, 14, 467-510. 30. Burke, J. P.; Rubin, E. Lab. Invest. 1979,41, 393400. 31. Shorey, R. L.; Pyle, B.; McAllister, M.; Miller, S. S.; Erickson, C. K.; Thornson, G. A. Biochem. Pharmcol. 1982,31 2447-2452. 32. Littleton, J. M.; John, G. R. J . Pharm. Pharmacol. 1977, 29, I
-
Fi'lLFiRn - . - - -.
33. Rubin, E.; Katz, A. M.; Lieber, C. S.;Stein, E. P.; Puszkin, S.Am. J . Pathol. 1976, 83,499-515. 34. Yamashita, S. Jap. Heart J . 1951, 12, 242-250. 35. Segel, L. D.; Rendin S. V.; Choquet, Y.; Chacko, K.; Amsterdam, E. A,; Mason, T. Cardiovasc. Res. 1975,9,649-653. 36. Mattfeld, T.; Mall, G.; Volk, B. J . Mid. Cell. Cardiol. 1980, 12, 1311-1319. 37. Morland, J.; Christofferson, T.; Osnes, J. B. Acta Physiol. Scand. 1973.89, 61-67. 38. Wallin, B.; Morland, J.; Fikke, A. M. Acta Pharmacol. Toxicol. 1981,49, 134-140. 39. Scheig, R.; Alexander, N.; Klastskin, G. J . Lipid Res. 1966, 7, 188-196. 40. Rubin, E. N . Eng. J . Med. 1979,301, 28-33. 41. Chan, T. C. K.; Sutter, M. C. Can. J . Physiol. Pharmacol. 1982, 60, 777-782. 42. Koch-Weser, J. Cardiouasc. Clin. 1974, NII), 63-75.
%:
Acknowledgments This aper was adapted from a dissertation submitted by D.J. Gole to the e r a d u a t e School of Wayne State University in artial fulfillment of the Doctor of Philosophy degree requirements. !his work was supported in part by the Wayne State University Faculty Biomedical Research Award received by J . B. Nagwekar.
AND
JANARDAN B. NAGWEKAR*'
Received June 27, 1989, from the "Department of yha!maceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State 'Present address: Mediventures, Inc., Research and Development Accepted for publication April 13, 1990. Universit Detroit, MI 48202. Center, &5 Phoenix Drive, Ann Arbor, MI 48108.
Abstract 0 Blood level studies were carried out in rats to determine the effects of chronic ethanol ingestion on the distribution pharmacokinetic parameters and tissue steady-state partition coefficients of procainamide. The ethanol-treated rats received 4g/kg of ethanol daily for 28 days in Treatment A and 4 g/kg of ethanol for an initial 7 days, followed by 8 gikg of ethanol for the subsequent 21 days in Treatment 8; the control rats received isocaloric sucrose in the respective groups. As determined from two-compartment analysis of the blood level data, both ethanol treatments significantly decreased the distribution clearance (CL,; k,?Vd,) and the apparent first-order rate constant for drug transfer from the centralCompartment to the tissue compartment (k,*) of procainamide without affecting the total body clearance of drug (CL) or the apparent volumes of distribution of drug in the body at steady state (Vd,,) and at pseudo-equilibrium ( Vdp).Additionally, the apparent volume of distribution of the drug in the central compartment (Vd,) was 57-62% greater due to both ethanol treatments. Furthermore, the steady-state partition coefficients of the drug were found to be significantly lower in heart and kidneys and greater in fat of the ethanol-treatedrats (Treatment B) as compared with those in the control rats. Possible mechanisms are proposedto account for these various effects in light of the known effects of chronic ethanol ingestion on the chemical composition of cell membranes of tissues and organs.
In recent years, chronic ingestion of ethanol has been shown to increase the concentration of cholesterol, to decrease the synthesis of protein, a n d to alter the fatty acid composition of phospholipids in the membranes of a variety of tissues (heart, liver, muscles) of animals, including rats.1-4 Since cholesterol, phospholipid, a n d protein constituents of membranes act as barriers for the transport of drugs through biological membranes and since one of the major mechanisms responsible for the localization of drugs in tissues is their reversible binding to cellular membrane components, the purpose of t h i s study w a s t o determine the effects of chronic ethanol ingestion on the distribution pharmacokinetic parameters a n d t h e steady-state partition coefficients of procainamide, an antiarrhythmic drug, i n rats. Although m a n y studies reported in the literature have demonstrated the effects of chronic ethanol o n drug metabolism due to the induction of microsomal drug metabolizing enzymes, this study appears t o be the first to investigate t h e effects of chronic ethanol exposure on the kinetics of d r u g distribution in the body a n d on the steady-state partition coefficients of d r u g in various tissues. We considered procainamide to be suitable for the study because its metabolism, which occurs primarily by non-microsomal enzymes, w a s not expected to be affected by chronic ethanol treatment, thereby m a k i n g i t possible to isolate the effect of chronic ethanol treatment on the distribution pharmacokinetic parameters of t h e drug. Furthermore, since the pK, of this basic drug is 9.2, it was expected t o remain almost entirely i n the cationic form at the physiological pH of 7.4. 232 I Journal of Pharmaceutical Sciences Vol. 80,No. 3, March 1991
Experimental Section Chemicals-Procainamide HCl (mp 167 "C) was purchased from Sigma Chemical Company (St. Louis, MO) and N-acetylprocainamide HC1 (mp 185 "C) was purchased from Aldrich Chemical Company (Milwaukee, WI).Ethanol and sucrose were of U.S.P. grade and the reagents used in the analytical procedures were of analytical grade. Animals and Ethanol Treatment-Sprague-Dawley male rats with initial body weights of 200-220 g were used in the study. The rats were treated in the following manner Ethanol Treatment A-The rats in this group received 4 g/kg of ethanol orally per day for 28 days. Ethanol was administered as a 20% (vlv) solution in water. The corresponding control group received 5 g/kg of sucrose orally per day for 28 days. Sucrose was administered as a 20% (w/v) solution in water. Ethanol Treatment B-The rats in this group received 4 g/kg of ethanol orally per day for the initial 7 days and 8 g/kg of ethanol for the subsequent 21 days. Ethanol was administered as 20% (v/v) and 40% (v/v) solutions in water, respectively. The 8-g/kg ethanol dose was administered in two equally divided doses, -2 h apart, to avoid gastric discomfort. The control rats received 5 g/kg of sucrose orally per day for the initial 7 days and 10 g/kg of sucrose for the subsequent 21 days. Sucrose was administered as 20% (wlv) and 40% (w/v) solutions in water, respectively. The 10-g/kg sucrose dose was administered in two equally divided doses, -2 h apart. The volume of ethanol or sucrose solution administered to each rat was 25 mL/kg. The sucrose dose received by the control rats was isocaloric with the ethanol dose received by the ethanol-treated rats, thereby maintaining similar caloric content in both groups. The oral administration of sucrose or ethanol solution was accomplished in -15 s using a syringe equipped with round tip oral needle. The body weights of rats were recorded daily before sucrose or ethanol administration. After sucrose or ethanol administration, each rat was given five to six Purina Chow pellets and free access to water. Each pellet weighed -3 g and contained 22.5% protein, 4.5% fat, and a negligible amount of cholesterol (220 ppm), and provided 12 Kcal. The rats were housed in individual cages. Procainamide Administration-For the iv administration of procainamide HCl (hereafter referred to as procainamide), either as a single dose or by continuous infusion, the external jugular vein of each rat was surgically cannulated on the 28th day of the treatment -4 h prior to the administration of the last dose of ethanol or sucrose. Food was withheld from the rat for 12 h prior to drug administration. Intravenous Single-Dose Studies-On the 29th day, a 20-mg dose of procainamide, contained in 1mL of normal saline and adjusted to pH 7.4, was administered to each rat via the jugular vein cannula. A blank blood sample (0.3 mL) was obtained from the rat immediately prior to the administration of the drug. Blood Level Pharmacokinetics (Treatment A Rats)-The pharmacokinetics of procainamide was studied in the ethanol-treated rats (n = 5) and the corresponding control rats (11 = 5). Following the iv administration of the dose of the drug, and by taking necessary precaution, uncontaminated blood samples (0.3 mL each) were withdrawn from the rat at 3, 6 , 10, 15, 25,40, 50, 60, and 80 min, using 1-mL heparinized syringes. The blood samples were stored at - 10 "C until they were analyzed for procainamide. Blood Level Pharmacokinetics (Treatment B Rats)-Following the iv administration of the single dose of procainamide to the ethanoltreated rats (n = 24) and the corresponding control rats (n = 24) the
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0022-3549/91/0300-0232$07.00/0 0 1997, American Pharmaceutical Association
rats were sacrificed a t predetermined times; only one blood sample was obtained per rat. The rats were lightly anesthetized with ether prior to their decapitation. Three rats were sacrificed at each given time (3,6, 10, 15,25, 40,60, and 80 min). The blood of each rat was collected separately in the heparinized beakers. The 0.3-mL blood samples of individual rats were stored a t -10 "C until they were analyzed for procainamide. (This approach of obtaining only one blood sample per rat was adopted because we were interested in collecting various tissues of the rats for another purpose which is not a part of this report. It also allowed comparison of the drug pharmacokinetics in the control rats of Treatments A and B, as discussed later.) Urinary Excretion Study (Treatment E Rats)-The overall urinary excretion studies of procainamide were carried out in a separate group of ethanol-treated rats (n = 5) and the corresponding control rats (n = 5). The effect of ethanol treatment on the extent of procainamide metabolism was determined by measuring the total recovery of the intact drug and its major metabolite, N-acetylprocainamide. Following the iv administration of a single dose of the drug, the rats were transferred to individual urine-collection cages. Urine samples were carefully collected over a period of 28 h and the urine volumes were recorded. All urine samples were stored a t - 10 "C until they were analyzed for procainamide and N-acetylprocainamide. Although no food or water was given to the rats during the initial 7-h period of urine collection, two pellets ofrat chow and access to water were allowed during the 7-28-h period. Intravenous Infusion Studies (Treatment E Rats)-Tissue Partition Coefficients-The steady-state partition coefficients of procainamide in various tissues were determined in ethanol-treated rats (n = 5) and the corresponding control rats (n = 5) after iv infusion of the drug. On the 29th day, after withdrawing 0.3 mL of blank blood from the rat, a freshly prepared pH 7.4 solution of procainamide (20 mg/mL) was infused into each rat at a rate of 0.4 mg/min; the average infusion time was 407 ? 47 min. These infusion conditions provided the steady-state concentration of the drug as determined from the preliminary studies. Immediately prior to decapitation, the rat was lightly anesthetized with ether, the infusion was discontinued, and the residual drug solution remaining in the cannula was promptly withdrawn in <5 s with a 1-mL tuberculin syringe. Immediately after decapitation, blood was collected in a heparinized beaker and heart, liver, kidneys, and portions of thigh muscle and fat were excised. The excised tissues were promptly rinsed with ice-cold saline and blotted dry. Each tissue was minced and -500 mg of fat and 200300 mg of each of the other tissues were transferred to pre-weighed 17 x 60-mm sample vials and weighed accurately. The accurately measured 0.3 mL of blood and plasma from a given rat were transferred to sample vials. The plasma was obtained after centrifuging part of the blood a t 3000 rpm for 15 min. The blood, plasma, and tissue samples were stored a t - 10 "C until they were analyzed for procainamide. Blood and Plasma Binding-The binding of procainamide to whole blood or plasma was studied using the equilibrium dialysis method. The blood and plasma samples (containing procainamide) used in the binding studies were obtained from the decapitated ethanol-treated rats (n = 4) and the corresponding control rats (n = 4) employed in the partition coefficient studies described above. Plasma was diluted with an equal volume of isotonic Sorensen's pH 7.4 buffer before using it in the binding studies; this was done to provide the realistic concentration of plasma proteins present in blood. The 1-mL dialysis cells were set up for equilibrium dialysis study a t 37 "C as described elsewhere.5 One side of the dialysis cell contained 1 mL of pH 7.4 Sorensen's phosphate buffer and the other side contained 1 mL of blood or plasma (containing procainamide) from a single rat. The equilibrium was achieved in 12 h, as deterwere noted mined from a preliminary study. Volume shifts of 4% during dialysis. As determined separately, N-acetylprocainamide, the metabolite of procainamide, did not hydrolyze under the equilibrium dialysis conditions employed in this study. The fractions of free drug in blood (f,) or in plasma of the control and ethanol-treated rats were calculated according to the following equation:
100 - % Bound fB =
100
Extraction Procedure for Blood, Tissue, and Urine Samples-To each blood or tissue sample (except fat), 0.4 mL of 1M NaOH solution was added and the samples were vortexed for 10 s. Then, 2 mL of
1-octanol was added to each sample, the blood samples were vortexed for -1 min, and the tissue samples were homogenized for 2 min using a model I" 10135 Brinkman homogenizer. The samples were then centrifuged to separate the octanol phase. An accurately measured 1.4-mL aliquot of supernatant octanol phase from a given sample was transferred to a test tube and was extracted with 0.2 mL of 0.1 M HCI by vortexing for 1 min; a clear acidic aqueous phase was obtained by centrifuging the sample for 15 min. The aqueous phase samples were then subjected to the HPLC analysis of procainamide. To each fat sample, 2 mL of 0.1 M HCI (containing 22% NaCI) was added and the samples were homogenized for 2 min and then centrifuged to separate the aqueous phase. An accurately measured 1.4-mL volume of the aqueous phase from each sample was transferred to test tubes. To each tube was then added 0.1 mL of 4 M NaOH and the whole mixture was extracted with 2 mL of octanol. The drug was extracted from the octanol phase by employing the procedure described previously for the blood samples. The procedure used for the extraction of procainamide and N-acetylprocainamide from the urine samples was essentially the same as that described for the blood samples. High-Performance Liquid Chromatography Analysis-A Perkin-Elmer Series 10 liquid chromatograph, equipped with LC 85 variable wavelength detector and model R 100 microprocessor chart recorder was used for the quantitative determination of procainamide and its major metabolite, N-acetylprocainamide. A Perkin Elmer HS-C,, reversed-phase column was used. The mobile phase consisted of 20% (v/v) methyl alcohol, 5 8 (v/v) acetonitrile, and 75% (vlv) phosphate buffer; the phosphate buffer solution contained 0.1 M sodium dihydrogen phosphate and 0.1 M disodium hydrogen phosphate. The flow rate of mobile phase was 1.5 mumin. The chromatograms of procainamide and N-acetylprocainamide were obtained a t a wavelength of 280 nm. The volume of the aqueous extract of each blood and tissue sample injected into the C,, reversed-phase column was 15 fiL. The retention time for procainamide was 2 min and 10 sand that forN-acetylprocainamide was 3 min and 30 s. The concentrations of procainamide in blood, tissue, and urine samples were calculated with reference to the corresponding standard curves of peak height versus amount of procainamide; the standard solutions of the drug were appropriately spiked with rat blank blood, urine, or a given tissue. The concentrations of procainamide in these standard solutions ranged from 2.5 to 160 pg/mL. The standard curves of procainamide obtained for the blood, urine, and tissue samples were identical. The standard curves were also similarly prepared for N-acetylprocainamide. Treatment of Pharmacokinetic Data-The blood concentration (C) versus time data of procainamide, which were normalized on the basis of an 80-mg iv dose per kilogram of body weight of rats, were fitted to a polyexponential equation:
(2) where A, and A, are the coefficients and constants, respectively. Curve fitting was initially performed with the ESTRIP computer program and was refined by the PCNONLIN7 computer program. Statistical Treatment of Data-An unpaired t test was used to determine significant differences (p < 0.05) between the data obtained in the ethanol-treated rats and the corresponding control rats.
Results Selection of Ethanol Dose(s1 and Duration of Treatment and Their Effects on Body W e i g h t d i n c e pretreatment of animals (including rats) with 2.5 to 14 glkg of ethanol per day for 8 to 35 days has been shown3.&10 to alter the membrane composition of tissues, the ethanol pretreatment schedules described in Treatments A and B were utilized because of the objective of the study. The reason for using the mixed doses of ethanol in Treatment B, instead of an ethanol schedule of 8 g/kg P r day for 28 consecutive days, was that when the latter treatment schedule was used, >50% ofthe rats became ill, lost considerable body weight, and eventually died. Journal of Pharmaceutical Sciences I 233 Vol. 80, No. 3, March 1997
The body weight versus time data obtained for the Treatment B ethanol-treated rats (n = 29) and their corresponding control rats (n = 29) used in the blood level kinetic study and urinary excretion study are presented in Figure 1. Figure 1 indicates that while the body weights of the control rats increased with time during the entire treatment period of 28 days, the body weights of the ethanol-treated rats increased initially with time up to 8 days, decreased with time during the 8-12-day period, and then increased with time during the 12-28 day period.The loss in body weights occurred soon after the treatment with the higher dose ofethanol (8glkglday) was initiated and this effect lasted for the next 4 days, mainly due to the reduced consumption of food by these rats. It was noted that although the body weights of the ethanol-treated rats were lower (by 5 to 7%) than those of the control rats during the 12- to 28-day period, the rate of gain in the body weight of the ethanol-treated rats (2.6 ? 0.7) was similar to that of the control rats (2.8 ? 0.3), apparently indicating normal physiological behavior of the ethanol-treated rats. The body weights of the ethanol-treated rats (Treatment A) were similar to those of their respective control rats during the entire treatment period. Procainamide Pharmacokinetics (Treatment A)Computer analysis of the blood level data (Figure 2) indicated that the disposition of procainamide in all five control rats and in four out of five ethanol-treated rats was best described by a two-compartment open model, with drug elimination occurring from the central compartment. Procainamide disposition in untreated rats has been described by a two-compartment open model by several investigators.11-13 The distribution of most drugs from blood into the highly perfused tissues, such as liver and kidneys, is known to be quite rapid and that into the moderately to poorly perfused tissues, such as muscles and fat, is rathef slow. Therefore, in a two-compartment open model, it is considered that the liver and kidneys are part of the central compartment and muscles and fat are part of the peripheral tissue compartment. As mentioned earlier, we also studied the concentration-time profiles of procainamide in the individual tissues of the ethanol-treated and control rats of Treatment B for another report. In that study, we observed that the drug uptake curve noted in the muscles and fat was absent in the kidneys, heart, and liver, indicating that the distribution of procainamide from blood to the liver, kidneys, and heart was indeed rapid and that to the muscles and fat was slow (unpublished results). Therefore, liver, kidneys, and heart are included in the central compartment, while muscles and fat are included in the peripheral tissue compartment. The elimination of procainamide is known to occur due to its metabolism and urinary excretion. The metabolism of procainamide occurs14 in the rat liver due to N-acetylation,
A
5 W
s
I-
U <
-
200[.'
0
" ' . ' " 5
'
10
.
'
'
"
"
l
'
'
l
15
20
" . "
25
. ' ' I
j0
DAYS
Figure l-hnparative body weight versus time profiles of Treatment B rats. Key: ( 0 )ethanol-treated; (0)Control. 234 I Journal of Pharmaceutical Sciences Vol. 80. No. 3, March 1997
TIUE BIN1
.A
m
I
d
Table CAverage Values (* SD)of the Two-Compartment Open Model Pharmacoklnetic Parameters Determined for Procainamlde In Control and Ethanol-Treated Rats
k12,min-‘
hl,min-‘
k,,, min-’ A’. min-’ A,, min-’
Vd,, mUkg Vd, mUkg Vd,, mUkg CL, mUmin/kg CL,,, mL/min/kg
Control
Control (n = 5)
Treatment A (n = 4)a
0.2108 f: 0.1069 0.1494 f 0.0349 0.0667 f: 0.0288 0.4030f 0.1422 0.0239f 0.003 787.9f 274 1767 f 251 1967 t 247 46.4f 1.2 144.3 f 25.6
0.0655f: 0.0581 0.0758f:0.0254’ 0.0472f 0.0195 0.1661 f 0.0975b 0.0224f 0.004 1237 f 322 2073 f 109 2583 * 798 58.5 k 24 67.7f 44.6’
Parameter
(n
’
=
24)
0.2412f 0.0972 0.1486f 0.0223 0.0549f 0.0139 0.4255f: 0.1262 0.0192f:0.0022 787.9f 190 2064 ? 657 2253 2 829 43.2f 15 190.0f 18.2
Treatment B (n = 24) 0.0873f 0.0703’ 0.1446f 0.08 0.0357f 0.0079’ 0.24662 0.1509’ 0.02092 0.0039 1280 f 239’ 2048 f 207 2186 f 753 45.7f 13 111.7f 18.7’
stated in the text, one of the five rats displayed one-compartmentopen model pharmacokinetics for procainamide, with Vd = 2615 mL, 0.0217 min-’, and CL = 56.8 mUmin/kg. ’Significantly different from the corresponding control (p < 0.05).
a As
A2
=
ponentially (Figure 2) according to the one-compartment open model equation (C = C,e-’*‘), where all terms have the usual meaningsl.17 The apparent volume of distribution (Vd),the elimination rate constant (A,), and the CL of the drug were calculated using the PCNONLIN program. The values of these parameters are listed in Table I. Procainamide Pharmacokinetics (Treatment Bt-Since the blood samples from the ethanol-treated rats (Treatment B) and the corresponding control rats were collected by sacrificing three rats at each of the eight time periods, the blood level data of rats of a given group (n = 24) were pooled for the pharmacokinetic analysis and are presented in Figure 3. The computer analysis indicated that the disposition of procainamide in both groups of rats was best described by a two-compartment open model. The values of various pharmacokinetics parameters are listed in Table I. The standard deviations of k12, k,,, A,, A,, and k,, were provided by the
100
10
I
I
I
I
20
40
60
80
TIME [MINI Flgure +Comparative
concentration-time biexponential profiles of procainamide obtained in blood of the control (0)and ethanol-treated(W) rats of Treatment B after the administrationof an 80-mg iv dose to each rat. The solid lines are the computer predicted profiles and the symbols are average of three concentrations (with standard errors) of procainamide obtained in three rats sacrificed at a given time.
PCNONLIN Program, while those of Vd,, Vd,,, Vd,, CL, and CLd were calculated as described by others.ls.19 The distribution pharmacokinetic parameters A,, k,,, Vd,, and CL, observed in the ethanol-treated rats (Treatment B) were significantly different from those observed in the corresponding control rats. The elimination rate constant (klo)of the drug was also found to be significantly affected by the ethanol treatment. Overall Urinary Excretion of Procainamide (Treatment B k T h e overall urinary excretion data of procainamide were measured only in terms of the intact procainamide and its major metabolite (N-acetylprocainamide). It was observed that the amount of the intact drug excreted in the urine of the control rats (9.0 ? 0.5 mg) was not significantly different from that of the Treatment B ethanol-treated rats (9.2 ? 0.5 mg). Similarly, the amount of N-acetylprocainamide recovered in the urine of the ethanol-treated rats (3.8 2 0.4 mg) was not significantly different from that of the control rats (4.8 ? 1.1 mg). The total urinary recovery of procainamide and N-acetylprocainamide observed in this study was comparable to that reported by others.11.20 Partition Coefficients of Procainamide in Tissues (Treatment B k T h e apparent partition coefficient of procainamide in a given tissue (R,)was calculated by dividing the steadystate drug concentration in a given tissue CC), by the steadystate drug concentration in pooled blood (CB),according to the following equation:17
The contribution of the residual blood drug concentration to the total tissue drug concentration was considered to be negligible. The mean partition coefficients of the drug determined in heart (RH), liver ( R J ,kidneys (RK), thigh muscle (RTM),and fat (RF) are listed in Table 11. It was noted that the apparent partition coefficients of the drug in ethanol-treated rats (Treatment B) were significantly different from those in control rats only for heart, kidneys, and fat; in ethanol-treated rats, the R H and R K values were lower by 24 and 13%, respectively, and the RF value was higher by 38%. The normalized average steady-state concentrations of procainamide in blood (72.0 -C 13.6 pg/mL) and plasma (70.4 2 12.4 pg/mL) of the control rats were not significantly different from the normalized average concentrations of procainamide in blood (72.2 IT 13.3 pg/mL) and plasma (71.0 5 18.5 pg/mL) of the ethanol-treated rats (Treatment B). Furthermore, the drug concentration in blood was not significantly different from that in plasma either in control rats or the ethanol-treated rats. Journal of Pharmaceutical Sciences I 235 Vol. SO,No. 3, March 1991
Table ICAverage Steadystate Partitlon Coefficlents of Procainamlde Determined In Various Tlssues of Control and Ethanol-Treated Rats (Treatment B)
Tissue Partition Coefficient (4)
Rats Ethanol-Treated
Control
Heart (I?,,) Liver (RL) Kidney (RK)
2.472 0.27" 3.172 0.81" 6.36 t 0.94" 3.09 2 0.39" 0.13t 0.02'
Thigh muscle (&)
Fat ( 4 )
1.872 0.34" 3.35 2 1.22a 5.53 2 0.52' 2.75 2 0.72" 0.18? 0.02'
P
<0.05 NS
<0.05 NS
C0.05
" Standard deviation based on n = 7. Standard deviation based on n
3.
=
Binding of Procainamide to Whole Blood and Plasma (Treatment B)-The extent of binding of procainamide to blood (7.3 +. 2.7%) or plasma (8.5 2 7.8%) of the ethanoltreated rats (Treatment B) was not found to be significantly different from that for blood (7.9 +. 5.3%)or plasma (8.0 7.8%)of the control rats. The extent of binding of procainamide to whole blood was similar to that for plasma in both the control and ethanol-treated rats. The average of f B values determined for the ethanol-treated and control rats are listed in Table 111. Fractions of Free Procainamide in Tissues-The fractions of free procainamide in various tissues ( f T ) of the control and ethanol-treated rats (Treatment B) were calculated according to the equation:17
*
(4) The f B and RT values were known, as shown in Tables I1 and 111. The fH and fK values of the drug in ethanol-treated rats were found to be significantly greater than those in control rats by 32 and 21%, respectively.
Discussion It should be noted that, although the approach we used for rat blood sampling in pharmacokinetic studies related to Treatment A (blood samples were taken serially from each rat) was different from that we used in those studies related to Treatment B (only one blood sample per rat was obtained), the magnitudes of the pharmacokinetic parameters of procainamide determined in the control rats of Treatment A were similar to the control rats of Treatment B (Table I). Therefore, it can be inferred that the removal of 3.0 to 3.5 mL of blood from the individual rats of Treatment A had no effect on the pharmacokinetics of the drug and that the effects observed in the ethanol-treated rats of Treatments A and B were primarily due to chronic ethanol exposure. Effects on Procainamide Pharmacokinetics-Body Clearance and Metabolism-The ethanol treatments employed in Table Ill-Average Fractions of Free Procalnamlde in Blood (f,) and Various Tlssues (fT) of the Control and Ethanol-Treated Rats (Treatment B)
Tissue ~~~~~
~~~
Blood (&) Heart (f,) Liver (fL) Kidney (f,) Thigh muscle ( f m ) a
n
~~
P
~
0.922 0.05' 0.38 t 0.04' 0.31 t 0.07' 0.14t 0.02' 0.30 t 0.04b
Standard deviation based on n
=
Rats Ethanol-Treated
Control
= 4.
0.93t 0.03" 0.502 O . l l b 0.30 5 0.11 ' 0.17t 0.02' 0.362 0.12'
NS
<0.05 NS <0.05 NS
Standard deviation based on
7.
236 I Journal of Pharmaceutical Sciences Vol. 80, No. 3, March 7997
this study did not affect the CL (k,,Vd,) of procainamide (Table I), indicating that the irreversible removal of the drug from the central compartment was not affected. However, the k,, value of the drug was significantly lower in the ethanoltreated (Treatment B) rats than in the control rats (Table I). The decrease in k,, cannot be attributed to the direct effect of the ethanol Treatment B on the hepatic or renal elimination functions of the rats because the extent of urinary excretion of the intact drug and its major metabolite, N-acetylprocainamide, observed in the ethanol-treated and control rats were similar. However, in view of the fact that the ethanol treatment had no effect on CL of the drug, the decrease in k,,, which is a dependent variable,'l is most probably the consequence of the significant increase in Vd, of the drug caused by the ethanol treatment. Distribution Parameters-Table I shows that the elimination-independent distribution pharmacokinetic parameters k,,, A,, Vd, and, more importantly, the distribution clearance (k12 Vd,) were significantly affected by the ethanol Treatment B. The same distribution pharmacokinetic parameters, except Vd,, were also significantly affected by ethanol Treatment A. Although the Vd, of the drug in the ethanol-treated rats (Treatment A) was not significantly different from that observed in the corresponding control rats, the average Vd, value of the drug in these ethanol-treated rats was -57% greater than that in the control rats. This percentage increase in Vd, was quite comparable to the 62% increase in Vd, observed in ethanol-treated rats (Treatment B). The fact that one of the five ethanol-treated rats (Treatment A) exhibited one-compartment characteristics for procainamide appeared to be indicative of the effect of ethanol (Treatment A) on the distribution kinetics of procainamide. Thus, this study demonstrated that both ethanol treatments significantly decreased the distribution clearance (intercompartmental clearance) of procainamide. Since the tissue transport or uptake rate of drugs of molecular weights similar to that of procainamide is limited not only by their transport across the tissue cell membranes but also by the tissue perfusion rates,22 the decrease in procainamide distribution clearance observed in this study might be due to the alteration in blood flow andlor permeability of the tissues. Moravi et al.23 demonstrated that rats treated with 11.512.5 glkg of ethanol per day in a liquid diet for 4 weeks experienced a decrease in cardiac output as well as in blood flows of myocardium, kidneys, skin, and carcass by -2630%. Ituriaga e t al.24observed that the rats, which were fed ethanol in a liquid diet in doses similar to those used in our study, experienced a 28% decrease in hepatic blood flow. Although we did not measure the tissue blood flows, we cannot eliminate the possibility that the ethanol treatments in our study might have also lowered the cardiac output and the blood flows in various tissues, especially in the tissues (muscles) of the peripheral compartment, and thereby contributed to the reduction of the distribution clearance of procainamide in the ethanol-treatedrats. However, the increase in Vd, of the drug that we observed in the ethanol-treated rats cannot be explained by a reduction in tissue blood flows.Therefore, it seems that the permeability characteristics of tissue cell membranes were substantially affected by the chronic ethanol treatment. It is recognized that the large size of the apparent volume of distribution of the central compartment of procainamide is clearly indicative of the fact that it exceeds both the blood volume and extracellular space and that it includes not only the highly perfused tissues, but also part of the moderately perfused peripheral tissue which is in rapid equilibrium due to its close proximity to the capillary network of the tissue. Since chronic ethanol ingestion affects the concentration of cholesterol and phospholipid-associated fatty acids in the cell
membranes of a variety of tissues,1-' it is conceivablethat the ethanol treatment might have rendered additional parts of the tissues of the peripheral compartment (which largely consists of muscles) more readily accessible or permeable to the drug, thereby bringing it into rapid equilibrium with the central compartment, making it kinetically a part of the central compartment, and consequently yielding a higher value of Vd, in the chronically ethanol-treated rats. This mechanism appears to be consistent with our findings that Vd, and Vd, of procainamide (Table I), as well as the fractions of free procainamide in blood and muscles, remained unaffected by the ethanol treatments. It is noted in Table I that the body clearance and the total apparent volume of distribution of procainamide in the only ethanol-treated rat (Treatment A) which displayed the onecompartment characteristics for procainamide were similar to those in other ethanol-treated and control rats. It appears that the ethanol treatment in this rat rendered a large part of the tissues of the peripheral compartment readily permeable to the drug and brought it into rapid equilibrium with the central compartment, thereby rendering the tissue compartment kinetically indistinguishable. Although we did not measure the blood levels of ethanol when the pharmacokinetics of procainamide were studied, we assumed that the concentration of ethanol in blood was negligible at the time procainamide was administered. This assumption was considered reasonable because, at the time of procainamide administration, 24 h had already elapsed since the last dose of ethanol was administered either in Treatment A or Treatment B and, as demonstrated by others2627 in rats, the blood ethanol concentration becomes negligible even after 8 h following the last dose of ethanol in chronic ethanol treatments similar to those employed in our study. Therefore, the effects of chronic ethanol on the distribution pharmacokinetics of procainamide noted in the present study are attributed to the effects of ethanol on the composition and permeability characteristics of tissue cell membranes. Effect on Partition Coefficients-The fact that the R, and R, values of procainamide observed in the ethanol-treated rats were significantly lower than those in the control rats was rationalized by considering the alternative mathematical definition of tissue partition coefficient (R,) of drug which is expressed as follows:17 R T = fB/fT
where fT is a fraction of free procainamide in a given tissue. The usual assumption made in the derivation of this equation is that, a t steady state, the concentration of free drug in blood is the same as that in tissue fluids. This equation indicates that any alteration that may occur in the binding of drug to a tissue and/or blood would alter the partition coefficient of drug in that tissue. Since the f B values of procainamide observed in the ethanol-treated and control rats were similar, the lower RH and RK values of the drug noted for the ethanol-treated rats can be attributed to the increase in f H and f K values of the drug in their hearts and kidneys, respectively (Table 111); the increase in fH and f K may have resulted from the decreased binding of procainamide to membranes and intracellular constituents of these tissues. One of the major mechanisms known to be responsible for the localization of drugs in tissues is their reversible binding to the cellular membrane components and the intracellular constituents. The binding of the cationic forms of basic drugs, including procainamide, to phospholipids and contractile proteins (myosin and actin) of the heart and skeletal muscle have been demonstrated28.29 in vitro. Therefore, it seems reasonable to attribute the decrease in partition coefficientsof
procainamide in heart and kidneys of the ethanol-treated rats to the possible decrease in binding of the drug to proteins and phospholipids of these tissues. A decrease in the binding of procainamide to cell membranes and intracellular components might have resulted from the decreased protein synthesis,30 the modified acyl chains of cellular phospholipids,*,31-33 and/or the alterations in the protein conformations,33 all of which are brought about by chronic ethanol ingestion. Such ultrastructural changes in various organs have been shown to occur without causing hypertrophy of the organs. As noted by other workers3636 for ethanol treatment similar to that employed in our study, we also observed that the weights of hearts, livers, and kidneys of the ethanoltreated rats were very similar to those of the control rats. We also considered the possibility of the effect of chronic ethanol on the intracellular and/or extracellular pH of tissues which might affect the partition coefficients of procainamide in tissues. To our knowledge, liver is the only tissue that has been investigated37-3*and shown to have its intracellular pH lowered by -0.2 pH units due to a higher NADH:NAD+ratio resulting from a constant oxidation of ethanol. Since kidney and heart are not known to be the sites of metabolism for ethanol, chronic ethanol exposure would seem highly unlikely to alter the intracellular or extracellular pH of these tissues and affect the partition coefficientsof procainamide. It is interesting to note that the partition coefficient of procainamide was not affected in the liver of ethanol-treated rats, despite the possibility that the liver intracellular pH of these rats was affected due to chronic ethanol exposure. Furthermore, even if the tissue intracellular or extracellular pH were slightly altered by ethanol exposure, it is doubtful if it would influence the tissue partition coefficientof procainamide (pK, 9.2) which exists entirely in the cationic form at a physiological pH and would certainly remain in that form at a lower pH. While the ethanol treatment decreased RH and RK, the same ethanol treatment significantly increased RF (Table 11). The RF values of procainamide in both the control and ethanol-treated rats were much less than unity, indicating that the solubility of the drug in fat is small. In view of the report that chronic ethanol ingestion for a period as few as 4 weeks in doses similar to those used in our study lowered the triglyceride content of adipocytes due to decreased lipogenesis,39 it is conceivable that a decreased lipid content in adipocytes of the ethanol-treated rats might have resulted in increasing the solubility of procainamide in fat and consequently increasing its RF. Significance-One of the striking pharmacokinetic findings of this study which might be pharmacologically important is that the significant changes that may occur due to chronic ethanol exposure in the concentration profiles and partition coefficientsof drugs in individual tissues containing the sites of action may not necessarily manifest in exhibiting significant differences in their blood or plasma concentration profiles and body clearance values. This may result because the amounts of drugs involved in tissues containing the sites of action (i.e., heart and kidney) are usually too small (as compared with the total amount of drug present in the body) to influence their overall blood levels or clinically useful pharmacokinetic parameters, namely, body clearance and area under the blood level curves. In view of the results of this study, it becomes apparent that taking notice of subtle changes that might occur in the distribution pharmacokinetic parameters of drugs due to chronic ethanol ingestion might be useful in discovering potential changes that occur in drug concentration a t the site of action and in drug binding to receptors present in small organs. It has been recognized that chronic ethanol exposure affects the myocardial membrane and consequently impairs membrane-mediated functions in the heart.40.41 Therefore, since Journal of Pharmaceutical Sciences I 237 Vol. 80, No. 3, March 1991
procainamide has its site of action i n the membranes of myocardial fibers,42 a decrease in the binding ofprocainamide to myocardial tissue inferred in this s t u d y could be of clinical significance.
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238 / Journal of Pharmaceutical Sciences Vol. 80, No. 3, March 1991
299-305. 22. Gerlowski, L. E.; Jain, R. K. Microvasi:ularRes. 1986,31,228305. 23. Moravi, v.; Ungvary, G.; Varga, K.; Albert, ,K.; Foll , G. Acta Physiologrca Academiae ScientLarum Hungarmze 197{ 54,369379. 24. I t u r i p , , H . ; Bunout, D., Petermann, M.; Ugarte, G.; Israel, Y. Alco olism Clin. Exp. Res. 1981, 5, 221-224. 25. Khanna, J. M.; Israel, Y.; Kalant, H.; Mayer, J . M. Biochem. Pharmacol. 1982,31,3140-3141. 26. Jones, A. W.; Alling, C.; Becker, W.; Anngard, E. Pharmacol. Biochem. Behav. 1983,19, 115-119. 27. York, J. L. Pharmacol. Biochem. Beh.av. 1983, 19, 687-691. 28. Fichtl, B.; Bondy, B.; K u n , H. J . Phurmacol. Exp. Ther. 1980, 215.248-253. 29. K u n , H.; Fichtl, B. Drug Metab. Rers. 1983, 14, 467-510. 30. Burke, J. P.; Rubin, E. Lab. Invest. 1979,41, 393400. 31. Shorey, R. L.; Pyle, B.; McAllister, M.; Miller, S. S.; Erickson, C. K.; Thornson, G. A. Biochem. Pharmcol. 1982,31 2447-2452. 32. Littleton, J. M.; John, G. R. J . Pharm. Pharmacol. 1977, 29, I
-
Fi'lLFiRn - . - - -.
33. Rubin, E.; Katz, A. M.; Lieber, C. S.;Stein, E. P.; Puszkin, S.Am. J . Pathol. 1976, 83,499-515. 34. Yamashita, S. Jap. Heart J . 1951, 12, 242-250. 35. Segel, L. D.; Rendin S. V.; Choquet, Y.; Chacko, K.; Amsterdam, E. A,; Mason, T. Cardiovasc. Res. 1975,9,649-653. 36. Mattfeld, T.; Mall, G.; Volk, B. J . Mid. Cell. Cardiol. 1980, 12, 1311-1319. 37. Morland, J.; Christofferson, T.; Osnes, J. B. Acta Physiol. Scand. 1973.89, 61-67. 38. Wallin, B.; Morland, J.; Fikke, A. M. Acta Pharmacol. Toxicol. 1981,49, 134-140. 39. Scheig, R.; Alexander, N.; Klastskin, G. J . Lipid Res. 1966, 7, 188-196. 40. Rubin, E. N . Eng. J . Med. 1979,301, 28-33. 41. Chan, T. C. K.; Sutter, M. C. Can. J . Physiol. Pharmacol. 1982, 60, 777-782. 42. Koch-Weser, J. Cardiouasc. Clin. 1974, NII), 63-75.
%:
Acknowledgments This aper was adapted from a dissertation submitted by D.J. Gole to the e r a d u a t e School of Wayne State University in artial fulfillment of the Doctor of Philosophy degree requirements. !his work was supported in part by the Wayne State University Faculty Biomedical Research Award received by J . B. Nagwekar.