Acta pharrnacol. et toxicol. 1968, 26, 571-582

From the Research Institute of National Defence, Department 1, Sundbyberg 4, Sweden

The Fate of Atropine in the Dog

BY

L. Albanus, A. Sundwall, B. Vangbo and B. Winbladh (Received March 20, 1968)

A review of the literature reveals that there are important species differences both with regard to the metabolic disposition of atropine and the doses needed to produce its pharmacological effects. After the injection of 14C-labelled atropine, mice excrete 80-90 % of the label within 48 hr. Approximately 250/, of the radioactivity is in the form of un- changed atropine and over 50 % as conjugates with glucuronic acid (GOSSELIN et a/. 1955; GABOUREL & GOSSELIN 1958). In the guinea pig, on the other hand, most of the radioactivity in the urine is in the form of tropic acid (KALSER et crl. 1957). In man 50% is in the form of un- changed atropine. Little if any radioactivity was excreted as glucuronides and less than 2 % as tropic acid (GOSSELIN & GABOUREL 1958).

There are also large species differences with regard to the doses needed to produce a given pharmacological effect. Dogs are about ten times more sensitive with regard to the effects on the central nervous system than mice or rats (see e.g. LONGO 1966).

In connection with studies on the effects of atropine on the central nervous system of the dog (ALBANUS unpublished; ALBANUS et a/. 1967 & 1968 a), it became important to know the metabolic fate of the drug in this particular species.

Methods Tritiated atropine') (generally labelled) with a specific activity of 172 mCi/mmol was

used. The radiochemical purity was checked by descending paper chromatography using the upper phase of a solution of n-butanol, water, acetic acid ( 5 : 5 : 1) and by high voltage electrophoresis in 0.1 M borate buffer at pH 10. The radioactive spots were localized with

1) Purchased from the Radiochemical Centre, Amersham, England.

42 Acta pharmacologica. vol. 26. fax . 6

572 L. ALBANUS, A. SUNDWALL, B. VANGBO A N D B. WINBLADH

a Packard radiochromatograph. A stock solution in dilute HCI containing 1-5 mCi/ml was stored in the deep freze. The stability was frequently checked by paper chromatography.

The study was undertaken in 15 beagles of both sexes weighing 10-15 kg. All injections were administered subcutaneously in the nape of the neck (50 pCi corresponding to 0.1 ml/kg). Different doses of atropine were obtained by adding unlabelled atropine sulphate.

When the distribution of radioactivity between the diRerent parts of the brain was studied, the dogs were anesthetized with sodium pentobarbital and decapitated at the end of the experiment. After perfusion of the brain with 400 ml of saline through the carotid arteries, the different brain structures were dissected out, frozen in liquid nitrogen and ground to a fine powder.

The distribution of radioactivity between the plasma and the cerebrospinal fluid (CSF) was further studied in three dogs anasthetized with halothane in a mixture of 0 2 and NO2 (1 : 1). Cannulas were inserted into one lateral ventricle (using a stereotaxic frame) and into the cisterna magna. In the epicerebral subarachnoid space a small catheter (PEIO) was in- troduced through a hole in the rostra1 part of the skull roof and the CSF was allowed to flow freely. From the other compartments samples of 0.1 ml were collected at different time intervals. Blood samples were taken from the saphenous vein. The urinary bladder was catheterized.

Assay of radioactivity in tissues and body fluids 1 g of ground tissue (or 1 ml of heparinized plasma) was extracted with 2 ml acid

ethanol (0.2% acetic acid in 96% ethanol) at room temperature for 30 min. and then centrifuged. The pellet was then re-suspended in 1 ml acid ethanol (0.15 % acetic acid in 70% ethanol), extracted for 30 min. and centrifuged. The latter procedure was repeated once. The combined supernatants were concentrated to 1 ml and assayed by liquid scintil- lation in a Nuclear Chicago model 720 Liquid Scintillation Counter. T o 0.5 ml of the ex- tract were added 0.5 ml water and 14 ml of a scintillation solution containing 960 ml dioxane, 5.12 g PPO, 0.128 g POPOP and 102.4 g naphthalene. Quench corrections were made by the channels’ ratio procedure. The efficiency and reproducibility of the extraction procedure was checked by adding known amounts of radioactive atropine to heparinized human plasma. The recovery was 89.5 % and the reproducibility 5 1.8 % (standard error of the mean of 14 experiments).

Urine and CSF samples were assayed by adding 10400 pI of fluid to water (total volume 1 ml) followed by 14 ml of the scintillation solution. Labelled metabolites in the urine were separated by paper chromatography and high voltage electrophoresis.

The binding of atropine to plasma proteins was determined by centrifuging plasma, t o which a known amount of radioactive atropine had been added, by means of bags made of “Visking” dialysis tubing (Union Carbide Co., Chicago, mean pore size 24 A). Leakage of protein was checked by UV analysis according to the method of ZAMENHOF (1959). The leakage was found to be less than 5 %. The ultrafiltrate was assayed for radioactivity as described above.

Results Absorption of atropine .following subcutaneous injection

Atropine is rapidly absorbed into the blood following subcutaneous injection. Following a dose of 0.5 mg/kg (expressed as atropine sulphate) a maximum concentration in the plasma of about 0.2 pg/ml is reached

T H E FATE O F A T R O P I N E I N T H E DOG 573

0 20

0 10

si 00s

1 L-

60 120 180 240 SOU m m

Fig. 1. Absorption of atropine following subcut,ineous administration of 3H-atropine. 0.5 rng/kg body weight (vertical bars indicate extreme viilues in five dogs), 0.3 nig/kg

and A 0.1 rng/kg body weight. The doses are expressed as atropine sulphate.

after about 25 min. (fig. 1). Half this concentration is reached within ten min. The elimination from the blood is slow and appears to be composed of two exponential functions as seen in fig. 1. Following a lower dose (0.3 mg/kg) elimination is more rapid due to the absence of the slow second phase.

Metabolic t r a y format ions of atropine After the subcutaneous injection of 0.5 mg/kg about 30% of the

injected radioactivity is recovered in the urine after 2 hr., and 50% is excreted within 6 hr. (fig. 2). Paper chromatography of the urine in n-butanol :water: acetic acid ( 5 : 5 : 1) and elution of the radioactive spots with water reveals that, after 2 hr., 81-93 % of the excreted radioactivity

Fig. 2. Excretion of radioactivity in the urine following subcutaneous injection of tritium labelled atropine into two dogs.

42'

574 L. ALBANUS, A. SUNDWALL, B. V A N C B O A N D B. WINBLADH

Fig. 3. Paper chromatography of radioactive metabolites in the urine from dog and mouse following injection of tritium labelled atropine (2 hr. urine).

is in the form of unchanged atropine (Rf = 0.70-0.77) (fig. 3). The composition of the urine with regard to radioactive metabolites is shown in table 1. It can be seen that the major metabolite (5-6% of the radio- activity after 2 hr.) has an Rf-values of 0.25-0.32 (Metabolite 1). In addition, varying amounts of metabolites with Rf-values 0.494.55 (Metabolite 11), 0.66-0.69 (Metabolite 111) and 0.89 (Metabolite 1V) are found.

As seen in fig. 3, a radioactive glucuronide with the same Rf-value as Metabolite 1 is excreted in mouse urine following the injection of 3H- atropine. However, Metabolite I is not split by P-glucuronidase under these conditions when the glucuronide in mouse urine is split (fig. 3).

Metabolites I1 and IV are probably identical with tropine and tropic

T H E FATE O F ATROPINE IN T H E DOG 575

. . . . . . . . . . . I 2 . . . . . . . . . . . 3 . . . . . . . . . . . 4 . . . . . . . . . . .

Table I. Per cent o f radioactive atropine and metabolites in dog urine at diff'erent times after sub- cutaneous injection o f 0.5 mg/kg (expressed as atropine sulphatc) o f 3H-atropine (50 pCi/kg).

I ~ 0-30 - ' 5.1

0-120 6.6 93.4 0-120 ~ 5.3 ' 0.9 - - 8 92.0 0-360 9.8 ~ 2.8 5.9 , - ~ 67.0 ' i

I Mctabolite I

~~ ~~~~ Atropine

I 11 1 1 1 IV i R r =

Collection Dog time after No. ~ injection , = i R e = 1 Re= ~ Re = 1 0.70 0 . 7 7

Figures indicate per cent o f the radioactivity spotted on the chromatogram.

acid, respectively, since they are chromatographically and electrophore- tically indistinguishable from the products formed by alkaline hydrolysis of the tritiated atropine.

Distribution

The distribution of radioactivity to different parts of the brain was studied at three different time intervals: after 0.5 hr. when according to a previous study (ALBANUS unpublished), the atropine effects on behaviour are not yet evident; after 2 hr. when the CNS symptoms are maximal; and after 6 hr. when the symptoms have disappeared. The results are summarized in table 2 which shows the concentration of radioactivity at different time intervals. The data are expressed as ratio:

dpm in tissue per g dpm injected per g

.. x 100

The dose was 0.5 mg/kg. As seen in table 2, the concentration of radio- activity in the brain tissue at the different time intervals does not differ much. However, the results indicate a relatively slow penetration into the brain. Thus, after 30 min. the concentration of radioactivity in the brain is only about 3 of the plasma concentration, while the concentration in the heart is four times higher. After 2 hr. the concentrations in the brain and plasma are the same. It is also evident from the table that none of the brain structures studied contains a remarkably high or low concentra- tion of radioactivity.

576 L. ALBANUS, A. S U N D W A L L , B. VANGBO A N D B. W I N B L A D H

Table 2. Concentration of radioactivity in different tissues following subcutaneous injection of

tritium labelled atropinc (0.5 mg/kg s.c.). (Observations i n seven dogs).

Tissue

Cerebral cortex., . . . . . . Caudate nucleus. . . . . . . Diencephalon . . . . . . . . . Mesencephalon. . . . . . . . Metencephalon

pons. . . . . . . . . . . . . . . cerebellum. . . . . . . . . .

Myelencephalon . . . . . . . Medulla spinalis.. . . . . . Choroid plexus. . . . . . . . Liquor . . . . . . . . . . . . . . . Mandibular g l a n d . . . . . Retina . . . . . . . . . . . . . . . Ciliary b o d y . . . . . . . . . . Plasma. . . . . . . . . . . . . . . Blood. . . . . . . . . . . . . . . . Heart muscle. . . . . . . . . Liver.. . . . . . . . . . . . . . . . Rile.. . . . . . . . . . . . . . . . .

Tissue concentration rat io ' )

0.5 hr.

17 22

28

I9 26 24 12

-

- - - - -

0 0 )

I00 24 I

-

-

7 23 18 17

13 21 27

7 - -

- - -

-

62 232 460

-

2 h r .

24 27 32 27 27 26 30 3 3

22 18 32 28 24 19 IS 10 - - - -

- -

-

- - ( 2 5 ) - 24 22 36 38

321 268 2700 2500

I

The plasma values given in brackets are means from five dogs. 1) dpni in tissue per g

dpm injected per g -~ ~ > 102.

I S 22 18 20

14 8 8 6 - - -

-

- - 4

14 56

21 -

25 20 21 -

19 -

13 -

18 - 17 --

13 - - 31 - 26 - 65 - 122 - 164 - 13 9 -

14 -

85 -

14250 15500 -

It is interesting to note that, while after 6 hr. the highest concentrations of radioactivity in the brain are about 2 times that in plasma, the concentra- tions in the salivary glands, retina and ciliary body are 5 to 10 times greater than the plasma concentration.

Atropine enters the cerebrospinal fluid (CSF) and after 2 hr., the con- centration in the CSF is the same as in the plasma (fig. 4). After 10 hr. it is about three times that of the plasma concentration. The concentra- tions were the same in all the three compartments studied.

The amount of radioactivity excreted in the bile is rather small. Thus 0.4% of the injected radioactivity was found in the gall bladder after 30 min., 2.1 and 2.6% after 2 hr., and 12.0 and 16.7% after 6 hr.

Ultrafiltration of heparinized dog plasma to which labelled atropine was added in vitro showed that, at a plasma concentration of 0.03 pg/ml, less than 10% is bound to plasma proteins.

T H E F A T E O F A T R O P I N E IN T H E DOG 577

In urlne in urlne

L- -12-2 .A-U 60 180 300 420 540 660min

Fig. 4 . Distributioii of rntlioactivily bctween plasma and C S F following subcutaneous in- jection of 3H-atropine (0.5 mg/kg). 0 plasma. A lat. ventricle. = cisterna magna. 4- epi- cerebral subarachnoid space. The excretion of radioactivity in the urine is expressed in per

cent o f the amount injected.

Urinary excretion The renal elimination was studied at different plasma concentrations.

As seen in fig. 5 the amount of atropine excreted in the urine always exceeds the amount filtered by the glomeruli. Thus, the drug is secreted by the renal tubules. No Tm was reached at plasma concentrations up to 0.2 pg/nil. Higher plasma concentrations could not be studied since the urine flow was strongly inhibited. I n addition, the dogs became unmanage- able because of the pharmacological effects of the drug.

At plasma concentrations below 0.1 pg/ml, there is a linear relationship between the plasma concentration and the rate of elimination. At higher plasma concentrations excretion is less efficient.

In three experiments, the effect of varying the pH of the urine on

sL a, 0.2 0.3

PlQSnO cmc.dlollm UQlrnl

Fig. 5. Urinary excretion of atropine (radioactivity) a t different plasma levels.

578 L. ALBANUS, A. SUNDWALL, B. VANGBO A N D B. WINBLADH

Time min.

I

I Atropine ~~~~~ GFR

Urine Urine Bow

PH

50 60 7.0 8.0 Urinary pH

Fig. 6. Effect of urinary pH on atropine clearance.

55-70 . . . . 1 7.8 1 1.50 70-85 . . . . . 8. I 5.13 85-102 . . . . 1 8.1 1 2.94 102-117.. . I 8.1 2.87

atropine clearance was studied. The results are summarized in fig. 6. A full record of a typical experiment is shown in table 3. The dogs were pretreated with 3 g NH4CI orally for three days before the experiment in order to bring the urine to a pH of about 5.0. The urinary pH could then be gradually increased by the infusion of NaHC03. The plasma concentrations in all cases were well below 0.1 pg/ml. As seen in fig. 6 and table 3, atropine clearance is markedly influenced by the urinary pH. Net tubular transport is completely inhibited by pH levels in the urine of about 8.

65.0 0.028 1.82 2.58 0.76 58.3 0.026 1.51 1.49 -0.02 53.0 0.026 1.38 1.32 -0.06 65.1 0.024 1.56 1.47 -0.09

Table 3. Effect of' urine p H U I I tubular secretion of atropine.

The dogs were pretreated with 3 g NHdCI daily for three days. Alkalinization with sodium bicarbonate. GFR = creatinine clearance.

THE FATE OF ATROPINE IN THE DOG 579

Discussion According to the present study, the major path of elimination of

atropine in the dog appears to be urinary excretion of the unchanged drug. However, plasma concentrations over 0. I pg/ml produce a marked decrease in the net tubular secretion of the drug and this may explain the differences in the rate of elimination from the plasma after 0.3 and 0.5 mg/kg shown in fig. 1.

Relatively small amounts were found in the bile as compared with results obtained in rats where 50% of the radioactivity is excreted in the bile within 4 hr. (KALSER ef a/ . 1965). These authors also showed that the biliary excretion is markedly increased following nephrectomy. This may explain the high increase in the biliary excretion seen in the dog between 2 and 6 hr. following 0.5 mg/kg when urinary excretion is diminished.

In mice, the identity of the metabolites excreted in the urine has been tentatively established (GOSSELIN et d. 1955; GABOUREL & GOSSELIN 1958; WERNER 1961). The alkaloid is hydroxylated in the benzene ring in the para position (probably also to a certain extent in the meta position) and glucuronides are formed from both atropine and the hydroxylated atropine. In addition small amounts of tropine, tropic acid and tropine- modified atropine are present. We have made no attempt to establish the identity of the four metabolites in dog urine. By comparing Rf-values with the data in the literature it would appear that no qualitative differences exist between atropine metabolism in mice and dogs. One fact, however, which provides some doubts regarding the validity of these superficial comparisons is that, while metabolite I has the same Rf-values as a glucuronide of atropine found in mouse urine, the former is not split by P-glucuronidase under conditions prevailing when the latter is split.

Since not more than 10 % of the radioactivity in the urine is in the form of metabolites during the first two hr. tissue concentrations of atropine have been calculated without any corrections being made for the meta- bolites. Furthermore in the mouse, where atropine is extensively meta- bolized, chromatographic seperation of radioactive metabolites in different tissues, has shown that the salivary glands, lung, brain and blood contain only a small percentage of metabolites as compared with the urine (ALBANUS et a/ . 1968b). It is thus reasobable to assume that the error introduced into the calculations of tissue concentrations is less than I0 %.

The finding that the renal elimination of atropine is markedly influenced by the pH of the urine, that it is secreted into the stomach (ALBANUS e t a / . 1968b), and that it is absorbed from the stomach only when the contents are made alkaline (T0NNESEN 1948), show that pH gradients are important

580 L. ALBANUS, A. SUNDWALL, B. VANGBO A N D B. WINBLADH

for the distribution and excretion of the drug. The fact that the urinary excretion is favoured by a low pH in the urine may have a practical application in cases of atropine intoxications. It must be remembered, however, that we do not know what other effects acid-base changes might have on the distribution of the drug in the body. Thus, the shift of the blood towards the acid side following ammonium chloride administration is, in the CSF, at the same time followed by a shift to the alkaline side (WINTERSTEIN & GOKHAN 1953).

In the brain, the differences between the concentrations of atropine in different regions are relatively small. The results are in agreement with those of VEIT & VOGT (1935) who used a bioassay technique.

The differences may be due to differences in the regional blood supply. If our figures are compared with those of KETY (1966) for the regional blood supply in the cat, a direct correlation is indicated. There is no reliable evidence that atropine has any action which significantly alters the circulation through the brain (SOKOLOFF 1959).

The presence of atropine in the CSF after subcutaneous injection was already found in 1921 by STERN & CAUTIER by bio-assay. The rapid appear- ance of a high concentration of radioactivity in the epicerebral subarach- noidal space in our experiments can not be explained by the bulk flow but indicates a passage of the drug from the meningeal vessels or from the extracellular fluid of the brain. The remarkably constant concentra- tion of atropine in the different compartments of the CSF shown in fig. 4 shows that the relation between in- and outflux of the drug is the same between two and ten hr. after the subcutaneous injection. No simple explanation can be offered for this finding. However, the CSF concen- tration is about the same as that in the brain. It thus seems possible that the concentration in the CSF (higher than in plasma) is the result of an equilibration with the extracellular space of the brain. A decreased CSF production caused by atropine would also tend to maintain a con- stant drug concentration in the CSF. A saturated active transport from plasma into the CSF via the choroid plexus would also explain the con- centration plateau. However, in vitro experiments with choroid tissue rather suggest a reverse direction of active transport for some quaternary and primary amines ((TOCHINO & SCHANKER 1965a & b).

The results of the present work show that the differences between mice and dogs regarding the sensitivity towards atropine can, to a great extent, be explained by the difference in the rate of metabolism. The fact that the pharmacological effects on the central nervous system are seen only after doses much higher than those which block peripheral cholinergic receptors, is compatible with the uneven distribution of the drug.

T H E FATE OF ATROPINE I N T H E DOG 58 1

Summary The absorption, metabolism, distribution and excretion of 3H-labelled

atropine have been studied in the dog. Following subcutaneous injection, 25% of the injected radioactivity was recovered in the urine after 2 hr. and 90% of the excreted amount was in the form of unchanged atropine. The results explain the relative sensitivity of the dog towards the effects of atropine.

Atropine was secreted by the renal tubules and the urinary elimination was markedly pH-dependent. The latter fact may have practical applica- tion in the treatment of atropine intoxications.

In the brain substance, atropine appeared only slowly and only low concentrations were found. Particular attention has been paid to the distribution of atropine in different regions of the brain. However, the relatively small disrepancies found might be related to differences in regional blood supply. The atropine concentrations in the ventricular, cisternal and epicerebral subarachnoid space were found to be the same.

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