Naunyn-Schmiedeberg's Arch. Pharmacol.

285, 273--292 (1974)

9 by Springer-Verlag 1974

Pharmacokinetics in the Rat

of the Hallucinogenic Alkaloids Harmine and Harmaline
G. Zetler, G. B a c k a n d H. I v e n

Institut fiir Pharmakologie dcr Medizinisehcn Hochsehule L/ibeck

Received June 24 / Accepted August 22, 1974

Summary. 1. After i. v. injection in rats, harmine and harmaline were distri-

buted in the organism within a few seconds. In spite of the close chemical relation-
ship, both alkaloids revealed significant pharmacokinetic differences.
2. Highest concentrations occurred for harmine in the lung and for harmaline
in the kidney. The uptake into brain was for harmaline slower than for harmine.
The rate of elimination was in general smaller for harmaline than for harmine.
3. Harmaline was excreted into chymus and urine to a greater extent than
harmine.
4. The binding to proteins of rat plasma was 94.50/0 for harmine and 52%
for harmaline. Concentrations of free drug in plasma water were used to assess
the binding to various tissues.
5. The extent of tissue binding and the rate of its development were different
for both drugs.
6. The duration of tremor and the strength and decline of bradyeardia were
determined to compare the distribution of the drugs with their effects.
7. The drug concentration (in brain and heart) at the termination of the effect
(tremor and bradycardia) was used as a parameter connecting the pharmacokinetic
and the pharmacodynamic events.
Key words: I-Iarmine -- Harmaline -- Pharmacokinetics -- Bradycardia --
Tremor.

I n mice, t h e c h e m i c a l l y n e a r l y i d e n t i c a l h a r m a l a a l k a l o i d s h a r m i n e
a n d h a r m a l i n e a r e e q u i p o t e n t (EDs0) in i n d u c i n g t r e m o r b u t differ
w i t h r e g a r d t o d u r a t i o n o f t h e effect a n d to t h e i r p h a r m a c o k i n e t i e s
(Zefler et al., 1972). A f t e r t h e i n t r a v e n o u s i n j e c t i o n o f 10 m g / k g of either
h a r m i n e or harmMine, t h e t r e m o r l a s t e d for 25 a n d 96 rain, r e s p e c t i v e l y .
Correspondingly, h a r m a l i n e d i s a p p e a r e d f r o m t h e b r a i n m o r e slowly
(biological half-life t 0 . 5 = 5 3 . 4 r a i n ) t h a n h a r m i n e (to.5 = 10.1 rain).
H a r m a l i n e was r a p i d l y t a k e n u p i n t o t h e brain, whereas its d e c a y f r o m
this o r g a n was u n e x p e c t e d l y d e l a y e d . This p o i n t e d t o t h e p o s s i b i l i t y
t h a t t h e b r a i n was n o t o n l y a n i m m e d i a t e r e c i p i e n t o f t h e d o n o r com-
p a r t m e n t " b l o o d " (ef. Riggs, 1972). T h e p a n c r e a s was discussed to be
Send oJ]print requests to: G. getler, Institut f/it Pharmakologie der Medizinisehen
I-Iochschule, D-2400 Ltibeck, Ratzeburger Allee 160, Federal Republic of Germany.
274 G. Zetler et al.

HaCO~~/N H a C O ~ N
CH3 CH3
Harmine Harma[ine
another compartment acting as a donor to the brain, since Villeneuve
and Sourkes (1966) had found in rats that this organ takes up much
more harmaline than the brain and releases it more rapidly. However,
these authors determined the first concentrations as late as 30 rain
after administration and, thus, gave no information about the rate of
uptake into organs for harmine or harmaline.
Therefore, we found it necessary to compare these alkaloids with
regard to their pharmaeokinetie behaviour in the rat. I n addition to
the problems mentioned, this study is concerned with the fundamental
question (Riegelman et al., 1968a) of whether, and to what extent, a
small change in chemical structure of a given drug would influence the
pharmaeokinetic parameters and the kinetics of pharmacologic effects
(Levy, 1966). This influence was to be expected since the distribution
coefficient in the system n-heptane/phosphate buffer (1/15M, p H 7.4) has
been found to be 0.305 for harmine and 0.046 for harmaline (Zetler et al.,
1972). Differences in pharmacological properties of both alkaloids are
not of purely academic interest since these drugs are well-known halluci-
nogens (Hoffer and 0smond, 1967; Sehultes, 1969), harmaline probably
having greater activity in this respect than harmine (Naranjo, 1967).

Methods
1. Animals and Treatments
Male Wistar rats weighing 145--175g (~-~ 162g; P. B~umler/Wolfrats-
hausen) were kept at 23~ and maintained on Altromin pellets and tap water ad
libitum. Harmine and harmaline were dissolved in 0.1 N ~I~SO4 and injected
(10 mg/kg each) within 10 see into a tail vein in a volume of 3 ml/kg. The animals
were killed by decapitation.

2. Pharmacological Observations
The duration of the tremor was measured by observing the animals seated
each in a glass cylinder on a petri dish covered with filter paper.
The experiments on cardiac effects were performed in rats anaesthetized with
urethane (1.2 g/kg i. p.). The rectal temperature was kept at 36.5~ with the aid
of a heated operation table, a rectal thermoeouple and the YSI tele-thermometer 73
(Yellow Springs Instrument Co., Yellow Springs, Ohio). The drugs were injected
into the right femoral vein as described above. The heart frequency was determined
by means of lead II of the electrocardiogram ("Simpliseriptor", Hellige/Freiburg).
The mean arterial blood pressure was monitored from the left common carotid
artery using a Statham pressure transducer and a "Heleoseriptor" (Hellige).
 1)harmacokinetics in the Rat; 275

3. Sampling and Sample Processing

Blood from the carotids (1.7 ml) was centrifuged in heparinized plastic tubes
for 4 rain at 12 000 rpm (Centrifuge Eppendorf/Hamburg). In separate experiments,
erythrocytes were isolated by centrifugation after blood coagulation. The sizes
of samples were: Plasma 0.4 ml, erythrocytes plus blood cells (mean :j: standard
deviation, n -~ 20) 716 • 56.1 rag, right half of brain 802 • 56.2 rag, total lung
965 :L 115.6 rag, total heart 509 ~ 38.1 rag, piece of liver 506 • 3.6 rag, one kidney
639 j : 60.4 rag, total pancreas 732 • 99.0 mg, total retroperitoneal fat 287 ~ 113.1 mg;
for chymus and urine see Table 5. Mincing and extraction was performed as de-
scribed previously (Zetler et al., 1972). However, for heart, lung and kidney the
hand homogenizer was replaced by the "Ultra-Turrax" (IKA-Werk, Staufen/Brsg.)
which was applied for 0.5 rain at 10,000 rpm. Furthermore, 12 g NaCI was now
added to all homogenates to improve extraction into the organic phase. In the
final extracts, harmine and harmaline were determined fluorometrically (Zetler
et al., 1972) using the Aminco-Bowman fluorometer (American Instrument Co.).
Internal standards were first worked out for each organ or fluid to be extracted.
With both alkaloids recovery was about 90o/o.

4. Determination o/Plasma-Protein Binding

Binding of harmine and harmaline to plasma proteins was determined by means
of the Sephadex-gel filtration technique (Krieglstein and Kuschinsky, 1969; Kriegl-
stein, 1973a). 0.7• cm columns were packed with Sephadex G-25 )~ equili-
brated in 0.02 M phosphate buffer (pH 7.4) containing 9 g/100 ml NaCl(w/v).
The temperature was kept at 37~ the flow rate was 18 ml/hr, fractions of 1.2 ml
each were collected. Harmine and harmaline were added to heparinized rat plasma
to give final concentrations of 2 tzg/ml. I n the case of harmine 3 ml, and that of
harmaline 7 ml plasma was run through the column. In each fraction the concen-
tration of harmine or harmaline was determined directly (see above). Protein con-
centrations were estimated using the biuret reaction.

5. Thin-Layer Chromatography
Final extracts made by shaking the alkaloids from the organic phase into
0.1 N HC1 were evaporated in a desiccator over 1)205. The residue was redissolved
in 10 tzl of 0.1 n HCI, of which 1--3 tL1 was applied to 0.3 mm pre-coated silica-gel
plates (hi. Woelm/Eschwege). The plates were developed for 180 rain with the
solvent system chloroform-methanol-isopropanol-25 ~ ammonia 90:10: 95: 5, v/v
(He et al., 1971). Reference standards of harmine, harmaline, harmol, and harmalol
were run in parallel. At 366 nm, harmine and harmol appeared as blue spots and
harmatine and harmalol as yellow spots having the following Rt values: harmine
0.81, harmol 0.67, harmaline 0.53, harmalol 0.24. ~ 0.01 ~g of each alkaloid was
detectable.

6. Calculations and Statistics

The pharmacokinetio parameters were calculated using the equations summa-
rized in Table 1. The data presented in Figs. 1 and 2 indicated whether a given
concentration-time curve would be fitted with a monoexponential (C ~ - B e -~t)
or an biexponential equation of either the type C ----Ae -at -~ Be -~t or the type
C ~ Be-~t--Ae -~t due to a delayed uptake. The drug concentrations y were trans-
formed into In y, and the least-squares method was used to calculate the equations
276 G. Zetler et al.

Table 1. Pharmacokinetie parameters of the open two-compartment model, and

methods of calculation as described by Dost (1968), Gibaldi et al. (1969), Jusko and
Gibaldi (1972), Riggs (1972) and Riegelman et al. (1968a, b)

Param- Dimension Meaning Method of

eter calculation
Curve peeling
(regression lines
A ~g/ml Intercept of line from residuals by the method
B ~g/ml Intercept of terminal linear segment of least squares:
(% m i l l -1 Slope of line from residuals time -----x,
f min-i Slope of terminal linear segment concentration
-----In y)
t0.5, ~r rain Half-life of the fast a-phase of In 2/a
drug disposition
to.5, min Half-life of the slow f-phase of in 2If
drug disposition
Ezg/ml Zero-time concentration of drug A + B=Do/V~
in plasma a
rain-1 First-order rate constant of drug A B ( f - a)~/C~~ + Ba)
transfer from central to peripheral
compartment
rain-1 First-order rate constant of drug (Aft + Ba)/Opo
transfer from peripheral to central
compartment
ka min-X Sum of simultaneous first-order C~~ + B/f)
processes of metabolism and
excretion
ml Volume of central compartment b Do/C~~
ml Volume of peripheral compartment V1ka2//c21
ml Pseudoequilibrium (fl-phase) Vl k~z/~
volume of distribution
fe traction of drug located in VI
AUC (~g.min)/ Total area trader plasma AIo~+ ~lf =C~0/ke~
ml concentration-time curve
Cltot ml/min Total clearance (sum of all V1 ka
clearance processes)
a Do is the dose applied (rag/individual).
b C~ expressed as mg/ml.

of the regression lines yielding the coefficient B and the rate constant f of these
functions. "Curve peeling" led to residual values of in y from which regression
lines and the parameters A and ~ were obtained. I n the case of an immediate biex-
ponential decline of the concentration-time curve, the two-compartment open-
system model was used to cMcula+~ the kinetic parameters. As regards the curves
 Pharmaeokinetics in the Rat 277

for both harmine and harmaline in pancreas and harmaline in adipose tissue, only the
terminal linear segment was used for calculation. The rate of increase of the
quotient T/P during the initial rapid phase of its development (Table 5) was deter-
mined by transforming the differences from maximum into natural logarithms and
then calculating regression lines versus time to obtain t0.~.
Statistics. In generM, arithmetic means and standard deviations or confidence
limits for P = 0.05 were calculated. In the case of heart frequency, geometric
means were used because of the large variation of the results. Differences with
P < 0.05 were considered statistically significant.

7. Drugs and Chemicals

Harmine hydrochloride and harmol hydrochloride (Fluka, Buehs/Switzerland),
harmaline (Carl Roth, Karlsruhe), harmalol hydrochloride (Aldrich Chemical Co.,
Milwaukee, Wis./U.S.A.); n-heptane p. a., isoamyl alcohol p. a., methanol p.a.,
chloroform p. a., isopropyl alcohol p. a., urethane DAB 6 (all fl'om E. Merck, Darm-
stadt). -- With the aid of thin-layer chromatography, gel filtration on Sephadex
G-25, and direct fiuorometric determination it was found that the preparation
of harmaline contained about 5% harmine, and that of harmine less than 1 ~
harmaline. These contaminations were too low as to interfere with the quantita-
tive results.
Results
1. General Features
Changes in drug concentration developed more rapidly for harmine
t h a n for harmaline (Figs. 1 and 2). This applies especially to the slow
fl-phas e. I n contrast to the rather m o n o t o n o u s B-phase, for b o t h drugs
the rapid c~-phase revealed i m p o r t a n t differences between organs.

2. Distribution in Plasma and Erythrocytes

Time-concentration curves of both harmine a n d harmaline fitted a
biexponential decline and permitted an evaluation according to the
open t w o - c o m p a r t m e n t model (Table 2). As regards plasma, differences
between both alkaloids were negligible only during the rapid co-phase
b u t clearly present with m o s t parameters preferably due to the slow
fl-phase, e.g. t0.5.~, V2, and m o s t obviously V~,Z. The differences be-
tween ki2 and k~l indicate for harmaline a rapid transfer from V1 to V~
a n d a slow r e t u r n from Vu to V1- This would explain w h y for harmaline
Vu was v e r y large a n d / c v e r y small. Figs. 1 a n d 2 d e m o n s t r a t e t h a t the
erythrocy~es contained less harmine b u t more harmaline t h a n plasma.
t0.5, ~ was the same for plasma and erythrocytes and again revealed the
strong difference between the alkaloids. However, with harmMine to.5,
was larger for e r y t h r o c y t e s t h a n for plasma, which did n o t occur with
harmine a n d was perhaps a consequence of the cellular accumulation
of harmaline (Figs. 3 a n d 4). The sum of kinetic parameters indicates
t h a t erythrocytes behaved like a c o m p a r t m e n t different from plasma.
278 G. Zetler et al.

50 i 30
i,,~Fl A l~.l'41NE

10

0.5

0.~ 84
0 10 2.0 30 /+O 5"0 60 70 80 90 100 HO . 12.0
~ttl, t

Fig. 1. Time course of harmine concentrations in plasma (| erythrocytes (+),

adipose tissue (o), liver (• heart (.), pancreas (,), brain (A), kidney (~), and
lung (D), following an intravenous injection of 10 mg/kg of the alkaloid. Shown are
means and confidence limits for P = 0.05 (n ~ 5 for each point). Abscissa: time
past injection (rain). Ordinate: concentration of the alkaloid in plasma (~g/ml) or
tissues t~g/g, wet weight). Insert: brain concentrations of harmine and harmaline

3. Distribution in Organs
Biexponential decline curves pointing to the open two-compart-
m e n t system occurred with harmine for heart, liver, k i d n e y a n d brain,
b u t with harmaline for heart a n d liver only (Table 3). to.~,~ of harmine
was the same for these organs, a n d considerably less t h a n t h a t of harma-
line. W i t h one exception (harmaline-liver) these values of t0.s, ~ are n o t
statistically different from those obtained f r o m plasma. This applies
also to to.5,~ shown in Table 4 (exception: kidney). Thus, in general
the time factor of the fl-phase was the same for the organs as for plasma,
concentrations of harmaline declining more slowly t h a n those of harmine.
This is in accord with the view t h a t the slope of the fl-phase is a function
of all rate constants of the complex system. The t0.a. ~ of the rapid initial
phase (Table 3) was with harmine for heart a n d k i d n e y the same as
for plasma. F o r brain it was (significantly) larger and for liver even more
 Pharmacokinetics in the Rat 279

~50~ . 9

0A , , , , ~ . . . . . .
0 :;'0 /+0 60 80 100 12.0 ]gO IbO 180 200 2.20 .Zzl-O
ir
Fig.2. Time course of harmaline concentrations in plasma (| erythrocytes (+),
adipose tissue (o), liver (x), heart (,), pancreas (,), brain (A), kidney (~), and
lung (~), following an intravenous injection of 10 mg/kg of the alkaloid. Shown are
means and confidence limits for P = 0.05 (n _> 5 for each point). Abscissa: time
past injection (rain). Ordinate: concentration of the alkaloid in plasma (~xg/ml)
or tissues (~g/g, wet weight). It should be noticed that the abscissa has a calibration
different from that of Fig. 1

so. Interestingly, to.a, ~ of harmaline was v e r y large for liver, b u t for

h e a r t n o t v e r y different from t h a t for plasma.
E i g h t of the time-concentration curves shown in Figs. 1 a n d 2 did
n o t allow to a p p l y the open t w o - c o m p a r t m e n t model. A n immediate
monoexponential decline occurred only with harmaline in lung a n d
kidney, whereas in lung harmine showed a slow decline during the first
15rain with t0.5 = 66.1 rain (Table4). The largest difference in the
t y p e of curves between the two alkaloids was found for kidney, harmine
declining biexponentiMly a n d harmMine monoexponentially. A n o t h e r
remarkable difference resulted with brain: harmine declined biexponenti-
ally b u t harmaline m o n o e x p o n e n t i a l l y after a preceding phase of de-
layed u p t a k e with the rate constant ka -~ 0.1312 a n d t0.5 = 5.3 min. The
consideration of short time intervals between injection a n d determination
280 G. Zetler et al.

Table 2. Pharmacokinetie parameters as obtained from concentration-time curves

(Figs. 1 and 2) for harmine and harmaline in plasma and ery~hrocytes after i. v.
injection of 10 mg/kg
Plasma Erythrocytes
Harmine Harmaline Harmine Harmaline
A t~g/ml 11.39 7.34 5.24 8.56
B ~tg/ml 5.76 0.84 3.19 2.21
c~ rain -1 0.4075 0.5249 0.5139 0.1703
fl min - i 0.0349 0.0112 0.0490 0.0095
to.5, ~ rain 1.7 1.3 1.4 4.1
(1.02-- 2.38) a (0.89-- 1.76) (0.48-- 2.22) (2.45-- 5.60)
to.~, ~ rain 19.9 61.8 14.2 72.9
(12.43--27.29) a (50.83--72.70) (7.77--20.53) (59.86--86.01)
C~~ tzg/ml 17.15 8.18 8.43 10.78
ka~ min -x 0.1935 0.3801 0.2261 0.0992
k~, min -1 0.1601 0.0640 0.2248 0.0425
ka min -1 0.0889 0.0920 0.1120 0.0381
V1 ml 94.84 198.02 192.20 150.31
V2 ml 114.19 1175.89 193.37 350.70
Va, ~ ml 240.52 1623.92 439.45 601.92
/c 0.393 0.122 0.437 0.250
AUC (~g. rain)/ 192.97 88.89 75.27 283.19
ml
Cltot ml/min 8.395 18.22 21.52 5.72
a Confidence limits for P ~ 0.05.

Table 3. Pharmacokinetic parameters obtained from time-concentration curves

(see Figs. 1 and 2) fitting a biexponential decline, and evaluated according to the
open two-compartment model
Co to.~, ~ to.5,
[~g/g rain rain
Harmine Heart 35.6 2.7 21.4
(1.40-- 3.91) a (13.38--29.44)a
Liver 7.0 12.3 22.5
(7.47--17.11) (8.82--36.08)
Kidney 37.0 4.0 22.8
(1.10-- 6.83) (17.12--28.55)
Brain 27.3 6.3 21.3
(5.07-- 7.59) (16.48--26.17)

Harmaline Heart 38.2 2.5 69.1

(1.66-- 3.37) (61.95--76.20)
Liver 17.8 31.1 85.7
(22.80-- 39.43) (61.86-- 109.45)
C o ~ Zero-time concentration.
Confidence limits for P ~ 0.05.
g Table 4. Ph~rmacokinetic parameters obtained from time-concentration, curves (see Figs. 1 a n d 2) fitting a monoexponential decline occur-
~. ring immediately (Type A; open o n e - c o m p a r t m e n t model), after a slow penetration (Type B) or a slow penetration followed b y a fast
initial decline (Type C)

Type A Type B Type C

Lung Kidney Adipose tissue B r a i n Pancreas AdiposeTissue r

Harmine a Harmaline tIarmaline ttarmine ttarmaline Harmine Harmaline Harmaline
9
B tzg/g 26.6 23.4 26,9 5.2 11.9 10.3 10.03 2.45

fl rain -1 0.0305 0.0105 0.0078 0.0309 0.0095 0.0328 0.0100 0.0132

to,5, r rain 22,7 65.8 88,9 22.4 72.6 21.1 69.6 52.4
(15.71-29,69) b (59.82-71.77) (82.73-95.13) (16.94-27.94) (66.41-78.88) (16.07-26.14) (55.47-83.76) (32.14-72.60) o

tm~ rain <__ 0.25 ~ 0.25 ~ 0.25 4.3 ~ 21.6 c ~ 5.0 ~ 5.0 ~ 5,0

B = i n t e r c e p t of extrapolated (terminal) linear segment.

traax = Time to reach peak concentration; calculated for type B, otherwise t a k e n from Figs. 1 a n d 2.
After a slow initial segment (see Fig. 1).
b Confidence limits for P = 0.05.
o Calculated (Dost, 1968): t ..... = (1/[c~--//]) In (c~/r
282 G. Zetler et al.
cx. 150-

~14o
130-
/
IZ0- /
110-

100-

?0- /
80J /

~0, ~...... ~ % ~ , ~ -- ...... : . , . : ; . .................. ,

3o / ............ I
!

,o~-::/~
0 T . . . . . . . . " . . . . . . . . . . . , . , .

0 10 ZO 30 ~0 SO 60 70 80 90 100 110 .IZO

1'~tllr
Fig. 3. Time course of the quotient tissue/plasma water (T/P) of harmine for erythro-
cytes (+), adipose tissue (o), liver (• heart (.), pancreas (,), brain (~), kidney
(~), and lung (0). Abscissa: time following i. v. injection of 10 mg/kg of harmine.
Ordinate: the quotient T/P

(insert of Fig. 1) revealed t h a t harmine reached its maximal concen-

tration within 0.25 rain but harmaline after 10 min. 30 min after in-
jection, the concentration of harmaline was still nearly maximal whereas
t h a t of harmine had decreased to one seventh of its peak value.
The curve of the harmine concentration in adipose tissue belonged
to the same type as t h a t of harmaline in brain (Table 4). Harmaline
in fat tissue, however, showed after a delayed penetration phase a decline
which was somewhat more rapid than the final fl-phase and indicated
a redistribution. This type of curve occurred much more clearly with
pancreas for both alkaloids, which separates this gland from the other
organs.
4. Binding to P l a s m a Proteins and Tissues
Undiluted rat plasma bound 94.50/0 of the added harmine and 52~
of harmaline. Therefore, the quotient tissue/plasma water (T/P) was
calculated and used to determine the time course of binding to tissues
{Figs. 3 and 4). Since at higher drug concentrations the percentage of
free drug can be expected to be a little higher (cf. Kriiger-Thiemer,
 Pharmacokiaeties in the R a t 283

/
t:l. 150-
)140
130-
12.0-
f
J
110- ~J
100-

90-

80-

70-

60-
50-

30
I
2.0:

I0~

05
0 zo 4o 60 so ~oo lzo 14-o 1,so 18o zoo zzo z~o
"m,i~
Fig. 4. Time course of the quotient tissue/plasma water (T/P) of harmaline. For
details see Fig. 3. Abscissa different from t h a t of Fig. 3

1967), T/P-values at times before concentration in plasma had reached

2 ~g/ml (see "Methods") are somewhat overestimated and thereafter
somewhat underestimated. Both alkaloids rapidly penetrated into eryth-
rocytes and organs, which led to T / P > 1 within 15 see after i. v. in-
jection. In general, there was an initial phase of rapidly growing T/P,
lasting 15--20 min and followed b y either a further increase or a slow
decrease. Clear-cut differences between both alkaloids occurred in two
respects: (a) the magnitude of T/P, i. e. intensity of binding especially
in lung, kidney, liver, and fat; (b) the time course of T/P, which showed
an initial peak and then a decline for harmaline but not, or less markedly
for harmine in kidney, brain, pancreas, and heart. Furthermore, T / P
developed at different rates as demonstrated in Table 5 for five tissues.
Differences between tissues were more marked for harmine than for
harmaline. Significant differences between both drugs occurred with
brain, liver and kidney, binding of harmine developing in brain more
rapidly than in fat tissue but t h a t of harmaline in reverse order.
Finally, it m a y be pointed to the fact that T/P of erythrocytes con-
stantly decreased for harmine but increased for harmaline during the
observation time.
20*
284 G. Zetler et al.

Table 5. Rate of increase of the quotient T/P during the rapid initial phase of its
development (see Figs. 3 and 4). Shown are values of t0.5 (rain) and confidence limits
for P = 0.05. t0.5 is the time necessary to reach the half-maximal value of T/P.
The maximum was for each organ the highest value obtained within the first 60rain,
or for harmine in lung and fat tissue within the first 30 min
ttarmine Harmaline

Brain 0.3 5.5

(0.03-- 0.53) (4.51--6.40)
Lung 5.8 3.5
(4.89-- 6.73) (0.07--6.84)
Liver 7.2 3.3
(4.22-- 10.20) (1.56--4.93)
Kidney 0.15 4.2
(0 -- 0.78) (2.37--6.04)
Fat tissue 3.7 1.7
(2.93-- 4.40) (0 --3.76)

Table 6. Harmine and harmaline in urine and chymus from small intestine, deter-
mined 2 hrs after injection (10 mg/kg i. v.). Shown are arithmetic means and con-
fidence limits for P ~ 0.05
Harmine Harmaline

Urine n~5 n~5

Volume collected (ml) 1.12 (0.43-- 1.83) 2.80 (1.74-- 3.86)
Amount of alkaloid extracted (f~g) 7.74 (4.49--10.98) 76.59 (69.80--83.38)
Fraction of applied dose (~ 0.47 (0.28-- 0.67) 4.63 (4.19-- 5.06)

Chymus /rom small intestine n=5 n:5

Amount collected (g) 3.65 (3.17-- 4.13) 4.29 (3.93-- 4.65)
Amount of alkaloid extracted (~g) 11.27 (7.87--14.68) 72.83 (68.10--77.56)
Fraction of applied dose (~ 0.75 (0.51-- 0.99) 4.45 (4.03-- 4.87)

5. Excretion into Chymus and Urine

B o t h materials c o n t a i n e d m u c h less h a r m i n e t h a n h a r m a l i n e (Table 6),
the w a y of excretion into the small i n t e s t i n e being u n k n o w n . W i t h
h a r m i n e as well as h a r m a l i n e there was no difference b e t w e e n the a m o u n t s
of drug excreted b y the g u t a n d the kidney. Thus, these alkaloids are
n o t preferably e l i m i n a t e d b y the kidney. P r o d u c t i o n of u r i n e w i t h i n
2 hrs was significantly larger after h a r m a l i n e t h a n after harmine. I t
is u n k n o w n whether this was a consequence of the high r e n a l h a r m a l i n e
a c c u m u l a t i o n which, on the other h a n d , would explain the greater
a m o u n t f o u n d i n urine.
 Pharmaeokinetics in the Rat 285

6. Kinetics o/Pharmacological E/leers

a) Heart Frequency. A f t e r b o t h a l k a l o i d s b r a d y c a r d i a d e v e l o p e d
with t h e s a m e r a p i d i t y as d r u g a c c u m u l a t i o n in c a r d i a c tissue (Fig. 5):
15 see a f t e r t h e e n d o f i. v. a d m i n i s t r a t i o n , f r e q u e n c y was r e d u c e d b y
84 b e a t s a f t e r h a r m i n e (peak effect - - 1 0 5 b e a t s a f t e r 0.5 rain) a n d b y
123 b e a t s a f t e r h a r m a l i n e (peak effect --161 b e a t s a f t e r 1 rain). A f t e r
b o t h a l k a l o i d s decline of b r a d y c a r d i a d u r i n g t h e first 20 rain was m u c h
slower t h a n t h a t of cardiac d r u g c o n c e n t r a t i o n s . L a t e r on, the h a r m a l i n e
effect d i s a p p e a r e d s o m e w h a t m o r e slowly, a n d t h e h a r m i n e effect m u c h
m o r e r a p i d l y t h a n t h e c o r r e s p o n d i n g d r u g c o n c e n t r a t i o n s in t h e h e a r t .
T h e a b r u p t change of b r a d y c a r d i a b e t w e e n 35 a n d 40 rain p a s t h a r m a -
line i n j e c t i o n c a n n o t be explained. B l o o d pressure r e m a i n e d c o n s t a n t

r 50

-zoo
Z 0.5= 30. 3 ~i,,
"q ( 2~-.5-56.1 )
-100- r 10

-50

(.117 3
:;] "

§ .

-10

-5 ,,, TO.5=#.Sm/.v,. "'... 0.5

~] (0-13.9) ",, , ,

-1 lO 2 ' 0 - ~ 5 ~ ' o ~ E - 6 b 7b 8'0 9'0 1do 1,'o ~zo~

u,,d~

Fig. 5. Bradycardia of ~naesthetized rats following an intravenous injection of

10 mg/kg of harmine (+) or harmaline (.). 10 animals each were used in both ex-
periments. Shown are geometric means with their standard errors, and the cal-
culated regression lines. The interrupted lines indicate the cardiac concentrations
of harmine (e) and harmaline (o). The values of t0.5 (with confidence limits for
P = 0.05) refer to the decline of bradycardia. Abscissa: time after the injection.
Left ordinate: bradycardia, i. e. difference in number of heart beats from normal
rate which was 394 • 12 (2 • s~; n = 20). Right ordinate: cardiac concentration
of the alkaloids in ~zg/g (wet weight)
286 G. Zetler et al.

throughout with the following values (mm H g ; 2 and confidence limits

for P = 0.05): normal 86.1 (80.08--92.12 ; n = 20), 5 min after injection
of harmine 89.8 (83.65--95.95 ; n = 10) and 5 rain after t h a t of harma-
line 89.7 (84.83--94.57; n --~ 10).
b) Tremor. Intense tremor occurred about 15 sec after the i . v . in-
jection of 10 mg/kg of each alkaloid. Duration of tremor due to harmine
was 32.2 rain (confidence limits for P =- 0.05:30.6--33.8 rain) and of
t h a t due to harmaline 144.7 (140.6--148.8) min. The corresponding
drug concentrations in brain were calculated using the following equa-
tions: for harmine C = 16.5876 e -~176 + 10.7025 e -~176 and for
harmaline C = 11.9336 e ~176176 e 0.1312t. The resulting con-
centrations were for harmine 4.3 (4.54--3.98) 1 vg/g and for harmaline
3.0 (3.14--2.90)1 ~g/g. This indicates t h a t (a) both alkaloids had nearly
identical tremorigenic power, and (b) duration of tremor depended
solely on the concentration in brain of these tremorigenic agents. The
validity of statement (b) was strengthened b y the results of the following
experiment. 5 mg/kg i . v . of harmaline produced tremor lasting for
65.0 (59.95--70.05) rain. 5 animals each were killed 40 and 90 rain after
this treatment, and had 4.3 (4.00--4.57) ~g/g and 2.6 (2.36--2.77) ~zg/g
of harmaline in the brain. Interpolation resulted in a tremor-end con-
centration of 3.3 (3.55--3.15) 1 lzg/g which was the same as t h a t after
the double dose.

7. Results o] Thin-Layer Chromatography

Extracts of organs from animals killed 1 hr after the i. v. injection
of an alkaloid in general showed two spots of which the larger one be-
longed to the drug injected, and the smaller one to the impurity de-
scribed in "Methods". Liver was the only organ yielding a third spot
which in the case of harmine had the Rr of harmol, and in the case of
harmaline t h a t of harmalol. These demethylated derivatives of harmine
and harmaline have previously been found in organs of the r a t by Ville-
neuve and Sourkes (1966), Slotkin et at. (1970) and Ho et al. (1971).
I t is important t h a t the extracts of both kidney and lung, in spite of
the clearly visible high accumulation of the alkaloid in question, showed
no trace of either harmol or harmalol.

Discussion
Our present results agree with previous findings in mice (Zetler et al.,
1972) in the following respects: (a) the alkaloids rapidly penetrate into
the brain, (b) the time factors (t0.5) for harmaline are larger than those
1 Figures in brackets are concentrations corresponding with lower and upper
confidence limits of tremor-end time.
 Pharmacokinetics in the Rat 287

for harmine, and (e) the tremorigenie potency is nearly equal as con-
cluded from the cerebral drug concentrations at the termination of
tremor. According to the tissue distribution pattern, harmine and harma-
line belong to the group of lipophilic organic bases preferably taken
up by lung and kidney (Eichelbaum et al., 1970).

The rapid brain uptake of the drugs is reminiscent of that of morphine and
related analgesics found by Oldendorf et al. (1972). These authors suggested to
determine the first brain concentrations not later than 30 sec after an i. v. injection
since " . . . a delay of some minutes before measuring brain concentration may
give a spurious impression of the rate of distribution to the brain". In fact, we
would have missed important differences between harmine and harmaline not only
in brain but also in other tissues if we had taken the first samples 30 rain after
administration, as done after i. p. injection by Villeneuve and Sourkes (1966; har-
mine and harmaline in rats) and Bosin et al. (1972; harmaline in mice).

As previously found in mice (Zetler et al., 1972), maximal brain

concentrations of harmaline were considerably lower and occurred much
later than those of harmine. A possible role of the pancreas as envisaged
in "Introduction" can be denied for many reasons. I n our experiments,
the curves of concentration in pancreas versus time were for both alkaloids
nearly identical during the important first 30 rain after administration
and, thus, did not indicate a special kinetic behaviour of harmaline.
Furthermore, between 5 and 15 rain past injection, torn.1 brain content
of harmaline increased by 2.64 ~zg, and that of pancreas decreased by
3 ~zg. However, the latter amount would pass through liver, heart and
lung to be eventually distributed not only to the brain but also to the
kidneys and musculature. The amount of harmaline present in and
released from pancreas is too small as to influence drug concentrations
in the organs mentioned. The very high concentration of harmaline
found by Villeneuve and Sourkes (1966) in the pancreas was perhaps
not so much a consequence of the large dose (50 mg/kg) but rather one
of the intraperitoneal route of administration. -- We conclude that
harmaline penetrates the blood-brain barrier less easily than harmine.
Obviously, the negative influence of low lipid solubility on passage
through the blood-brain barrier was not overcome by the higher per-
centage of free harmaline in plasma water (cf. Oldendorf, 1974).
For both ~lkaloids V1 of plasma was much larger than the normal
plasma volume of the rat (39 ml/kg = 6.3 ml/162 g; Wang, 1959). Thus,
the central compartment includes the group of highly perfused lean
tissues as heart, liver, lung and kidneys (Riegelman et al., 1968a; Jusko
et al., 1972). However, time-concentration curves for these organs in-
dicated by no means a biexponential decline as the plasma curves did.
This deviation from theory is obvious for the organs summarized in
Table 4 and probably caused by an intense uptake during the rapid
288 G. Zetler et al.

a-phase (Figs. 3 and 4). I n the case of harmaline, the large accumulation
in tissues also enhanced kl~ for plasma and, consequently, V, and V~. ~.
The great difference in these parameters between both alkaloids might
further be caused by the binding to erythrocytes which must not be
neglected (Dost, 1968; pp. 96 and 291; J/thnehen et al., 1971): during
the time of observation T/P of erythrocytes declined for harmine but
increased for harmaline.

Lipid solubility of a drug promotes its binding to proteins of plasma and tissues
as well as to erythrocytes, and the penetration into lipid-rich organs (for literature
see Kurz, 1970; gaaflaub, 1970; Jghnchen et al., 1971; Kolassa and Pfleger, 1973;
Krieglstein, 1973b). Thus, the more hydrophobie nature of harmine may be the
cause of many differences from harmaline as regards protein binding, accumulation
in lung, kidney and fat, and excretion in chymus and urine. However, other factors
are also involved. For example, it is obvious why the hydrophobic harmine pene-
trates into liver to at least the same extent as into adipose tissue. Harmaline, on
the other hand, in spite of a considerably less affinity to fat, accumulated in liver
much more than harmine. This might indicate: (a) higher concentrations of harmine
were prevented by metabolic destruction in loco, or/and (b) harmaline was bound
in a watery and protein-rich liver compartment different from that of drug meta-
bolism. A very intense destruction of harmine in the liver would help to explain
why, in spite of being only poorly excreted in chymus and urine, this drug had a
shorter to.5,~ than harmaline. Differences in binding to erythrocytes were surpri-
singly little. This is reminiscent of the view that besides non-polar forces ionic
bonds may be operative in drug binding to erythrocytes (ef. Wassermann, 1972).

Binding to plasma proteins could be expected to prolong t0.a,~ (Keen,

1971). However, to.5,~ was even shorter for harmine than for harmaline,
which means that for this drug binding to plasma proteins should be
considered as transport rather than storage mechanism (Gillette, 1973).
Binding to tissues can be correctly evaluated only if plasma binding
is accounted for (Brodie and Hogben, 1957; Martin, 1965). Fig.6 de-
monstrates that considering the quotient tissue/plasma instead of tissue/
plasma water would lead to the conclusion of the hydrophilic harmaline
being preferentially taken up by lipoid-rich tissues, which would deviate
from theory. This confirms the view of Liillmann et al. (1969) who worked
on an in-vitro system. Thus, the finding that the benzothiophene analog
of harmaline has a lipid solubility 200-times higher than that of harmine
but a much lower tissue/plasma quotient, is difficult to be understood
as the extent of plasma binding is unknown (Bosin et al., 1972). The
rates at which tissue binding (T/P) developed (Table 5) do not point
to a dominating role of either lipohilic or hydrophilic nature of drug.
Puzzling is not only the inverted relation of rates for brain and fat as
regards harmine on the one hand and harmaline on the other hand,
but also the difference between these alkaloids as regards liver and
kidney. This points to important factors besides physical properties
 Pharmacokinetics in the Rat 289

120-

I00-

90-

80

70

60-

50

40.

30

2_0.

10

A B AB AB AB A B A B
B R.AIN LUNG FAT
Fig. 6. Comparison os quotients tissue/plasma water (empty columns) and tissue/
plasma (black columns) for harmine (A) and harmaline (B). The resu!ts refer to
wlues obtained 60 min after the administration

of a drug, as e. g. permeability of cell membranes, circulatory differences

between organs, and unequal binding forces of tissue proteins (cf. Cohen,
1971).
Blood flow to tissues and in organs strongly influences pharmaco-
kinetics (Zaharko and Dedrick, 1973). Therefore, it is necessary to ap-
praise whether, and to what extent, the marked bradycardia might have
altered the blood flow to tissues. Blood pressure remained constant
even during very strong bradyeardia. I n previous experiments in ana-
esthetized rats, a dose of harmine reducing heart rate by 32~ did not
significantly change cardiac output and blood pressure since stroke
volume and peripheral resistance were enhanced (Strubelt et al., 1971).
Hence, a general deterioration of circulation during the bradycardia
is improbable. However, it cannot be excluded t h a t the difference be-
tween the alkaloids during the a-phase of the brain curves was at least
in p a r t caused by the greater strength of harmaline bradycardia. This
influence, then, should also be detectable for other highly perfused organs
as e.g. kidney. The time-concentration curve of harmaline for kidney
was, however, unlike t h a t for brain in t h a t it immediately reached a
m a x i m u m and then declined in a purely monoexponential way. In this
organ, the intense uptake (Fig. 4~) could have compensated for an adverse
circulatory influence on the kinetic net result.
290 G. Zetler et al.

L e v y (1966) pointed out t h a t in general the pharmacologic effect

declines at a constant rate indicating apparent zero-order kinetics. How-
ever, the bradyeardia due to both harmine or harmaline declined ex-
ponentially at changing rates which were different from those of cardiac
drug concentrations. L e v y (1966) discussed numerous reasons for devia-
tion from theory but did not mention the possibility of a drug causing
also an indirect effect which counteracts its direct one. This could apply
to harmine which releases eatecholamines from the guinea-pig myo-
cardium (Zetler, 1974). This interaction could prevent maximal brady-
cardia during the first 20 rain of the experiment, and hasten the
decline of effect later on. Pilot experiments have shown harmaline
also to cause a harminelike indirect effect which was perhaps
somewhat weaker, and underwent taehyphylaxis more rapidly.
I t would be helpful for this discussion to know the drug concentra-
tions just high enough to cause a defined effect, thus linking kinetics
of a drug with t h a t of its effect. To be sure not to refer to inadequately
high concentrations, minimal drug effects should be used for this purpose.
Hence, the concentration at 100~ termination of effect (CTElo0) was
determined in the case of tremor, and t h a t for an 80~ decline (CTEs0)
in the case of bradycardia. Of harmine and harmaline, CTElo o for tremor
was 4.3 and 3.0 ~g/g (wet weight) of brain, respectively, and CTEs0
for bradyeardia was 3.6 and 5.8 ~g/g (wet weight) of heart, respectively.
Thus, in spite of very different kinetics, both alkaloids had in brain
and heart equieffective concentrations which were of the same order
of magnitude. The CTE can be expected to be constant for a given drug
and a given organ unless the susceptibility of the ]after changes, and
it does not depend on the dose as demonstrated for the tremor. The
CTE of a given drug is indicative of the true pharmacological potency
at interaction with specific structures since for a group of seven indole
alkaloids the tremor-CTEl0 o in mouse brain was highly correlated with
the subcutaneous EDs0 but not at all with the distribution coefficient
(Zetler et al., 1972). Considering t h a t the CTE100 is the threshold con-
centration in brain for both the origin and disappearance of tremor,
it can be understood why there was no difference between the
alkaloids in their rate of onset of tremor. Therefore, the difference
in shapes of brain-concentration curves over the first 2 0 m i n
following t r e a t m e n t is of pharmacokinetic rather than pharmaco-
dynamic importance.

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