Acta pharmacol. et toxicol. 1975, 36, 415-426.
From the Department of Pharmacology, ACO Lakemedel AB, Box 3026, S-17103 Solna, Sweden, and Department of Pharmacology, Apotekens Centrallakoratorium, Apoteksbolaget AB, Box 3045, S-17103 Solna, Sweden
Effect of Stress on the Pharmacokinetics of Sodium Salicylate and Quinidine Sulphate in Rats* BY Uno Otto and Lennart Paalzow (Received October 29, 1974; Accepted December 6, 1974)
Abstract: Studies on the influence of foot shock stress on the absorption, distribution and overall serum elimination of sodium salicylate and quinidine sulphate were performed in rats. Foot shock stress was produced by grid floor electrical stimulation. The animals were stressed 1 hr before administration of the drugs and then throughout the experimental period. A significant induced stress was demonstrated by increased blood glucose levels and increased whole brain turnover of noradrenaline. Gastric emptying and intestinal transit as well as tissue levels of the drugs in the serum, heart, brain, liver, muscle and fat were determined. Results were obtained indicating that stress produced a significant increase in the gastro-intestinal absorption of quinidine sulphate while no change in the distribution pattern or overall elimination was noted. On the other hand, sodium salicylate did not show any marked change in the pharmacokinetic parameters during a stressful situation.
Key-words: Stress - intestinal absorption - sodium salicylate - quinidine.
A variety of physical and emotional stimuli can induce stress which is associated with physiological alterations of the homeostasis. It is also known or assumed that physiological and pathological factors can influence the kinetics of drug absorption, distribution, metabolism or elimination (for review see PRESCOTT & NIMMO 1971). Of these physiological processes the absorption of drugs from the gastro-intestinal tract depends on many factors, such as gastric emptying, intestinal motility, blood flow, pH, volume, composition and presence of food, viscosity, osmolarity, and the like (RIEGELMAN & ROWLAND 1973). Since a stressful situation can be expected to * A preliminary report of this investigation was presented at the XXVIII Meeting of the Scandinavian Pharmacological Society, Odense, 1974.
416
UNO OTI'O AND LENNART PAALZOW
produce changes in some of these factors, it is important to investigate further the kinetics of drugs under the influence of stress. Furthermore, many drugs modify physiological or pathological functions which can then bring about an interaction with the kinetics of other drugs given at the same time. We have therefore now studied the absorption and distribution of sodium salicylate and quinidine in the rat subjected to electrical foot shock stress. The drugs were chosen because of their physico-chemical character, being an acid and a base, respectively. Consequently, the absorption kinetics can be expected to be influenced differently by physiological changes in the gastro-intestinal tract. Materials and methods Male Sprague-Dawley rats, weighing 130-180 g, were used throughout the study. For 16 hrs before the test, the rats were allowed water ad libitum,but no food. Rats in groups of four were placed on a grid floor in a cage of 30 X 30 X 30 cm. Foot shock stress was produced by electrical stimulation (4OV, 125 Hz,1.6 msec. pulse width, 1 sec. duration given each 10 sec.) from a square wave stimulator (Kistner Instruments 421). To ensure that the animals were subjected to significant stress, the blood glucose level was determined enzymatically with a commercial glucose oxidase preparation (Kabi Reagents, Stockholm, Sweden). In another experiment, the whole brain level of noradrenaline and dopamine was determined (SHELLENBERGER & GORDON 1971) after inhibition of tyrosine hydroxylase with a-methyl-p-tyrosine methylester (H 44/68) given 4 hrs before death according t o A N D ~ et N al. (1966). The time course of drug serum concentrations was followed during 6 hrs after oral and subcutaneous administration of sodium salicylate and quinidine sulphate. The dose of the drugs were chosen as low as possible, allowing adequate chemical analysis. In the distribution studies the animals were killed by decapitation 1 hr after the oral or subcutaneous administration of the drugs, and the blood was collected. The brain or heart, liver, fat of testicles and muscle tissue (hind leg) were rapidly excised, blotted and weighed. The different tissues were homogenized with an Ultra-Turrax TP-1ON in 0.2 M sodium hydroxide for the tissues from the quinidine treated animals, and 1 M hydrochloric acid from animals given salicylate. Gastric emptying and intestinal transit were determined by using phenol red as a marker (REYNELL& SPRAY 1956). The rats were killed at intervals after administration as indicated in the results and the stomach and intestine were ligated a t the cardia, while the pylorus and the small intestine were ligated into three equal segments down to the ileocaecal sphincter. During evisceration care was taken to prevent displacement of the small intestinal contents owing t o differences in level. The determination of phenol red in the different parts of the gastro-intestinal canal and the calculation of gastric emptying and intestinal transit rate were made according to FELDMAN & GIBALDI (1%8).
All drugs administered to the animals were dissolved in physiological saline solution. Fluorometrical analysis of the quinidine levels in the tissues was performed according to CRAMBR & ISAKSSON(1963) and salicylic acid according to VF~ESH et al. (1971). The statistical calculations were performed according to FISHER (1954).
417
STRESS AND PHARMACOKINETICS
Table 1 . Changes in blood glucose levels in 8 control and stressed rats. ~
hr Control Stressed Difference from control t P
100 % 100 %
1 hr 2 hrs 3 hrs M e a n f S . E. M. M e a n f S . E. M. M e a n f S . E . M. 101 147
5% 10 %
47
11 % 4.155 0.001
0.8
102 82
5% 15 %
-20 15 % - 1.310 > 0.2
Results
Significance of induced stress. The electrical parameters used to induce a foot shock stress were fundamentally the same as those used to induce a pain reaction in animals (PAALZOW1969a&b; PAALZOW& PAALZOW 1973). Due to the higher resistance of the feet of the rats compared to the intracutaneous electrodes used in the above-mentioned studies, it was found in a pretest that a standardized voltage of 40 V given every 10 sec. during 1 hr before the administration of the drugs was sufficient to produce stress in the animals. Table 1 shows that animals subjected to foot shock stress of 40 V gave a significant increase of blood glucose level following 1 hr of stress. During the following 2 hrs the levels showed a tendency to
Table 2. The effect of foot shock stress on brain noradrenaline and dopamine after inhibition of catecholamine synthesis with H44/68 given 4 hrs before death. The number in brackets indicates the number of animals. Brain noradrenaline ng/g k S.E.M.
Brain dopamine n d g f S. E. M.
Controls
192 f 3 (n = 8)
311 k 14 (n = 9)
1 hr of stress
168 f 5 (n=6)
288 f 141) (n = 6)
2 hrs of stress
157 f 6 : ~ (n =6 )
285 f 7 b (n=6)
Treatment
3.
1)
Significantly different from controls (P < 0.01). Not significantly different from controls (P > 0.05).
UNO OTTO AND LENNART PAALZOW
418
decline although not significantly as compared to control rats placed on the grid that did not give electrical shocks. By blocking the catecholamine synthesis with a-methyl-p-tyrosine the rate of depletion of noradrenaline and dopamine should be proportional to the impulse flow within the neurons i. e. the higher activity, the greater the depletion (ANDBNet al. 1966). Table 2 shows that 1 and 2 hrs of foot shock stress caused an increased depletion of whole rat brain noradrenaline, indicating an increase in the turnover of this transmitter. There was no significant difference between control animals and stressed rats with regard to dopamine depletion induced by enzyme inhibitor. Serum levels of salicylic acid and quinidine in stressed and control animals. Following 1 hr of foot shock stress, groups of rats were given 100 mgkg sodium salicylate or 50 mgkg quinidine sulphate subcutaneously or orally and subjected to foot shock stress for the following 6 hrs during which period blood was drawn and analysed for salicylates and quinidine. The decrease in log salicylic acid and quinidine concentrations in serum with respect to time appeared linear in all animals 2 hrs after dosing, regardless of the drug given and mode of administration. The overall elimination rate constant in serum and corresponding half-life were estimated from the
r!
-
rJs,
4.0
'a
4.0 Quinidine p.0.
'ha,
2.0
1.0
1 .o
0.5
0.5
0.25
0.25 1
I
I
I
1
2
3
4
Control animals Stressed animals Quinidine
2.0
I
-----
,I
II
I
6 h
$
1
1
I
I
I
2
3
4
S.C.
I
6 h
Fig. 1. The semi-logarithmic plot of the serum levels of quinidine after oral and subcutaneous administration of 50 mg/kg quinidine sulphate to control and stressed rats. Each point represents the mean of 12 animals after oral and 4 animals after subcutaneous administration.
P
t
Diff. from control
Stressed
Control
83.927.1
85.858.9
Liver Irdg
-2.8k5.4 -0.518 > 0.6
20.5f3.1
23.3k4.4
Pug
Fat p.g/ml
Serum
5.5k7.5 -7.6k14.2 0.735 0.536 > 0.4 > 0.5
48.3k4.8 227.3f10.3
42.825.8 234.9k9.8
VdS
Musc!e
Oral administration
4.0k3.6 -1.9k11.4 1.118 -0.166 > 0.2 > 0.8
25.8k2.3 (n=8) 29.8f2.8 (n = 12)
Ildg
Brain CLdg
Liver
7.7k2.0 3.952 < 0.01
5.0t4.2 1.205 > 0.2
41.0f1.1 116.3k2.6 (n=8) 48.7k1.6 121.3k3.2 (n = 8)
P.g/P
Brain
3.9f2.8 1.400 > 0.1
38.1k2.4
34.2k1.4
pdg
Fat
p.g/ml
Serum
1.5Z6.5 0.232 > 0.8
4.2k10.8 0.389 > 0.7
71.7k5.5 246.7k6.8
70.2k2.9 242.5k8.4
IrdS
Muscle
Subcutaneous administration
Mean tissue levels k S. E. M. of salicylate in control and stressed rats respectively 60 minutes after oral and subcutaneous administration of 100 m g k g sodium salicylate.
Table 3.
~
c
82
$
z>
50
3
P
Diff. from control t
Stressed
Control
0.7
12.1k1.6
11.3f1.8
PdP
3.4f1.1 3.038 < 0.02
7.2f0.9
3.8k0.7
Pdg
>
3.2k0.2
3.5k0.3
Pdml
Serum
0.3f1.2 -0.3k0.3 0.268 -1.045 > 0.7 0.3
13.4f1.0
13.150.7
Pdg
Subcutaneous administration Liver Fat Muscle
Mean tissue levels f S. E. M. of quinidine in control and stressed rats respectively 60 minutes after oral and subcutaneous administration of 50 m d k g quinidine sulphate.
Table 4.
2
8
G
2>
e
$
z
E;; 2
U
5
0
3
0
0
C
0
h)
P
421
STRESS AND PHARMACOKINETICS
P
11
300
-------
lll
C o n t r o l animals Stressed animals
300
200
100
100 Salicylic a c i d p.0.
Salicylic a c i d
50
S.C.
50 I
I
f
4
3
I
I
4
6
I
h
I
I
4
3
1
I
4
6
h
Fig. 2. The semi-logarithmic plot of the serum levels of salicylic acid after oral and subcutaneous administration of 100 mg/kg sodium salicylate to control and stressed rats. Each point represents the mean of 8 animals after oral and 6 animals after subcutaneous administration.
log concentration-time curves using the data from 2-6 hrs. Figs. 1 and 2 show the mean log serum concentration-time curves of salicylic acid and quinidine after oral and subcutaneous administration. The mean serum half-life for salicylic acid was 8.7 f 1.7 hrs (S. E. M.) in control animals and 9.0 f 2.6 hrs in stressed animals after oral dosing and 8.4 ? 1.7 and 9.1 -I 2.3 hrs, respectively, after subcutaneous administration. There was no significant difference between these half-lives. After oral administration of quinidine, the half-life in control animals was estimated to be 1.7 f 0.8 hrs and in stressed animals to be 2.5 -I 1.1hrs. The corresponding values after subcutaneous administration were 1.8 .+ 0.2 hrs and 1.7 f 0.1 hrs, respectively. As for salicylic acid, there was no statistically significant difference between the half-lives for quinidine. However, the serum levels of quinidine after oral dosing were increased 2-3 fold in the stressed animals (fig. 2) and an analysis of variance showed that this increase was significant (F=24.9; d.f.= 142/1; P < 0.001). The mean serum levels of salicylate, on the other hand, showed slightly increased levels in stressed animals (fig. 1) and this small difference (13 yo) was statistically significant (F=5.91; d. f.=94/1; P < 0.05).
422
UNO OTTO A N D LENNART PAALZOW
Since the overall eliminations from serum of the two drugs studied were unchanged regardless of the mode of administration or whether there was any subjection to stress or not; this could be an indication of an unchanged distribution, elimination or metabolism during stress. The increased serum level of quinidine after oral administration could be a consequence of, for example, a changed degree of absorption or tissue distribution pattern. Therefore, in the following experiments the distribution pattern of the two drugs was investigated. Tissue levels of salicylic acid in stressed and control rats. Following 1 hr of foot shock stress, rats were given 100 mgkg sodium salicylate per 0s or subcutaneously and then stressed for the following 1 hr. Thereafter the animals were killed by decapitation and tissue levels of salicylic acid were determined. As can be seen in table 3, foot shock stress does not change the tissue level of salicylate regardless of the mode of administration, except in the brain where, after subcutaneous injection, an increased level was seen in stressed rats compared to controls. As a measure of the tissue distribution pattern, the ratio between tissue and serum levels was calculated from table 3, and in that respect no significant differences (P > 0.05) in the distribution of salicylate were obtained in the stressed animals compared to the controls after oral administration. However, surprisingly elevated ratios were found in the brain and fat of stressed animals after parenteral injection. Tissue levels of quinidine in stressed and control animals. As in previous experiments, rats were subjected to stress for 1 hr and then given 50 mgkg quinidine sulphate subcutaneously or orally and the foot shock stress was continued for the following hour. One hour after drug administration the rats were killed and the tissue levels of quinidine were determined in the different tissues. Contrary to the previous experiment with sodium salicylate, about 2-3 fold higher levels of quinidine (P = 0.01-0.001) were found in all tissues after oral administration of this drug to the stressed animals compared to controls (table 4). However, when quinidine sulphate was given subcutaneously, there was no significant difference in tissue levels between the stressed and control rats except for fat tissue. As in the experiment with salicylate, the ratio of tissue to serum level of quinidine was calculated. A significant increase (0.05 > P > 0.01) in liverserum ratio was obtained for stressed animals after oral administration and in the fat-serum ratio after subcutaneous injection. No other significant differences were found regardless of whether stress was given or not. Thus the studies of the tissue distribution pattern of salicylic acid and quinidine seem on the whole to indicate an unchanged distribution of these drugs under stress conditions. Gastric emptying and intestinal transit rate in stressed and control animals.
STRESS AND PHARMACOKINETICS
423
From the previous experiments it was evident that foot shock stress increases the serum and tissue levels of quinidine after oral administration but probably not the distribution pattern. Some explanations of these findings might be offered, e. g. increased absorption from the intestinal canal resulting from changed gastric emptying, intestinal motility or intestinal blood flow. In order to investigate possible changes in gastric emptying or intestinal transit rate in the stressed animals, phenol red was used as a marker. The rats were pre-stressed for 1 hr and then given phenol red orally; 30 and 60 min. later the stomach and intestine were eviscerated, segmented and analysed for phenol red. From table 5 it is apparent that gastric emptying of phenol red showed a tendency to increase in the stressed rats although no significant differences were noted. However, as for transit rate, a decreased rate in the stressed animals was noted compared to controls, and this was significant (P < 0.05). Discussion
The absorption characteristics of weak acids or bases from the alimentary tract has been particularly investigated by BRODIEand co-workers (e. g. SCHANKER 1971). Most drugs are absorbed as unionised molecules by passive diffusion. An important factor for the transfer is the lipid solubility of the unionized drug and thus the pH will determine the fraction of the
Table 5. Gastric emptying and intestinal transit of phenol red in control and stressed rats. The results are expressed as mean values f S. E. M. Time after administration
NO. of animals
Gastric emptying %
Intestinal transit % Segment 1 Segment 2
30 60
12 19
59.2.f 5.1 72.4f3.4
87.9 ? 1.7 94.2 f0.9
24.6 ? 5.8 78.7 f 3.1
30 60
10 12
64.5 f 5.7 76.5 +4.3
75.9 k 8.1 87.9 f 2.8
8.554.1 27.6 f 7.8
5.3 f 7.7 4.1.55.5
- 12.0k 8.3 -5.3+_2.9&
- 16.1 k 7.1% -51.1f8.4a
Control
Stressed
Difference from control 30 60 a
Significantly different from control, P
< 0.05.
424
UNO OTTO AND LENNART PAALZOW
drug present in this form. This means that weak acids will be fairly well absorbed from the stomach while weak bases will be readily absorbed from the alkaline environment of the small intestines. However, salicylic acid is also fairly well absorbed from the small intestines, this presumably being due to the relatively large surface area of the latter. The rate of gastric emptying and intestinal motility will thus be important factors for the absorption of drugs from the alimentary canal. Furthermore, drugs are primarily removed from this canal by blood flow, and a reduced flow, for example, may decrease the rate of removal of the drug absorbed by passive diffussion (RIEGELMAN & ROWLAND 1973). A stressful situation can be expected to produce changes in those three physiological functions, e. g. by an increased sympathetic activity (THOMAS & BALDWIN1968). The animals, subjected to foot shock stress, showed a significant increase in blood glucose levels, and a significant increased acceleration of the depletion of the whole brain noradrenaline, induced by the enzyme inhibition. CORRODIet al. (1968) have shown that immobilization stress activates various noradrenergic nerve terminal systems in the rat brain, and if the immobilization was made more complete they also found a decreased turnover of dopamine as well as an increased noradrenaline turnover (CORRODI et al. 1971). Furthermore, THIERRY et al. (1970) have demonstrated that foot shock stress of weak intensity and short duration brings about important modifications of the noradrenaline metabolism in the brain. Rats given foot shock stress and injected subcutaneously with sodium salicylate or quinidine sulphate, respectively, showed about the same serum and tissue levels of these drugs as control animals. However, if the drugs were given orally, quinidine was obtained in a significantly higher concentration in all tissues of the rats subjected to stress. Animals given salicylic acid showed no such marked differences. The time course of the serum levels of salicylic acid and quinidine indicated no change in the overall elimination rate constant of these drugs during stress. The elimination of these two drugs are highly dependent on the pH of the urine and possible stress induced changes in pH should in our experiment have been detected in the elimination rate constant. Furthermore, the stress did not seem to change the distribution ratio of the tissue to serum. However, the ratio of the liver to serum after oral administration of quinidine in the stressed animals was markedly elevated. This latter finding as well as that of the higher tissue levels of quinidine after oral administration to stressed animals, may be due to an increased absorption induced by a changed physiological activity of the gastro-intestinal tract, e. g. in gastric emptying, motility or blood flow. Subsequent experiments showed that the intestinal transit rate was decreased in the stressed animals and there was a tendency of increased gastric emptying. Since quinidine is a base these physiological conditions
STRESS AND PHARMACOKINETICS
425
may favour its absorption. An acid such as salicylic acid is not so highly dependent on these two physiological factors since it is well absorbed from both the stomach and the intestines. However, one possible explanation for the different behaviour of the two drugs is that salicylic acid is completely absorbed and increases of it can therefore not be traced. In conclusion, the present study indicates an increased gastro-intestinal absorption of quinidine during stress. The therapeutic significance of the 2-3 fold increase found in tissue quinidine levels during a stressful situation has yet to be evaluated in human subjects. Preliminary studies in our laboratories have also shown that a minor tranquilizer such as meprobamate can prevent the stress-induced elevation of quinidine levels in tissue.
REFERENCES Andtn, N.-E., H. Corrodi, A. Dahlstrom, K. Fuxe & T. Hokfelt: Effects of tyrosine hydroxylase inhibition on the amine levels of central monoamine neurons. Life Sci. 1966, 5, 561-568. Corrodi, H., K. Fuxe & T. Hokfelt: The effect of immobilization stress on the activity of central monoamine neurons. Life Sci. 1968, 7 , 107-112. Corrodi, H., K. Fuxe, P. Lidbrink & L. Olson: Minor tranquillizers. Stress and central catecholamine neurons. Brain Res. 1971, 29, 1-16. Cramtr, G. & B. Isaksson: Quantitative determination of quinidine in plasma. Scand. J. Clin. Lab. Invest. 1963, 15, 553-556. Feldman, S. & M. Gibaldi: Effect of bile salts on gastric emptying and intestinal transit in the rat. Gastroenterol. 1968, 54, 918-921. Fisher, R. A.: Statistical methods for research workers, 12th ed. Oliver & Boyd, Edinburgh, 1954. Paalzow, L.: An electrical method for estimation of analgesic activity in mice. Part I. Methodological investigations. Acta Pharm. Suecica 1969a, 6, 193-206. Paalzow, L.: An electrical method for estimation of analgesic activity in mice. Part 11. Application of the method in investigations of some analgesic drugs. Acta Pharm. Suecica 1969b, 6, 207-226. Paalzow, G. & L. Paalzow: The effects of caffeine and theophylline on nociceptive stimulation in the rat. Acra pharmacol. et toxicol. 1973, 32, 22-32. Prescott, L. F. & J. Nimmo: Drug therapy. Physiological considerations. J . Mond. Pharm. 1971, 14, 253-260. Reynell, P. C. & G. H. Spray: The simultaneous measurement of absorption and transit in the gastro-intestinal tract of the rat. J. Physiol. (London) 1956, 131, 452-462. Riegelman, S. & M. Rowland: Effect of route of administration on drug disposition. J. Pharmacokin. Biopharm. 1973, 1, 419-434. Schanker, L. S.: Drug absorption. In: Fundamentals of drug metabolism and drug disposition. Eds.: B. N. LaDu, H. G. Mandel & E. L. Way. Williams & Wilkins Company, Baltimore, 1971. Shellenberger, M. K. & J. H. Gordon: A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas. Anulytical Biochemistry 1971, 39, 356-372.
426
UNO OTTO AND LENNART PAALZOW
Thierry, A.-M., G. Blanc & J. Glowinski: Preferential utilization of newly synthesized norepinephrine in the brain stem of stressed rats. Eur. J . Pharrnacol. 1970, 10, 139-142. Thomas, J. E. & M. V. Baldwin: Pathways and mechanisms of regulation of gastric motility. In.: Handbook of Physiology. Section 6. Alimentary Canal. Ed.: C. F. Code. American Physiological Society, Washington D. C., 1968. Veresh, S. A., F. S. Hom & J. J. Miskel: Spectrophotofluorometric determination of salicylamide in blood serum and urine. J. Pharm. Sci. 1971, 60, 1092-1095.
From the Department of Pharmacology, ACO Lakemedel AB, Box 3026, S-17103 Solna, Sweden, and Department of Pharmacology, Apotekens Centrallakoratorium, Apoteksbolaget AB, Box 3045, S-17103 Solna, Sweden
Effect of Stress on the Pharmacokinetics of Sodium Salicylate and Quinidine Sulphate in Rats* BY Uno Otto and Lennart Paalzow (Received October 29, 1974; Accepted December 6, 1974)
Abstract: Studies on the influence of foot shock stress on the absorption, distribution and overall serum elimination of sodium salicylate and quinidine sulphate were performed in rats. Foot shock stress was produced by grid floor electrical stimulation. The animals were stressed 1 hr before administration of the drugs and then throughout the experimental period. A significant induced stress was demonstrated by increased blood glucose levels and increased whole brain turnover of noradrenaline. Gastric emptying and intestinal transit as well as tissue levels of the drugs in the serum, heart, brain, liver, muscle and fat were determined. Results were obtained indicating that stress produced a significant increase in the gastro-intestinal absorption of quinidine sulphate while no change in the distribution pattern or overall elimination was noted. On the other hand, sodium salicylate did not show any marked change in the pharmacokinetic parameters during a stressful situation.
Key-words: Stress - intestinal absorption - sodium salicylate - quinidine.
A variety of physical and emotional stimuli can induce stress which is associated with physiological alterations of the homeostasis. It is also known or assumed that physiological and pathological factors can influence the kinetics of drug absorption, distribution, metabolism or elimination (for review see PRESCOTT & NIMMO 1971). Of these physiological processes the absorption of drugs from the gastro-intestinal tract depends on many factors, such as gastric emptying, intestinal motility, blood flow, pH, volume, composition and presence of food, viscosity, osmolarity, and the like (RIEGELMAN & ROWLAND 1973). Since a stressful situation can be expected to * A preliminary report of this investigation was presented at the XXVIII Meeting of the Scandinavian Pharmacological Society, Odense, 1974.
416
UNO OTI'O AND LENNART PAALZOW
produce changes in some of these factors, it is important to investigate further the kinetics of drugs under the influence of stress. Furthermore, many drugs modify physiological or pathological functions which can then bring about an interaction with the kinetics of other drugs given at the same time. We have therefore now studied the absorption and distribution of sodium salicylate and quinidine in the rat subjected to electrical foot shock stress. The drugs were chosen because of their physico-chemical character, being an acid and a base, respectively. Consequently, the absorption kinetics can be expected to be influenced differently by physiological changes in the gastro-intestinal tract. Materials and methods Male Sprague-Dawley rats, weighing 130-180 g, were used throughout the study. For 16 hrs before the test, the rats were allowed water ad libitum,but no food. Rats in groups of four were placed on a grid floor in a cage of 30 X 30 X 30 cm. Foot shock stress was produced by electrical stimulation (4OV, 125 Hz,1.6 msec. pulse width, 1 sec. duration given each 10 sec.) from a square wave stimulator (Kistner Instruments 421). To ensure that the animals were subjected to significant stress, the blood glucose level was determined enzymatically with a commercial glucose oxidase preparation (Kabi Reagents, Stockholm, Sweden). In another experiment, the whole brain level of noradrenaline and dopamine was determined (SHELLENBERGER & GORDON 1971) after inhibition of tyrosine hydroxylase with a-methyl-p-tyrosine methylester (H 44/68) given 4 hrs before death according t o A N D ~ et N al. (1966). The time course of drug serum concentrations was followed during 6 hrs after oral and subcutaneous administration of sodium salicylate and quinidine sulphate. The dose of the drugs were chosen as low as possible, allowing adequate chemical analysis. In the distribution studies the animals were killed by decapitation 1 hr after the oral or subcutaneous administration of the drugs, and the blood was collected. The brain or heart, liver, fat of testicles and muscle tissue (hind leg) were rapidly excised, blotted and weighed. The different tissues were homogenized with an Ultra-Turrax TP-1ON in 0.2 M sodium hydroxide for the tissues from the quinidine treated animals, and 1 M hydrochloric acid from animals given salicylate. Gastric emptying and intestinal transit were determined by using phenol red as a marker (REYNELL& SPRAY 1956). The rats were killed at intervals after administration as indicated in the results and the stomach and intestine were ligated a t the cardia, while the pylorus and the small intestine were ligated into three equal segments down to the ileocaecal sphincter. During evisceration care was taken to prevent displacement of the small intestinal contents owing t o differences in level. The determination of phenol red in the different parts of the gastro-intestinal canal and the calculation of gastric emptying and intestinal transit rate were made according to FELDMAN & GIBALDI (1%8).
All drugs administered to the animals were dissolved in physiological saline solution. Fluorometrical analysis of the quinidine levels in the tissues was performed according to CRAMBR & ISAKSSON(1963) and salicylic acid according to VF~ESH et al. (1971). The statistical calculations were performed according to FISHER (1954).
417
STRESS AND PHARMACOKINETICS
Table 1 . Changes in blood glucose levels in 8 control and stressed rats. ~
hr Control Stressed Difference from control t P
100 % 100 %
1 hr 2 hrs 3 hrs M e a n f S . E. M. M e a n f S . E. M. M e a n f S . E . M. 101 147
5% 10 %
47
11 % 4.155 0.001
0.8
102 82
5% 15 %
-20 15 % - 1.310 > 0.2
Results
Significance of induced stress. The electrical parameters used to induce a foot shock stress were fundamentally the same as those used to induce a pain reaction in animals (PAALZOW1969a&b; PAALZOW& PAALZOW 1973). Due to the higher resistance of the feet of the rats compared to the intracutaneous electrodes used in the above-mentioned studies, it was found in a pretest that a standardized voltage of 40 V given every 10 sec. during 1 hr before the administration of the drugs was sufficient to produce stress in the animals. Table 1 shows that animals subjected to foot shock stress of 40 V gave a significant increase of blood glucose level following 1 hr of stress. During the following 2 hrs the levels showed a tendency to
Table 2. The effect of foot shock stress on brain noradrenaline and dopamine after inhibition of catecholamine synthesis with H44/68 given 4 hrs before death. The number in brackets indicates the number of animals. Brain noradrenaline ng/g k S.E.M.
Brain dopamine n d g f S. E. M.
Controls
192 f 3 (n = 8)
311 k 14 (n = 9)
1 hr of stress
168 f 5 (n=6)
288 f 141) (n = 6)
2 hrs of stress
157 f 6 : ~ (n =6 )
285 f 7 b (n=6)
Treatment
3.
1)
Significantly different from controls (P < 0.01). Not significantly different from controls (P > 0.05).
UNO OTTO AND LENNART PAALZOW
418
decline although not significantly as compared to control rats placed on the grid that did not give electrical shocks. By blocking the catecholamine synthesis with a-methyl-p-tyrosine the rate of depletion of noradrenaline and dopamine should be proportional to the impulse flow within the neurons i. e. the higher activity, the greater the depletion (ANDBNet al. 1966). Table 2 shows that 1 and 2 hrs of foot shock stress caused an increased depletion of whole rat brain noradrenaline, indicating an increase in the turnover of this transmitter. There was no significant difference between control animals and stressed rats with regard to dopamine depletion induced by enzyme inhibitor. Serum levels of salicylic acid and quinidine in stressed and control animals. Following 1 hr of foot shock stress, groups of rats were given 100 mgkg sodium salicylate or 50 mgkg quinidine sulphate subcutaneously or orally and subjected to foot shock stress for the following 6 hrs during which period blood was drawn and analysed for salicylates and quinidine. The decrease in log salicylic acid and quinidine concentrations in serum with respect to time appeared linear in all animals 2 hrs after dosing, regardless of the drug given and mode of administration. The overall elimination rate constant in serum and corresponding half-life were estimated from the
r!
-
rJs,
4.0
'a
4.0 Quinidine p.0.
'ha,
2.0
1.0
1 .o
0.5
0.5
0.25
0.25 1
I
I
I
1
2
3
4
Control animals Stressed animals Quinidine
2.0
I
-----
,I
II
I
6 h
$
1
1
I
I
I
2
3
4
S.C.
I
6 h
Fig. 1. The semi-logarithmic plot of the serum levels of quinidine after oral and subcutaneous administration of 50 mg/kg quinidine sulphate to control and stressed rats. Each point represents the mean of 12 animals after oral and 4 animals after subcutaneous administration.
P
t
Diff. from control
Stressed
Control
83.927.1
85.858.9
Liver Irdg
-2.8k5.4 -0.518 > 0.6
20.5f3.1
23.3k4.4
Pug
Fat p.g/ml
Serum
5.5k7.5 -7.6k14.2 0.735 0.536 > 0.4 > 0.5
48.3k4.8 227.3f10.3
42.825.8 234.9k9.8
VdS
Musc!e
Oral administration
4.0k3.6 -1.9k11.4 1.118 -0.166 > 0.2 > 0.8
25.8k2.3 (n=8) 29.8f2.8 (n = 12)
Ildg
Brain CLdg
Liver
7.7k2.0 3.952 < 0.01
5.0t4.2 1.205 > 0.2
41.0f1.1 116.3k2.6 (n=8) 48.7k1.6 121.3k3.2 (n = 8)
P.g/P
Brain
3.9f2.8 1.400 > 0.1
38.1k2.4
34.2k1.4
pdg
Fat
p.g/ml
Serum
1.5Z6.5 0.232 > 0.8
4.2k10.8 0.389 > 0.7
71.7k5.5 246.7k6.8
70.2k2.9 242.5k8.4
IrdS
Muscle
Subcutaneous administration
Mean tissue levels k S. E. M. of salicylate in control and stressed rats respectively 60 minutes after oral and subcutaneous administration of 100 m g k g sodium salicylate.
Table 3.
~
c
82
$
z>
50
3
P
Diff. from control t
Stressed
Control
0.7
12.1k1.6
11.3f1.8
PdP
3.4f1.1 3.038 < 0.02
7.2f0.9
3.8k0.7
Pdg
>
3.2k0.2
3.5k0.3
Pdml
Serum
0.3f1.2 -0.3k0.3 0.268 -1.045 > 0.7 0.3
13.4f1.0
13.150.7
Pdg
Subcutaneous administration Liver Fat Muscle
Mean tissue levels f S. E. M. of quinidine in control and stressed rats respectively 60 minutes after oral and subcutaneous administration of 50 m d k g quinidine sulphate.
Table 4.
2
8
G
2>
e
$
z
E;; 2
U
5
0
3
0
0
C
0
h)
P
421
STRESS AND PHARMACOKINETICS
P
11
300
-------
lll
C o n t r o l animals Stressed animals
300
200
100
100 Salicylic a c i d p.0.
Salicylic a c i d
50
S.C.
50 I
I
f
4
3
I
I
4
6
I
h
I
I
4
3
1
I
4
6
h
Fig. 2. The semi-logarithmic plot of the serum levels of salicylic acid after oral and subcutaneous administration of 100 mg/kg sodium salicylate to control and stressed rats. Each point represents the mean of 8 animals after oral and 6 animals after subcutaneous administration.
log concentration-time curves using the data from 2-6 hrs. Figs. 1 and 2 show the mean log serum concentration-time curves of salicylic acid and quinidine after oral and subcutaneous administration. The mean serum half-life for salicylic acid was 8.7 f 1.7 hrs (S. E. M.) in control animals and 9.0 f 2.6 hrs in stressed animals after oral dosing and 8.4 ? 1.7 and 9.1 -I 2.3 hrs, respectively, after subcutaneous administration. There was no significant difference between these half-lives. After oral administration of quinidine, the half-life in control animals was estimated to be 1.7 f 0.8 hrs and in stressed animals to be 2.5 -I 1.1hrs. The corresponding values after subcutaneous administration were 1.8 .+ 0.2 hrs and 1.7 f 0.1 hrs, respectively. As for salicylic acid, there was no statistically significant difference between the half-lives for quinidine. However, the serum levels of quinidine after oral dosing were increased 2-3 fold in the stressed animals (fig. 2) and an analysis of variance showed that this increase was significant (F=24.9; d.f.= 142/1; P < 0.001). The mean serum levels of salicylate, on the other hand, showed slightly increased levels in stressed animals (fig. 1) and this small difference (13 yo) was statistically significant (F=5.91; d. f.=94/1; P < 0.05).
422
UNO OTTO A N D LENNART PAALZOW
Since the overall eliminations from serum of the two drugs studied were unchanged regardless of the mode of administration or whether there was any subjection to stress or not; this could be an indication of an unchanged distribution, elimination or metabolism during stress. The increased serum level of quinidine after oral administration could be a consequence of, for example, a changed degree of absorption or tissue distribution pattern. Therefore, in the following experiments the distribution pattern of the two drugs was investigated. Tissue levels of salicylic acid in stressed and control rats. Following 1 hr of foot shock stress, rats were given 100 mgkg sodium salicylate per 0s or subcutaneously and then stressed for the following 1 hr. Thereafter the animals were killed by decapitation and tissue levels of salicylic acid were determined. As can be seen in table 3, foot shock stress does not change the tissue level of salicylate regardless of the mode of administration, except in the brain where, after subcutaneous injection, an increased level was seen in stressed rats compared to controls. As a measure of the tissue distribution pattern, the ratio between tissue and serum levels was calculated from table 3, and in that respect no significant differences (P > 0.05) in the distribution of salicylate were obtained in the stressed animals compared to the controls after oral administration. However, surprisingly elevated ratios were found in the brain and fat of stressed animals after parenteral injection. Tissue levels of quinidine in stressed and control animals. As in previous experiments, rats were subjected to stress for 1 hr and then given 50 mgkg quinidine sulphate subcutaneously or orally and the foot shock stress was continued for the following hour. One hour after drug administration the rats were killed and the tissue levels of quinidine were determined in the different tissues. Contrary to the previous experiment with sodium salicylate, about 2-3 fold higher levels of quinidine (P = 0.01-0.001) were found in all tissues after oral administration of this drug to the stressed animals compared to controls (table 4). However, when quinidine sulphate was given subcutaneously, there was no significant difference in tissue levels between the stressed and control rats except for fat tissue. As in the experiment with salicylate, the ratio of tissue to serum level of quinidine was calculated. A significant increase (0.05 > P > 0.01) in liverserum ratio was obtained for stressed animals after oral administration and in the fat-serum ratio after subcutaneous injection. No other significant differences were found regardless of whether stress was given or not. Thus the studies of the tissue distribution pattern of salicylic acid and quinidine seem on the whole to indicate an unchanged distribution of these drugs under stress conditions. Gastric emptying and intestinal transit rate in stressed and control animals.
STRESS AND PHARMACOKINETICS
423
From the previous experiments it was evident that foot shock stress increases the serum and tissue levels of quinidine after oral administration but probably not the distribution pattern. Some explanations of these findings might be offered, e. g. increased absorption from the intestinal canal resulting from changed gastric emptying, intestinal motility or intestinal blood flow. In order to investigate possible changes in gastric emptying or intestinal transit rate in the stressed animals, phenol red was used as a marker. The rats were pre-stressed for 1 hr and then given phenol red orally; 30 and 60 min. later the stomach and intestine were eviscerated, segmented and analysed for phenol red. From table 5 it is apparent that gastric emptying of phenol red showed a tendency to increase in the stressed rats although no significant differences were noted. However, as for transit rate, a decreased rate in the stressed animals was noted compared to controls, and this was significant (P < 0.05). Discussion
The absorption characteristics of weak acids or bases from the alimentary tract has been particularly investigated by BRODIEand co-workers (e. g. SCHANKER 1971). Most drugs are absorbed as unionised molecules by passive diffusion. An important factor for the transfer is the lipid solubility of the unionized drug and thus the pH will determine the fraction of the
Table 5. Gastric emptying and intestinal transit of phenol red in control and stressed rats. The results are expressed as mean values f S. E. M. Time after administration
NO. of animals
Gastric emptying %
Intestinal transit % Segment 1 Segment 2
30 60
12 19
59.2.f 5.1 72.4f3.4
87.9 ? 1.7 94.2 f0.9
24.6 ? 5.8 78.7 f 3.1
30 60
10 12
64.5 f 5.7 76.5 +4.3
75.9 k 8.1 87.9 f 2.8
8.554.1 27.6 f 7.8
5.3 f 7.7 4.1.55.5
- 12.0k 8.3 -5.3+_2.9&
- 16.1 k 7.1% -51.1f8.4a
Control
Stressed
Difference from control 30 60 a
Significantly different from control, P
< 0.05.
424
UNO OTTO AND LENNART PAALZOW
drug present in this form. This means that weak acids will be fairly well absorbed from the stomach while weak bases will be readily absorbed from the alkaline environment of the small intestines. However, salicylic acid is also fairly well absorbed from the small intestines, this presumably being due to the relatively large surface area of the latter. The rate of gastric emptying and intestinal motility will thus be important factors for the absorption of drugs from the alimentary canal. Furthermore, drugs are primarily removed from this canal by blood flow, and a reduced flow, for example, may decrease the rate of removal of the drug absorbed by passive diffussion (RIEGELMAN & ROWLAND 1973). A stressful situation can be expected to produce changes in those three physiological functions, e. g. by an increased sympathetic activity (THOMAS & BALDWIN1968). The animals, subjected to foot shock stress, showed a significant increase in blood glucose levels, and a significant increased acceleration of the depletion of the whole brain noradrenaline, induced by the enzyme inhibition. CORRODIet al. (1968) have shown that immobilization stress activates various noradrenergic nerve terminal systems in the rat brain, and if the immobilization was made more complete they also found a decreased turnover of dopamine as well as an increased noradrenaline turnover (CORRODI et al. 1971). Furthermore, THIERRY et al. (1970) have demonstrated that foot shock stress of weak intensity and short duration brings about important modifications of the noradrenaline metabolism in the brain. Rats given foot shock stress and injected subcutaneously with sodium salicylate or quinidine sulphate, respectively, showed about the same serum and tissue levels of these drugs as control animals. However, if the drugs were given orally, quinidine was obtained in a significantly higher concentration in all tissues of the rats subjected to stress. Animals given salicylic acid showed no such marked differences. The time course of the serum levels of salicylic acid and quinidine indicated no change in the overall elimination rate constant of these drugs during stress. The elimination of these two drugs are highly dependent on the pH of the urine and possible stress induced changes in pH should in our experiment have been detected in the elimination rate constant. Furthermore, the stress did not seem to change the distribution ratio of the tissue to serum. However, the ratio of the liver to serum after oral administration of quinidine in the stressed animals was markedly elevated. This latter finding as well as that of the higher tissue levels of quinidine after oral administration to stressed animals, may be due to an increased absorption induced by a changed physiological activity of the gastro-intestinal tract, e. g. in gastric emptying, motility or blood flow. Subsequent experiments showed that the intestinal transit rate was decreased in the stressed animals and there was a tendency of increased gastric emptying. Since quinidine is a base these physiological conditions
STRESS AND PHARMACOKINETICS
425
may favour its absorption. An acid such as salicylic acid is not so highly dependent on these two physiological factors since it is well absorbed from both the stomach and the intestines. However, one possible explanation for the different behaviour of the two drugs is that salicylic acid is completely absorbed and increases of it can therefore not be traced. In conclusion, the present study indicates an increased gastro-intestinal absorption of quinidine during stress. The therapeutic significance of the 2-3 fold increase found in tissue quinidine levels during a stressful situation has yet to be evaluated in human subjects. Preliminary studies in our laboratories have also shown that a minor tranquilizer such as meprobamate can prevent the stress-induced elevation of quinidine levels in tissue.
REFERENCES Andtn, N.-E., H. Corrodi, A. Dahlstrom, K. Fuxe & T. Hokfelt: Effects of tyrosine hydroxylase inhibition on the amine levels of central monoamine neurons. Life Sci. 1966, 5, 561-568. Corrodi, H., K. Fuxe & T. Hokfelt: The effect of immobilization stress on the activity of central monoamine neurons. Life Sci. 1968, 7 , 107-112. Corrodi, H., K. Fuxe, P. Lidbrink & L. Olson: Minor tranquillizers. Stress and central catecholamine neurons. Brain Res. 1971, 29, 1-16. Cramtr, G. & B. Isaksson: Quantitative determination of quinidine in plasma. Scand. J. Clin. Lab. Invest. 1963, 15, 553-556. Feldman, S. & M. Gibaldi: Effect of bile salts on gastric emptying and intestinal transit in the rat. Gastroenterol. 1968, 54, 918-921. Fisher, R. A.: Statistical methods for research workers, 12th ed. Oliver & Boyd, Edinburgh, 1954. Paalzow, L.: An electrical method for estimation of analgesic activity in mice. Part I. Methodological investigations. Acta Pharm. Suecica 1969a, 6, 193-206. Paalzow, L.: An electrical method for estimation of analgesic activity in mice. Part 11. Application of the method in investigations of some analgesic drugs. Acta Pharm. Suecica 1969b, 6, 207-226. Paalzow, G. & L. Paalzow: The effects of caffeine and theophylline on nociceptive stimulation in the rat. Acra pharmacol. et toxicol. 1973, 32, 22-32. Prescott, L. F. & J. Nimmo: Drug therapy. Physiological considerations. J . Mond. Pharm. 1971, 14, 253-260. Reynell, P. C. & G. H. Spray: The simultaneous measurement of absorption and transit in the gastro-intestinal tract of the rat. J. Physiol. (London) 1956, 131, 452-462. Riegelman, S. & M. Rowland: Effect of route of administration on drug disposition. J. Pharmacokin. Biopharm. 1973, 1, 419-434. Schanker, L. S.: Drug absorption. In: Fundamentals of drug metabolism and drug disposition. Eds.: B. N. LaDu, H. G. Mandel & E. L. Way. Williams & Wilkins Company, Baltimore, 1971. Shellenberger, M. K. & J. H. Gordon: A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas. Anulytical Biochemistry 1971, 39, 356-372.
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Thierry, A.-M., G. Blanc & J. Glowinski: Preferential utilization of newly synthesized norepinephrine in the brain stem of stressed rats. Eur. J . Pharrnacol. 1970, 10, 139-142. Thomas, J. E. & M. V. Baldwin: Pathways and mechanisms of regulation of gastric motility. In.: Handbook of Physiology. Section 6. Alimentary Canal. Ed.: C. F. Code. American Physiological Society, Washington D. C., 1968. Veresh, S. A., F. S. Hom & J. J. Miskel: Spectrophotofluorometric determination of salicylamide in blood serum and urine. J. Pharm. Sci. 1971, 60, 1092-1095.
