Xenobiotica the fate of foreign compounds in biological systems
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Influence of Route of Administration on 14 Metabolism of [ C]Nicotine in Four Species David M. Turner To cite this article: David M. Turner (1975) Influence of Route of Administration on Metabolism 14
of [ C]Nicotine in Four Species, Xenobiotica, 5:9, 553-561 To link to this article: http://dx.doi.org/10.3109/00498257509056125
Published online: 14 Oct 2008.
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Date: 07 November 2015, At: 12:04
XENOBIOTICA,
1975, VOL. 5,
NO.
9, 553-561
Influence of Route of Administration on Metabolism of [14C]Nicotinein Four Species DAVID M. TURNER* Tobacco Research Council Laboratories, Otley Road, Harrogate, Yorkshire, U.K.
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(Received 28 December 1974)
1. The metabolism of [14C]nicotine has been studied in four species of animals, rabbit, rat, cat and squirrel monkey, after administration by different routes. 2. Intravenous injection of 4 pg/kg [14C]nicotine every 60 s for 1 h results in peak blood levels of approximately 100 ng/ml in all species but the rabbit. [14C]Cotininelevels in blood vary widely between species. 3. Subcutaneous injection of 0.4 mg/kg [14C]nicotine produces similar peak blood nicotine levels but the time course, for a given species, is different. 4. Intragastric instillation of 1 mg/kg [14C]nicotineto the cat and rabbit results in much lower levels of ['*C]nicotine in blood and relatively high levels of [14C]cotinine 5. Urinary excretion data indicate that, irrespective of route, the squirrel monkey excretes only a small proportion of the dose into urine during the period of experiment, of which the major proportion is [14C]nicotine. The cat, in contrast, excretes a relatively large proportion of the dose during the experimental period though only a minor proportion of the radioactivity is due to [14C]nicotine or ['*C]cotinine. 6 . All four species are potentially useful for model experiments with nicotine, though metabolism of nicotine by squirrel monkey is most similar to man.
.
Introduction Much is known concerning the principal pathways by which nicotine is metabolized in many animal species including man (Bowman, Turnbull & McKennis, 1959 ; McKennis et al., 1962 ; McKennis, Schwartz & Bowman, 1964) and many metabolites of the drug have been identified (Papadopoulos, 1964 ; Dagne & Castagnoli, 1972 ; Murphy, 1973). T h e principal aim of the majority of in v i m studies on nicotine has been to identify metabolites, and less attention has been given to correlating the blood and tissue levels of the drug with its pharmacological effects. Though some tissue distribution studies have been made (Ganz, Kelsey & Geiling, 1951 ; Fishman, 1963 ; Turner, 1969 ; Nakashima, 1972), the use of different species and different routes of administration makes correlation of data difficult. I n order to relate some aspect of nicotine pharmacology or biochemistry to smoking in man, the way the nicotine is administered, the dose and the distribution must bear some resemblance to that which occurs in man. It has been established (Armitage, 1965) that a series of suitably chosen intravenous injections of nicotine can mimic closely the pharmacological effects of intermittent
* Address correspondence to : Department of Biochemistry and Drug Metabolism, Hazleton Laboratories Europe Limited, Otley Road, Harrogate HG3 lPY, North Yorkshire, U.K. X.B.
20
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D. M . Turner
puffs of tobacco smoke. Thus a series of intravenous injections-usually 2-4 pg/kg every 30 or 60 s (Armitage, Hall & Morrison, 1968)-provides an acceptable substitute in terms of pharmacological effects and blood levels for inhaled nicotine in man (Armitage et al., 1974 a) and animals (Turner, 1970). There are limitations on its use for all types of study, however, not the least being the technical difficulties of cannulation encountered with some experimental animals, particularly when chronic dosage studies are planned. Numerous investigators have instead used the subcutaneous (Armitage et al., 1968 ; Rosecram & Schechter, 1972 ; Fisher et al., 1973) or intraperitoneal (Yamamoto, Inoki & Iwatsubo, 1968) routes, and yet others have administered nicotine in drinking water (Wenzel & Broadie, 1966 ; Schievelbein et al., 1970 ; Ruddon & Cohen, 1970). T h e purpose of this investigation was to compare the blood levels of [14C] nicotine and its principal metabolite [14C]cotinine in the squirrel monkey, the rabbit, the rat and the cat, after administration of [14C]nicotine by different routes. All four species have been used by various workers to study the effects of nicotine in relation to smoking.
Methods Nine male cats weighing 2.3-3.0 kg, bred in these laboratories, were used. Three were anaesthetized by intraperitoneal injection of diallyl barbitoneurethane mixture (Dial) at a dose of 0.8 ml/kg. Cannulae were inserted in the left femoral vein and the right carotid artery. T h e animals were given a series of intravenous injections of [14C]nicotine at a dose of 4 pg/kg every 60 s for 1 h, each in a volume of 0.3 ml. Blood samples were taken over this period and for a further 3 h. Three animals were similarly anaesthetized and cannulated but a dose of 0.4 mg/kg was given subcutaneously in the region of the upper left groin. T h e other three cats were anaesthetized with fluothane and a femoral arterial cannula as well as a femoral venous cannula implanted with exteriorization on the skull via two stainless steel valves (Hall, Gomersall & Heneage, 1968). After recovery from the surgery (approx. 2 weeks) each animal was anaesthetized with the smallest amount of intravenous thiopentone sodium and [14C]nicotine administered at a dose of 1 mg/kg in a total volume of 10 ml saline by tube directly into the stomach. Blood samples were taken over 4 h following nicotine administration. T h e animals were conscious for all but a few minutes of that time. T e n male hooded Lister rats, obtained from Oxfordshire Laboratory Animal Colonies, Bicester, England, weighing 3 5 0 4 5 0 g were anaesthetized with an intraperitoneal injection of 60 mg/kg pentobarbitone sodium and cannulae implanted in a femoral vein and a carotid artery. Four rats received [14C]nicotine at a dose of 4 pg/kg every 60 s for 1 h via the venous cannula. The volume of each injection was 10 pl. A further three animals were given [14C]nicotine in a dose of 0.4 mg/kg subcutaneously in the region of the upper left groin and the remaining three given 1 mg/kg [14C]nicotine via a blind-ended cannula inserted down the oesophagus into the stomach. T h e volume of subcutaneous injection was 0.4 ml/kg in buffered saline at p H 7.4 and the volume of each intragastric injection was 0.5 ml.
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iWetabolism of Nicotine
555
Six male squirrel monkeys weighing 750-1100 g, obtained from Tarpon Springs Zoo, Florida, U.S.A. were anaesthetized by intraperitoneal injection of 0.8 ml/kg Dial. T h e right femoral artery was cannulated for withdrawal of blood and for intravenous experiments the [14C]nicotine administered via a left femoral vein cannula. For subcutaneous experiments the [14C]nicotine was injected in the area of the left groin. T h e dosage volumes were the same as for the rat. Nine male New Zealand White rabbits weighing 1.9-2-2 kg obtained from H & E Rabbitry, Chadderton, Lancashire, England, were anaesthetized by intravenous injection of 25% w/v urethane (6 ml/kg). T h e right femoral artery was cannulated. Three animals received 4 pg/kg [14C]nicotine intravenously and the same number were given 1 mg/kg [14C]nicotine by catheter directly into the stomach in a total volume of 5 ml physiological saline. A further three animals were given a subcutaneous injection of 0.4 mg/kg [14C]nicotine in 1 ml buffered saline. I n all the animal experiments, except for the rats, after each blood sample, a similar amount of plasma substitute (Intradex ; Glaxo Laboratories, Greenford, Middlesex) was given intravenously. Rats were given a similar volume of saline. I n a majority of animals, at the end of each experiment the bladder contents were removed and the radioactivity analysed. Measurement of [l4C]nicotine and [14C]cotinine in blood was made using the method of Turner (1969). Blood samples (1 ml) were assayed for the cat, monkey and rabbit experiments and 0.5 ml samples (diluted 1 in 3 with saline) of rat blood were extracted. All samples were counted in a Model 3375 liquid scintillation spectrometer (Packard Instrument Ltd., Wembley, England) using a modified Bray solution (Evans, 1961) at a maximum efficiency of 83% and to 1 % accuracy. T h e L-( - )-[2’-14C]nicotine hydrogen tartrate at a specific activity of 19 mCi/mmol was synthesized (Decker, 1964) and resolved in these laboratories by Dr. T. H. Houseman.
Results Intravenous nicotine Table 1 shows the results for each species. T h e peak level of [14C]nicotine in the blood in rabbit, monkey and rat occurred at the end of injection and the levels in all three species were of the order of 100 ng/ml. T h e peak nicotine level in the rabbit was slightly less. I n the cat, however, an equilibrium level appeared to have been attained by 20-30 min. T h e decline in blood [14C]nicotine levels after end of injection was rapid in the cat and rabbit, much slower in the monkey and slower still in the rat. The time course of the blood levels of [l*C]cotinine in the four species shows wide variation. I n the cat, maximum blood [14C]cotinine levels occurred between 60 and 90 min whereas in the other species [14C]cotinine levels were greater at 120 min than at 60 min. I n the rat, even after 4 h [14C]cotinine levels were higher than after 2 h but metabolism to cotinine was slow, no significant blood level of cotinine being detectable until 20 min after start of injections. In the rabbit, cotinine was detectable after 5 min. T h e squirrel monkey produced extremely high levels of [14C]cotinine in the blood with a ratio of cotinine to nicotine of 2.5 : 1 after 120 min. T h e rabbit also showed high cotinine levels. 202
Rabbit (3)
Rat (4)
Cat (3)
25.4 f 7.4 8.0 f4.6 39.6 f4.5 2.4 ? 0.6 22.6 f 6.1 0 11.6 k 2.1 5.6 f 1.4
Squirrel monkey (3)
Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine
5
Species
20
90
120
65.0 6.8 97.6 5 15.6 166.1 f 16.2 154.0 f 8.4 30.6 f 3.9 106.2 k 2.7 45.9 i4.2 38.1 k 2.7 36.3 f 5.7 29.4 f 5.4 75.8 f4.4 106.6 f 1.8 16.4+ 6.1 25.0 5.2 78.1 rfI 6.9 30.5 f4.4 110.7 f 7.7 120.2 f 7.6
Time (min) 30 60 (ng/ml blood)
Numbers of animals are referred to in parentheses.
59.6 f 22.8 81.2 f 24.8 18.2 f 3.8 52.2 f 7.2 56.4 f 6.6 90.3 + 9.6 103.2 f 8.4 6.9 f 1.2 15.3 i 1.2 27.9f 1.5 50.2 k 8.0 82.2 f 12.2 0 3.2 f 1.2 34.2 f 3.6 52.4 f4.6 17.1 f 2.6 26-6 f 3.1
10
Results are expressed as ng/ml blood f SEM.
38.6 f4.6 148.8 f 14.4 15.6 f 5.1 17.7 f 3 . 0 57.2 f0.6 36.4 k 9.4
240
Table 1. Blood levels of [14C]nicotineand [14C]cotinineafter intravenous administration of 4 pg/kg [l*C]nicotineevery 60 s for 60 min
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Y
2
F
Y
g
P
m
fn 3
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Metabolism of Nicotine
557
Subcutaneous nicotine Table 2 shows that the peak blood levels were different in each species and the times at which these maxima occur were different. T h e peak levels for a given species, however, were very similar to those obtained after intravenous injection. T h e rise in [14C]nicotine in the squirrel monkey was very fast with a maximum at 10 min. T h e rat was somewhat slower but the peak level was maintained for about 40 min. T h e cat reached a maximum after 30 rnin and the rabbit at about 20 min. [14C]Cotinine levels reached their maximum in the cat after 60 min after which time they declined very slowly. I n the rat and squirrel monkey [14C]cotinine levels appeared to increase over the course of experiment, and in the rabbit the levels were stable by 60 min. As with intravenous injection, the cotinine/nicotine ratio at the end of the experiments was high in both monkey and rabbit though cotinine levels were higher in these two species after subcutaneous injection towards the end of experiment than after intravenous nicotine. Intragastric nicotine T h e data following administration of 1 mg/kg [14C]nicotine by intragastric instillation to the cat, the rat and the rabbit are shown in Table 3. I n the unanaesthetized cats the blood nicotine levels reached a peak at 40 min as did cotinine levels but at all times cotinine levels were higher than nicotine. I n the anaesthetized rabbit, similar results were obtained as regards nicotine levels but much more cotinine was produced. T h e ratio of cotinine to nicotine in blood after 4 h being almost 70 : 1. I n the rat, however, the levels of [14C]nicotine were significantly elevated 20 rnin after administration and increased until 60 min, after which time they levelled off. [14C]Cotinine levels increased steadily over 2 h exceeding the [14C]nicotine levels at that time. Only in the rat did [14C]nicotine levels approach and exceed those obtained using other routes of administration. T h e peak blood levels of [14C]nicotinein both rabbit and cat, at this dosage, were about one quarter of those obtained when nicotine was given by the other routes. Urinary excretion Urinary excretion data are illustrated in Table 4. p H was not controlled but did not vary widely within an individual species. Rabbit urine tended to be more acidic than the other three species. T h e volumes of bladder contents varied widely. Rabbits and cats tended to yield bladder urine volumes of 2030 ml whereas both rat and squirrel monkey had urinary volumes of 0.5-1.2 ml. T h e urinary excretion pattern of radioactivity was very different in each species. T h e squirrel monkey excreted the lowest proportion of the injected dose in both intravenous and subcutaneous experiments and the proportions of [14C]nicotine in those urines were larger than in any of the other species. For the subcutaneous and intravenous experiments the proportion of [14C]nicotine in urine was always greater than [14C]cotinine. I n the intragastric instillation experiments, for rabbits, the reverse was true. T h e overall urinary recoveries of radioactivity were much lower after intragastric administration than by other
Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine
70.8 f 20.5 8.6 f0.7 22.0 f 5.3 0.4 f0.2 88.0 f4.5 2.1 f 1.1 13.5 f 3.5 10.9 f 2.9
110.4f 13.6 43.2 f 8.6 51.2 f 5.9 2.8 f 1.7 91.3 f 2.9 11.5 f 3.1 36.0 f 17.2 23.2 f 8.9
10
+
93.4+ 10.1 134.9 13.3 75.1 f 8.5 8.2 f 3.6 101.9 f 8.5 19.9 ri: 3.6 80.2 f 9.0 81.3 f7.7 77.8 f 7.9 20.4 +_ 4.9 96.7 6.5 30.2 f 3.1
Time after dosage (min) 20 30 (ng/blood)
48.0 f4.0 226.4 & 19.3 72.5 & 9.8 32.1 f 2.2 91.4f3.6 38.7 f0.3 41.7 f 36.5 212.5 ri: 13.7
60
21.4f 1.3 253.8 k 22.2 36.41- 15.4 30.1 f4.9 73.5 f 1.4 51.1 f 10.2 34.5 f 38.3 194.7 f 14.2
120
Rat (3)
Rabbit (3)
Cat (3)
Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine
0 8.8 f4.7 3.8k0.2 1.4k0.7
5 20
120
240
24.6 f 6.7 7.2 f 1.6 3.7 f 0.8 31.2f1.9 13.7k3.0 5.2f1.3 23.7 f 3.9 17.1 f 3.5 5.3 f 0.7 373.5 f 38.6 343.9 f40.7 341.1 f 23.4 144.8f6.9 153.1 k4.7 154.7ri:ll.O 123.0f19.1 136.6f23.3 168.1 f27.9
25.4 f 5.2 43.6k5.2
Time after dosage (min) 40 60 (ng/ml blood)
15.3 f 3.2 23.5 2 2.0 5.0 f 2.1 26.3 4.4 54.4 f 19.6 289.9 f 9.0 29.0f8.8 90.1f19.1 15.4f3.8 55.6f5.2
10
Results are expressed as ng/ml blood f SEM.
Table 3. Blood levels of [14C]nicotineand [14C]cotinineafter administration of 1 mg/kg [l*C]nicotine by intragastric intubation
Rabbit (3)
Rat (3)
Cat (3)
Squirrel monkey (3)
5
Results are expressed as ng/ml blood f SEM.
Table 2. Blood levels of [14C]nicotineand [laC]cotinine after subcutaneous administration of 0.4 mg/kg [14C]nicotine
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ul
3
3
2
b
00
ul
559
Metabolism of Nicotine
Table 4. Urinary excretion of [14C]nicotine and [14C]cotinine after administration of ['*C]nicotine Results are expressed as means f SEM.
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pH of urine
%
dose excreted
%
%
[14C]nicotine
[14C]cotinine
Intraoenous : 4 pg/kg [14C]nicotineevery 60 min. 6.6 0.1 Squirrel monkey (3) 7.9 f 2.7 Cat (3) 6.5 f 0.3 44.6 k 2.7 Rat (3) 6.4 & 0.2 37.5 f 3.9 Rabbit (3) 6.3 k 0.2 37.1 k 4-7
74-6 f6.0 12.1 f 3.9 23.8 f 1.7 42.8 k 3.2
10.0 f 3.0 1.2f0.5 2.2 f 0.2 18.0fl-6
Subcutaneous : 0.4 mg/kg [14C]nicotine* 6.6 k 0.2 4.1 & 0.7 Squirrel monkey (3) 6.6 f 0.1 Cat (3) 23.6 f 1.8 Rat (3) 6.5 f 0.3 12.6 f 5.6 6.4 f 0.1 Rabbit (3) 25.0 f 3.4
66.7 k 4.7 8.3 f 2.2 18.6 f 3.3 31.5 k 240
2.6 f 2.2 0.5 f0-3 2.0 f 0-5 10.3 f 1.0
Intragustric : 1 mg/kg [14C]nicotine. 6.6 0.1 Rat (3) Rabbit (3) 6.2 k 0.1
17.1 f 3.1 12.0 f 2.6
10.4 f 2.0 30.1 f9.4
6.6 f 3.6 16.5 & 142
* Urine sampled 2 h after start of experiment. two routes. T h e cat excreted a higher proportion of the injected dose than other species irrespective of the route but the proportions of [14C]nicotine and [14C]cotinine were small. I n all species the mean percentage dose excreted was lower after subcutaneous injection but the time of sampling was different (2 h for subcutaneous [14C]nicotine, 4 h for intravenous [14C]nicotine).
Discussion The experiments described have shown some remarkable species similarities as well as differences in response to [14C]nicotine administration by different routes. I n all but the rabbit the serial intravenous injections produced virtually identical peak blood levels of [14C]nicotine (approx. 100 mg/ml) though the [14C]cotinine levels differed very widely. Since nicotine is believed to be the principal, central, pharmacologically active agent in tobacco smoke (Knapp & Domino, 1962 ; Yamamoto & Domino, 1965 ; Clarke, Rand & Vanov, 1965) and since such nicotine levels are very similar at comparable times to those obtained in man during smoking (Armitage et al., 1974 a), the squirrel monkey, cat and rat may be considered as useful model animals for pharmacological studies on nicotine. However, known species differences in sensitivity to the effects of nicotine may put additional constraints on the choice of a suitable animal model. T h e squirrel monkey is, for example, much more sensitive than the cat and the rat to some of the effects of nicotine (A. K. Armitage, unpublished observations). By constrast, the rabbit is very insensitive to nicotine in relation to plasma lipid changes (D. L. Topping, unpublished observations). T h e proportion of
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D . M . Turner
cotinine in human smokers’ blood after heavy smoking is believed to be large relative to nicotine (Langone, Gjika & Van Vunakis, 1973) and in this respect the blood profile produced by the squirrel monkey is perhaps closest of all to the human situation. Subcutaneous injection of 0.4 mg/kg in all four species produced a rapid rise in blood [14C]nicotine which reached the intravenous peak in rabbit, rat and squirrel monkey. T h e time at which this peak was attained however varied widely. I n the rat, for example, blood [14C]nicotine levels were approaching peak levels 5 min after injection, consistent with the rapid onset of behavioural changes observed after this dose, by Morrison and Stephenson (1972). [14C]Cotinine in blood, in general reached somewhat higher levels than at comparable times after intravenous injection. Intragastric instillation of 1 mg/kg [14C]nicotine, which is a route comparable to the administration of nicotine in drinking water at dose levels derived from data of Johnston (1942), resulted in very low blood levels of [14C]nicotine and relatively high levels of [14C]cotinine in both cat and rabbit. This presumably represents a ‘first pass’ effect of the liver. I n the rat, however, high levels of [14C]nicotine were obtained 20 min after nicotine administration, though [14C]cotinine levels were also high at this time. This is consistent with the relatively reduced rate of nicotine metabolism observed in this species and, even with the ‘first pass’ effect, oxidation at least, is slower than in the other two species examined. I n the cat it would appear that the further metabolism of cotinine is rapid since N-oxidation, which could represent an alternative metabolic route is not inordinately high (D. M. Turner, unpublished observations). T h e blood levels in the other three species indicate that C-oxidation is a significant route of nicotine metabolism and in the squirrel monkey, it appears to be the dominant one. If experimental data are to be interpreted in a smoking context then it would seem reasonable that the blood levels of the nicotine should be as closely similar, both in terms of the maximum actual concentration and the time course of distribution, to those in man (Isaac & Rand, 1972). We have recently shown that the blood levels of nicotine achieved in inhaling cigarette smoke may be mimicked closely by a suitable series of intravenous injections both in man (Armitage et al., 1974 a) and the cat (Turner, 1970 ; Armitage et al., 1974 b ) and therefore in model experiments, unless smoke inhalation is employed, the preferred route for administration is by intravenous injection. Subcutaneous administration in all four species appears to be a reasonable alternative and the dose of 0.4 mg/kg would appear to be equivalent in terms of blood peak levels to 4 pg/kg intravenous for 20 min. Intragastric instillation of nicotine appears to be less useful at least in the cat and rabbit, and so administration of nicotine via drinking water, which would result in a slow release of the compound into the hepatic portal circulation, might be expected to produce low levels of nicotine unless very large doses were given. Though nicotine is considered to be the principal pharmacological active agent of smoke, it is not yet known whether any metabolites are biochemically active (Gorrod et al., 1974) and therefore, in a particular animal model the presence of either large or small amounts of metabolites may make interpretation of data in terms of nicotine alone, and by extrapolation human smoking, difficult.
Metabolism of Nicotine
561
Acknowledgments I am grateful to Mrs J. Bedwell and Mr. P. S. B. Minty for their skilled technical assistance.
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References ARMITAGE, A. K. (1965). BY.J . Pharmac., 25, 515. ARMITAGE, A. K., HALL,G. H. & MORRISON, C. F. (1968). Nature, 217,331. C. F., HOUSEMAN, T. H., LEWIS,P. J. &TURNER, ARMITAGE, A. K., DOLLERY, C. T., GEORGE, D. M. (1974 a). BY.J. clin. Pharmac., 1, 18OP. ARMITAGE, A. K., HOUSEMAN, T. H. & TURNER, D. M. (1974 b). Q. J l . exp. Physiol., 59,55. BOWMAN, E. R., TURNBULL, L. B. & MCKENNIS, JR., H. (1959). J. Pharmac. exp. They., 127, 92. CLARK,M. S. G., RAND,M. J. & VANOV, S. (1965). Archs int. Pharmacodyn. They., 156, 363. DAGNE, E. & CASTAGNOLI, JR., N. (1972). J. med. Chem., 15, 840. DECKER, K. (1964). Proceedings of the Symposium on Biomedical Applications of Labelled Molecules, Venice, 38. EVANS, E. A. (1961). In Tritium and its Compounds, p. 221, London : Butterworths. FISHER, E. R., ROTHSTEIN, R., WHOLEY, M. H. & NELSON, R. (1973). Archs Path., 96, 298. FISHMAN, S. S. (1963). Archs int. Pharmacodyn. T k . , 145, 123. F. E. & GEILING, E. M. K. (1951). J. Pharmac. exp. They., 103, 209. GANZ,A., KELSEY, P., KEYSELL, G. R. & MIKHAEL, B. R. (1974). J . natl. Cancer Inst., GORROD, J. W., JENNER, 52, 1421. J. C. R. & HENEAGE, E. (1968). Physiol. Behav., 3, 20. HALL,G. H., GOMERSALL, ISAAC, P. F. & RAND,M. J. (1972). Nature, 236, 308. L. M. (1942). Lancet, ii, 742. JOHNSTON, KNAPP,D. E. & DOMINO, E. F. (1962). Int. J . Neuropharmac., 1, 33. LANGONE, J. J., GJIKA,H. B. & VANVUNAKIS, H. (1973). Biochemistry, 12, 5025. S. L. & BOWMAN, E. R. (1964). J. biol. Chem., 239, 3990. MCKENNIS, JR. H., SCHWARTZ, MCKENNIS, JR. H., TURNBULL, L. B., SCHWARTZ, S. L., TAMAKI, E. & BOWMAN, E. R. (1962). J. biol. Chem., 237, 541. J. A. (1972). B Y .J . Pharmac., 46, 151. MORRISON, C. F. & STEPHENSON, MURPHY, P. J. (1973). J. biol. Chem., 248, 2796. T. (1972). Folia pharmac. japon., 68, 29. NAKASHIMA, N. M. (1964). Archs Biochem. Biophys., 106, 182. PAPADOPOULOS, ROSECRANS, J. A. & SCHECHTER, M. D. (1972). Archs int. Pharmacodyn. They., 196, 46 RUDDON, R. W. and COHEN,A. M. (1970). Toxic. appl. Phurmac., 16, 613. V., GRUMBACH, H., REMPLIK,V. & SCHAUER, A. (1970). SCHIEVELBEIN, H., LONDONG, 2. klin. Biochem., 8, 190. TURNER, D. M. (1969). Biochem. J . , 115, 889. TURNER, D. M. (1970). B Y .J. Pharmac., 41, 521. WENZEL, D. G. & BROADIE, L. L. (1966). Toxic. appl. Pharmac., 8, 455. YAMAMOTO, I., INOKI,R. & IWATSUBO, K. (1968). Toxic. appl. Pharmac., 12,560. E. F. (1965). I n t . J . Neurophurmc., 4, 359. YAMAMOTO, K. & DOMINO,
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Influence of Route of Administration on 14 Metabolism of [ C]Nicotine in Four Species David M. Turner To cite this article: David M. Turner (1975) Influence of Route of Administration on Metabolism 14
of [ C]Nicotine in Four Species, Xenobiotica, 5:9, 553-561 To link to this article: http://dx.doi.org/10.3109/00498257509056125
Published online: 14 Oct 2008.
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Date: 07 November 2015, At: 12:04
XENOBIOTICA,
1975, VOL. 5,
NO.
9, 553-561
Influence of Route of Administration on Metabolism of [14C]Nicotinein Four Species DAVID M. TURNER* Tobacco Research Council Laboratories, Otley Road, Harrogate, Yorkshire, U.K.
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(Received 28 December 1974)
1. The metabolism of [14C]nicotine has been studied in four species of animals, rabbit, rat, cat and squirrel monkey, after administration by different routes. 2. Intravenous injection of 4 pg/kg [14C]nicotine every 60 s for 1 h results in peak blood levels of approximately 100 ng/ml in all species but the rabbit. [14C]Cotininelevels in blood vary widely between species. 3. Subcutaneous injection of 0.4 mg/kg [14C]nicotine produces similar peak blood nicotine levels but the time course, for a given species, is different. 4. Intragastric instillation of 1 mg/kg [14C]nicotineto the cat and rabbit results in much lower levels of ['*C]nicotine in blood and relatively high levels of [14C]cotinine 5. Urinary excretion data indicate that, irrespective of route, the squirrel monkey excretes only a small proportion of the dose into urine during the period of experiment, of which the major proportion is [14C]nicotine. The cat, in contrast, excretes a relatively large proportion of the dose during the experimental period though only a minor proportion of the radioactivity is due to [14C]nicotine or ['*C]cotinine. 6 . All four species are potentially useful for model experiments with nicotine, though metabolism of nicotine by squirrel monkey is most similar to man.
.
Introduction Much is known concerning the principal pathways by which nicotine is metabolized in many animal species including man (Bowman, Turnbull & McKennis, 1959 ; McKennis et al., 1962 ; McKennis, Schwartz & Bowman, 1964) and many metabolites of the drug have been identified (Papadopoulos, 1964 ; Dagne & Castagnoli, 1972 ; Murphy, 1973). T h e principal aim of the majority of in v i m studies on nicotine has been to identify metabolites, and less attention has been given to correlating the blood and tissue levels of the drug with its pharmacological effects. Though some tissue distribution studies have been made (Ganz, Kelsey & Geiling, 1951 ; Fishman, 1963 ; Turner, 1969 ; Nakashima, 1972), the use of different species and different routes of administration makes correlation of data difficult. I n order to relate some aspect of nicotine pharmacology or biochemistry to smoking in man, the way the nicotine is administered, the dose and the distribution must bear some resemblance to that which occurs in man. It has been established (Armitage, 1965) that a series of suitably chosen intravenous injections of nicotine can mimic closely the pharmacological effects of intermittent
* Address correspondence to : Department of Biochemistry and Drug Metabolism, Hazleton Laboratories Europe Limited, Otley Road, Harrogate HG3 lPY, North Yorkshire, U.K. X.B.
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puffs of tobacco smoke. Thus a series of intravenous injections-usually 2-4 pg/kg every 30 or 60 s (Armitage, Hall & Morrison, 1968)-provides an acceptable substitute in terms of pharmacological effects and blood levels for inhaled nicotine in man (Armitage et al., 1974 a) and animals (Turner, 1970). There are limitations on its use for all types of study, however, not the least being the technical difficulties of cannulation encountered with some experimental animals, particularly when chronic dosage studies are planned. Numerous investigators have instead used the subcutaneous (Armitage et al., 1968 ; Rosecram & Schechter, 1972 ; Fisher et al., 1973) or intraperitoneal (Yamamoto, Inoki & Iwatsubo, 1968) routes, and yet others have administered nicotine in drinking water (Wenzel & Broadie, 1966 ; Schievelbein et al., 1970 ; Ruddon & Cohen, 1970). T h e purpose of this investigation was to compare the blood levels of [14C] nicotine and its principal metabolite [14C]cotinine in the squirrel monkey, the rabbit, the rat and the cat, after administration of [14C]nicotine by different routes. All four species have been used by various workers to study the effects of nicotine in relation to smoking.
Methods Nine male cats weighing 2.3-3.0 kg, bred in these laboratories, were used. Three were anaesthetized by intraperitoneal injection of diallyl barbitoneurethane mixture (Dial) at a dose of 0.8 ml/kg. Cannulae were inserted in the left femoral vein and the right carotid artery. T h e animals were given a series of intravenous injections of [14C]nicotine at a dose of 4 pg/kg every 60 s for 1 h, each in a volume of 0.3 ml. Blood samples were taken over this period and for a further 3 h. Three animals were similarly anaesthetized and cannulated but a dose of 0.4 mg/kg was given subcutaneously in the region of the upper left groin. T h e other three cats were anaesthetized with fluothane and a femoral arterial cannula as well as a femoral venous cannula implanted with exteriorization on the skull via two stainless steel valves (Hall, Gomersall & Heneage, 1968). After recovery from the surgery (approx. 2 weeks) each animal was anaesthetized with the smallest amount of intravenous thiopentone sodium and [14C]nicotine administered at a dose of 1 mg/kg in a total volume of 10 ml saline by tube directly into the stomach. Blood samples were taken over 4 h following nicotine administration. T h e animals were conscious for all but a few minutes of that time. T e n male hooded Lister rats, obtained from Oxfordshire Laboratory Animal Colonies, Bicester, England, weighing 3 5 0 4 5 0 g were anaesthetized with an intraperitoneal injection of 60 mg/kg pentobarbitone sodium and cannulae implanted in a femoral vein and a carotid artery. Four rats received [14C]nicotine at a dose of 4 pg/kg every 60 s for 1 h via the venous cannula. The volume of each injection was 10 pl. A further three animals were given [14C]nicotine in a dose of 0.4 mg/kg subcutaneously in the region of the upper left groin and the remaining three given 1 mg/kg [14C]nicotine via a blind-ended cannula inserted down the oesophagus into the stomach. T h e volume of subcutaneous injection was 0.4 ml/kg in buffered saline at p H 7.4 and the volume of each intragastric injection was 0.5 ml.
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Six male squirrel monkeys weighing 750-1100 g, obtained from Tarpon Springs Zoo, Florida, U.S.A. were anaesthetized by intraperitoneal injection of 0.8 ml/kg Dial. T h e right femoral artery was cannulated for withdrawal of blood and for intravenous experiments the [14C]nicotine administered via a left femoral vein cannula. For subcutaneous experiments the [14C]nicotine was injected in the area of the left groin. T h e dosage volumes were the same as for the rat. Nine male New Zealand White rabbits weighing 1.9-2-2 kg obtained from H & E Rabbitry, Chadderton, Lancashire, England, were anaesthetized by intravenous injection of 25% w/v urethane (6 ml/kg). T h e right femoral artery was cannulated. Three animals received 4 pg/kg [14C]nicotine intravenously and the same number were given 1 mg/kg [14C]nicotine by catheter directly into the stomach in a total volume of 5 ml physiological saline. A further three animals were given a subcutaneous injection of 0.4 mg/kg [14C]nicotine in 1 ml buffered saline. I n all the animal experiments, except for the rats, after each blood sample, a similar amount of plasma substitute (Intradex ; Glaxo Laboratories, Greenford, Middlesex) was given intravenously. Rats were given a similar volume of saline. I n a majority of animals, at the end of each experiment the bladder contents were removed and the radioactivity analysed. Measurement of [l4C]nicotine and [14C]cotinine in blood was made using the method of Turner (1969). Blood samples (1 ml) were assayed for the cat, monkey and rabbit experiments and 0.5 ml samples (diluted 1 in 3 with saline) of rat blood were extracted. All samples were counted in a Model 3375 liquid scintillation spectrometer (Packard Instrument Ltd., Wembley, England) using a modified Bray solution (Evans, 1961) at a maximum efficiency of 83% and to 1 % accuracy. T h e L-( - )-[2’-14C]nicotine hydrogen tartrate at a specific activity of 19 mCi/mmol was synthesized (Decker, 1964) and resolved in these laboratories by Dr. T. H. Houseman.
Results Intravenous nicotine Table 1 shows the results for each species. T h e peak level of [14C]nicotine in the blood in rabbit, monkey and rat occurred at the end of injection and the levels in all three species were of the order of 100 ng/ml. T h e peak nicotine level in the rabbit was slightly less. I n the cat, however, an equilibrium level appeared to have been attained by 20-30 min. T h e decline in blood [14C]nicotine levels after end of injection was rapid in the cat and rabbit, much slower in the monkey and slower still in the rat. The time course of the blood levels of [l*C]cotinine in the four species shows wide variation. I n the cat, maximum blood [14C]cotinine levels occurred between 60 and 90 min whereas in the other species [14C]cotinine levels were greater at 120 min than at 60 min. I n the rat, even after 4 h [14C]cotinine levels were higher than after 2 h but metabolism to cotinine was slow, no significant blood level of cotinine being detectable until 20 min after start of injections. In the rabbit, cotinine was detectable after 5 min. T h e squirrel monkey produced extremely high levels of [14C]cotinine in the blood with a ratio of cotinine to nicotine of 2.5 : 1 after 120 min. T h e rabbit also showed high cotinine levels. 202
Rabbit (3)
Rat (4)
Cat (3)
25.4 f 7.4 8.0 f4.6 39.6 f4.5 2.4 ? 0.6 22.6 f 6.1 0 11.6 k 2.1 5.6 f 1.4
Squirrel monkey (3)
Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine
5
Species
20
90
120
65.0 6.8 97.6 5 15.6 166.1 f 16.2 154.0 f 8.4 30.6 f 3.9 106.2 k 2.7 45.9 i4.2 38.1 k 2.7 36.3 f 5.7 29.4 f 5.4 75.8 f4.4 106.6 f 1.8 16.4+ 6.1 25.0 5.2 78.1 rfI 6.9 30.5 f4.4 110.7 f 7.7 120.2 f 7.6
Time (min) 30 60 (ng/ml blood)
Numbers of animals are referred to in parentheses.
59.6 f 22.8 81.2 f 24.8 18.2 f 3.8 52.2 f 7.2 56.4 f 6.6 90.3 + 9.6 103.2 f 8.4 6.9 f 1.2 15.3 i 1.2 27.9f 1.5 50.2 k 8.0 82.2 f 12.2 0 3.2 f 1.2 34.2 f 3.6 52.4 f4.6 17.1 f 2.6 26-6 f 3.1
10
Results are expressed as ng/ml blood f SEM.
38.6 f4.6 148.8 f 14.4 15.6 f 5.1 17.7 f 3 . 0 57.2 f0.6 36.4 k 9.4
240
Table 1. Blood levels of [14C]nicotineand [14C]cotinineafter intravenous administration of 4 pg/kg [l*C]nicotineevery 60 s for 60 min
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2
F
Y
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P
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fn 3
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Subcutaneous nicotine Table 2 shows that the peak blood levels were different in each species and the times at which these maxima occur were different. T h e peak levels for a given species, however, were very similar to those obtained after intravenous injection. T h e rise in [14C]nicotine in the squirrel monkey was very fast with a maximum at 10 min. T h e rat was somewhat slower but the peak level was maintained for about 40 min. T h e cat reached a maximum after 30 rnin and the rabbit at about 20 min. [14C]Cotinine levels reached their maximum in the cat after 60 min after which time they declined very slowly. I n the rat and squirrel monkey [14C]cotinine levels appeared to increase over the course of experiment, and in the rabbit the levels were stable by 60 min. As with intravenous injection, the cotinine/nicotine ratio at the end of the experiments was high in both monkey and rabbit though cotinine levels were higher in these two species after subcutaneous injection towards the end of experiment than after intravenous nicotine. Intragastric nicotine T h e data following administration of 1 mg/kg [14C]nicotine by intragastric instillation to the cat, the rat and the rabbit are shown in Table 3. I n the unanaesthetized cats the blood nicotine levels reached a peak at 40 min as did cotinine levels but at all times cotinine levels were higher than nicotine. I n the anaesthetized rabbit, similar results were obtained as regards nicotine levels but much more cotinine was produced. T h e ratio of cotinine to nicotine in blood after 4 h being almost 70 : 1. I n the rat, however, the levels of [14C]nicotine were significantly elevated 20 rnin after administration and increased until 60 min, after which time they levelled off. [14C]Cotinine levels increased steadily over 2 h exceeding the [14C]nicotine levels at that time. Only in the rat did [14C]nicotine levels approach and exceed those obtained using other routes of administration. T h e peak blood levels of [14C]nicotinein both rabbit and cat, at this dosage, were about one quarter of those obtained when nicotine was given by the other routes. Urinary excretion Urinary excretion data are illustrated in Table 4. p H was not controlled but did not vary widely within an individual species. Rabbit urine tended to be more acidic than the other three species. T h e volumes of bladder contents varied widely. Rabbits and cats tended to yield bladder urine volumes of 2030 ml whereas both rat and squirrel monkey had urinary volumes of 0.5-1.2 ml. T h e urinary excretion pattern of radioactivity was very different in each species. T h e squirrel monkey excreted the lowest proportion of the injected dose in both intravenous and subcutaneous experiments and the proportions of [14C]nicotine in those urines were larger than in any of the other species. For the subcutaneous and intravenous experiments the proportion of [14C]nicotine in urine was always greater than [14C]cotinine. I n the intragastric instillation experiments, for rabbits, the reverse was true. T h e overall urinary recoveries of radioactivity were much lower after intragastric administration than by other
Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine
70.8 f 20.5 8.6 f0.7 22.0 f 5.3 0.4 f0.2 88.0 f4.5 2.1 f 1.1 13.5 f 3.5 10.9 f 2.9
110.4f 13.6 43.2 f 8.6 51.2 f 5.9 2.8 f 1.7 91.3 f 2.9 11.5 f 3.1 36.0 f 17.2 23.2 f 8.9
10
+
93.4+ 10.1 134.9 13.3 75.1 f 8.5 8.2 f 3.6 101.9 f 8.5 19.9 ri: 3.6 80.2 f 9.0 81.3 f7.7 77.8 f 7.9 20.4 +_ 4.9 96.7 6.5 30.2 f 3.1
Time after dosage (min) 20 30 (ng/blood)
48.0 f4.0 226.4 & 19.3 72.5 & 9.8 32.1 f 2.2 91.4f3.6 38.7 f0.3 41.7 f 36.5 212.5 ri: 13.7
60
21.4f 1.3 253.8 k 22.2 36.41- 15.4 30.1 f4.9 73.5 f 1.4 51.1 f 10.2 34.5 f 38.3 194.7 f 14.2
120
Rat (3)
Rabbit (3)
Cat (3)
Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine
0 8.8 f4.7 3.8k0.2 1.4k0.7
5 20
120
240
24.6 f 6.7 7.2 f 1.6 3.7 f 0.8 31.2f1.9 13.7k3.0 5.2f1.3 23.7 f 3.9 17.1 f 3.5 5.3 f 0.7 373.5 f 38.6 343.9 f40.7 341.1 f 23.4 144.8f6.9 153.1 k4.7 154.7ri:ll.O 123.0f19.1 136.6f23.3 168.1 f27.9
25.4 f 5.2 43.6k5.2
Time after dosage (min) 40 60 (ng/ml blood)
15.3 f 3.2 23.5 2 2.0 5.0 f 2.1 26.3 4.4 54.4 f 19.6 289.9 f 9.0 29.0f8.8 90.1f19.1 15.4f3.8 55.6f5.2
10
Results are expressed as ng/ml blood f SEM.
Table 3. Blood levels of [14C]nicotineand [14C]cotinineafter administration of 1 mg/kg [l*C]nicotine by intragastric intubation
Rabbit (3)
Rat (3)
Cat (3)
Squirrel monkey (3)
5
Results are expressed as ng/ml blood f SEM.
Table 2. Blood levels of [14C]nicotineand [laC]cotinine after subcutaneous administration of 0.4 mg/kg [14C]nicotine
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3
3
2
b
00
ul
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Metabolism of Nicotine
Table 4. Urinary excretion of [14C]nicotine and [14C]cotinine after administration of ['*C]nicotine Results are expressed as means f SEM.
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pH of urine
%
dose excreted
%
%
[14C]nicotine
[14C]cotinine
Intraoenous : 4 pg/kg [14C]nicotineevery 60 min. 6.6 0.1 Squirrel monkey (3) 7.9 f 2.7 Cat (3) 6.5 f 0.3 44.6 k 2.7 Rat (3) 6.4 & 0.2 37.5 f 3.9 Rabbit (3) 6.3 k 0.2 37.1 k 4-7
74-6 f6.0 12.1 f 3.9 23.8 f 1.7 42.8 k 3.2
10.0 f 3.0 1.2f0.5 2.2 f 0.2 18.0fl-6
Subcutaneous : 0.4 mg/kg [14C]nicotine* 6.6 k 0.2 4.1 & 0.7 Squirrel monkey (3) 6.6 f 0.1 Cat (3) 23.6 f 1.8 Rat (3) 6.5 f 0.3 12.6 f 5.6 6.4 f 0.1 Rabbit (3) 25.0 f 3.4
66.7 k 4.7 8.3 f 2.2 18.6 f 3.3 31.5 k 240
2.6 f 2.2 0.5 f0-3 2.0 f 0-5 10.3 f 1.0
Intragustric : 1 mg/kg [14C]nicotine. 6.6 0.1 Rat (3) Rabbit (3) 6.2 k 0.1
17.1 f 3.1 12.0 f 2.6
10.4 f 2.0 30.1 f9.4
6.6 f 3.6 16.5 & 142
* Urine sampled 2 h after start of experiment. two routes. T h e cat excreted a higher proportion of the injected dose than other species irrespective of the route but the proportions of [14C]nicotine and [14C]cotinine were small. I n all species the mean percentage dose excreted was lower after subcutaneous injection but the time of sampling was different (2 h for subcutaneous [14C]nicotine, 4 h for intravenous [14C]nicotine).
Discussion The experiments described have shown some remarkable species similarities as well as differences in response to [14C]nicotine administration by different routes. I n all but the rabbit the serial intravenous injections produced virtually identical peak blood levels of [14C]nicotine (approx. 100 mg/ml) though the [14C]cotinine levels differed very widely. Since nicotine is believed to be the principal, central, pharmacologically active agent in tobacco smoke (Knapp & Domino, 1962 ; Yamamoto & Domino, 1965 ; Clarke, Rand & Vanov, 1965) and since such nicotine levels are very similar at comparable times to those obtained in man during smoking (Armitage et al., 1974 a), the squirrel monkey, cat and rat may be considered as useful model animals for pharmacological studies on nicotine. However, known species differences in sensitivity to the effects of nicotine may put additional constraints on the choice of a suitable animal model. T h e squirrel monkey is, for example, much more sensitive than the cat and the rat to some of the effects of nicotine (A. K. Armitage, unpublished observations). By constrast, the rabbit is very insensitive to nicotine in relation to plasma lipid changes (D. L. Topping, unpublished observations). T h e proportion of
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cotinine in human smokers’ blood after heavy smoking is believed to be large relative to nicotine (Langone, Gjika & Van Vunakis, 1973) and in this respect the blood profile produced by the squirrel monkey is perhaps closest of all to the human situation. Subcutaneous injection of 0.4 mg/kg in all four species produced a rapid rise in blood [14C]nicotine which reached the intravenous peak in rabbit, rat and squirrel monkey. T h e time at which this peak was attained however varied widely. I n the rat, for example, blood [14C]nicotine levels were approaching peak levels 5 min after injection, consistent with the rapid onset of behavioural changes observed after this dose, by Morrison and Stephenson (1972). [14C]Cotinine in blood, in general reached somewhat higher levels than at comparable times after intravenous injection. Intragastric instillation of 1 mg/kg [14C]nicotine, which is a route comparable to the administration of nicotine in drinking water at dose levels derived from data of Johnston (1942), resulted in very low blood levels of [14C]nicotine and relatively high levels of [14C]cotinine in both cat and rabbit. This presumably represents a ‘first pass’ effect of the liver. I n the rat, however, high levels of [14C]nicotine were obtained 20 min after nicotine administration, though [14C]cotinine levels were also high at this time. This is consistent with the relatively reduced rate of nicotine metabolism observed in this species and, even with the ‘first pass’ effect, oxidation at least, is slower than in the other two species examined. I n the cat it would appear that the further metabolism of cotinine is rapid since N-oxidation, which could represent an alternative metabolic route is not inordinately high (D. M. Turner, unpublished observations). T h e blood levels in the other three species indicate that C-oxidation is a significant route of nicotine metabolism and in the squirrel monkey, it appears to be the dominant one. If experimental data are to be interpreted in a smoking context then it would seem reasonable that the blood levels of the nicotine should be as closely similar, both in terms of the maximum actual concentration and the time course of distribution, to those in man (Isaac & Rand, 1972). We have recently shown that the blood levels of nicotine achieved in inhaling cigarette smoke may be mimicked closely by a suitable series of intravenous injections both in man (Armitage et al., 1974 a) and the cat (Turner, 1970 ; Armitage et al., 1974 b ) and therefore in model experiments, unless smoke inhalation is employed, the preferred route for administration is by intravenous injection. Subcutaneous administration in all four species appears to be a reasonable alternative and the dose of 0.4 mg/kg would appear to be equivalent in terms of blood peak levels to 4 pg/kg intravenous for 20 min. Intragastric instillation of nicotine appears to be less useful at least in the cat and rabbit, and so administration of nicotine via drinking water, which would result in a slow release of the compound into the hepatic portal circulation, might be expected to produce low levels of nicotine unless very large doses were given. Though nicotine is considered to be the principal pharmacological active agent of smoke, it is not yet known whether any metabolites are biochemically active (Gorrod et al., 1974) and therefore, in a particular animal model the presence of either large or small amounts of metabolites may make interpretation of data in terms of nicotine alone, and by extrapolation human smoking, difficult.
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Acknowledgments I am grateful to Mrs J. Bedwell and Mr. P. S. B. Minty for their skilled technical assistance.
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