GANGLION BLOCKING PROPERTIES OF THIAMINE AND ITS RELATED COMPOUNDS

GANGLION

BLOCKING ITS

PROPERTIES

RELATED

IWAO YAMAMOTO,

OF

THIAMINE

AND

COMPOUNDS

KIHEI OTORI,

SEKIZO KOJIMA

AND KOJI MIZOGUCHI Department of Pharmacology, OsakaUniversity DentalSchool, Kita-ku,Osaka Receivedfor publication February 10, 1965

In the course of investigation Yamamoto

and

his co-workers

of antagonistic

actions of thiamine

led to a conclusion

that a main

against

antagonistic

nicotine, site in the

peripheral region is the nervous ganglion and the antagonistic action is involved in the competition at the receptor site (1-6). The antagonism between nicotine and thiamine dealt with the investigation vitamin.

was by no means referable

In its thiazole ring structure

thiamine

to the action of thiamine

has a quaternary

ammonium,

as a

as other

ganglion blockers have. The ganglion blocking action of thiamine on the cat's nictitat ing membrane has been previously demonstrated by Yamamoto and his co-workers (3-6) and Mazzella macological

et al. (7). properties

The present of thiamine

nium and tetraethylammonium

investigation

was undertaken

and its related compounds

to compare

the phar

with those of hexametho

with special reference to the ganglion

blocking

activity.

MATERIALS Thiamine hydrofurfuryl

hydrochloride disulfide

(thiamine),

(TTFD),

thiamine

propyl disulfide (TPD), thiamine

3-(4-amino-2-methyl

tetra

pyrimidyl-5-methyl)-4-methyl-5

methyl thiazolium chloride (TM), provided by Research Laboratories, Takeda Chemical Industries, Ltd., were used. Chemical structures of those compounds are shown in Fig. 1. As the reference substances bitartrate

(C6) were used.

in 0.9% saline solution.

tetraethylammonium

The compounds

bromide

were used immediately

The other compounds

hydrochloride

chloride

torazoline

sulfate (atropine),

after being dissolved

used were as follows : acetylcholine

ride (ACh), l-nicotine (nicotine), l-adrenaline (Norad), atropine

(TEA) and hexamethonium

(Ad), dl-noradrenaline

chloride

chlo hydro

(tolazoline).

METHODS 1) Effects on blood pressure Blood pressure

was recorded

from

the unilateral

common

carotid

artery

on kymo

graph via a mercury manometer in pentobarbitalized dogs and cats. The vagus dissected in the neck and the splanchnic nerve extraperitoneally. The peripheral 山本

巌 ・大 鳥

喜 平 ・小 島

碩 蔵 ・溝 口

幸二

was cut

FIG.

1.

Chemical

structure

of

thiamine

and

its

related

compounds.

end of each nerve was stimulated with bipolar electrode. Sub and supramaximum stimulation with 1 millisecond duration, frequency of 20 per second were used for 5 seconds. Injection of each test compound was made into the femoral vein with a con stant speed for 10 seconds. 2) Effects on the contractionresponseof the cat's nictitating membrane Cats, weighing 2 to 4 kg, were anesthetized with intraperitoneal pentobarbital sodium (30 mg/kg). Contractions of the nictitating membrane were recorded with a frontal witing point lever as usual. At the lower part of the neck one preganglionic cervical sympathetic nerve was separated from the vagus. Supramaximum stimuli were given on the pre ganglionic sympathetic nerve with the frequency of 20 per second and submaximum stimuli on the postganglionic nerve with the frequency of 10 per second. Stimuli with 1 millisecond duration were given using bipolar electrode for 6 seconds every 5 minutes. Intraarterial injections of ACh and nicotine were made through the cervical end of the lingual artery into the blood supply of the nictitating membrane or the superior cervical ganglion, following the method of Trendelenburg (8). To obtain a dose-response curve, graded doses of a test compound solved in 0.2 ml of saline solution, were injected. The extracarotid artery was clamped and supramaximum stimulation of the pregang lionic nerve was made 1 minute after the injection. The clamp was removed 20 seconds after the preganglionic stimulation. The interval between each series of arterial injec

tion was 10 to 30 minutes. In other experiments, intravenous injection of thiamine and its related compounds was made at a constant speed for 120 seconds. Decentralized and denervated preparations were made 2 to 3 weeks before by the aseptical removal of about 2 cm of the appropriate cervical sympathetic nerve or the superior cervical ganglion. RESULTS 1)

Effects of test compounds on blood pressure a)

Blood pressure in dogs

A decrease of blood pressure or more of thiamine,

was observed

TPD and TTFD.

following the administration

Thiamine

(Fig. 2). Immediately after the administration response due to peripheral vagal stimulation However, a hypotensive of thiamine.

The

2).

TPD and

TTFD

the vagal stimulation

was more potent than TPD or TTFD

of 5 mg/kg of thiamine a hypotensive became about 60% of the initial level.

response to 2 ng/kg of ACh was not affected by the same dose

inhibitory

tion lasted in parallel

of 5 mg/kg

effect of thiamine

with the pressure did not produce

lowering

on the response

due to vagal stimula

effect caused by thiamine

such inhibitory

alone (Fig.

effects on the response

due to

and ACh, even in a dose of more than 5 mg/kg.

FIG. 2. Blood pressure in dog : Effects of thiamine and TTFD on hypotensive responses to peripheral vagal stimulation and intravenous 2 Itg/kg of ACh.

Thiamine,

in a dose of more than

to a peripheral. splanchnic of thiamine

10 mg/kg, inhibited

nerve stimulation.

this hypertensive

response

a hypertensive

response due

Following the administration

of 30 mg/kg

was completely

,"g/kg of Ad was significantly potentiated (Fig. 3). thiamine was diminished following preadministration sure lowering

effect of TPD and

TTFD

was hardly

blocked

and the response

to 2

The pressure lowering effect of of 2 mg/kg of C6j while the pres affected

following

pretreatment

with C6 or atropine. b) Blood pressure in cats Thiamine,

in a dose of 10 mg/kg,

lowered _the blood pressure.

TTFD,

in a dose

FIG. 3. Blood pressure in dog : Effects of thiamine and TPD on hypertensive responses to peripheral splanchnic nerve stimulation and intravenous 2 E.ig/kg of Ad or Norad.

FIG.

4.

Blood

pressure

in

cat

: Hypertensive

effect

of

TTFD.

of more than 5 mg/kg, tension

(Fig. 4).

and TTFD

were not influenced

administration

of tolazoline,

Ad, the hypertensive slight hypotensive 2)

produced

a transient

hypertension

TPD also showed a similar

by the preadministration

in the dose enough

action

action

of each compound

followed

effect to TTFD.

by a slight hypo

These effects of TPD

of Cs.

However,

to block the response was completely

after the

to 2 ug/kg of

abolished,

and only a

was observed.

Effects of test componuds on the contraction response of the cat nictitating membrane a)

Effects on contraction response due to preganglionic supramaximum electric stimulation

Thiamine depressed a contraction response due to the preganglionic stimulation, in a dose of more than 5 mg/kg. As shown in Fig. 5, 30 mg/kg of thiamine depressed the response by 30 to 60% of the initial level and such depression after the administration the response, whereas mg/kg, TM depressed served that thiamine

of the drug. more than

TM, in a dose of 5 mg/kg, transiently

10 mg/kg

FIG.

5.

almost the same as thiamine

response

stimulation

of

of

nictitating

cervical

effect.

effect by repeated

in contraction

membrane.

sympathetic

nerve

5 minutes,

augmented It was ob

administration

More than 60 mg/kg

with a transient

Preganglionic every

1 hour

In a dose of 30

did in the same dose.

replaced for the effect.

showed a slight depression

Contraction

electric

caused a depressive

and TM caused an accumulating

and that either of them could be mutually of TPD or TTFD

lasted about

supramaximum

lowering

of the basal line. When 4 mg/kg of TEA or 1.5 mg/kg of C6 was administered, the contraction response was depressed at almost the same potency and duration as in the case of 30 mg/kg of thiamine (Fig. 5). b) Effects on contractionresponsedue to the injectionof 100 ,ug of ACh or 30 jcg of nicotine into the bloodsupply of the superior cervicalganglion The contraction response of the nictitating membrane elicited by the repeated ad ministration of ACh every 10 minutes was depressed by 30 mg/kg of thiamine and recovered usually 1 hour after the administration (Fig. 6). Four milligrams per kilo gram of TEA or 1.5 mg/kg of Cs showed almost the same depression of the response to ACh as in the case of 30 mg/kg of thiamine. Having confirmed the fact that 30 ag of nicotine which is approximate to a dose of 100% reactivity according to Jones (9) showed no inhibition of contraction response due to preganglionic stimulation, the effect of thiamine was examined. Thiamine showed a depressive effect on the response to nicotine as shown in Fig. 7.

FIG. 6. Contraction response of nictitating membrane. Injection of 100pg of ACh into blood supply of the acute decapitated superior cervical ganglion every 10 minutes.

FIG. 7. Contraction response of nictitating membrane. Injection of 30 pg of nicotine into blood supply of the acute decapitated superior cervical ganglion every 30 minutes.

c) Effects on contraction response due to the injection of 100 ag of ACh into the blood supply of the decentralized superior cervical ganglion It was observed that that obtained

this contraction

by the intact

mg/kg of thiamine

animal

response to ACh was usually

(Fig. 8).

but in a dose of 60 mg/kg.

in a dose of 4 mg/kg.

This response was hardly

stronger depressed

TEA also showed a transient

But C6 showed only an augmenting

effect even

than by 30

depression

in a dose of 8

mg/kg (Fig. 8).

Fir. 8. Contraction response of nictitating membrane. Injection of 100 tog of ACh into blood supply, of the decentralized superior cervical ganglion every 15 minutes.

d) Effects of contractionresponsedue to pestganglionicsubmaximumelectricstimulation Five to 10 mg/kg of thiamine augmented the contraction response due to post ganglionic stimulation transiently and the dose more than 30 mg/kg caused a slight depression. TEA, CE, TM, TPD and TTFD hardly affected the response (Fig. 9). e) Effects of contractionresponsedue to 3 1tg1kgof Ad administeredintravenously Acutely ganglionectomized animals were used. Either 50 mg/kg of thiamine or 30 mg/kg of TM caused 130 to 170% increase of the initial contraction in height 5 minutes after the administration and more than 200% increase after 20 minutes. TEA showed about 120% increase in a dose of 8 mg/kg, but Cs scarcely showed the increasing effect. It was also observed that TPD and TTFD, in a dose of 60 mg/kg, caused an increasing effect respectively (Fig. 9). f)

Effects on con!ra.-lionresponsedue to the injectionof 100 ,rig of ACh into the bloodsupply of the nictitating membrane Acutely ipsilateral superior cervical ganglionectomized animals were employed. The contraction response due to the repeated injection of ACh in every 15 minutes was depressed slightly by 60 mg/kg of thiamine or 8 mg/kg of TEA but hardly depressed

Fir. 9. Contraction response of nictitating membrane. Postganglionic submaximum electric stimulation of cervical sympathetic nerve every 5 minutes. O : 3 fig/kg Ad, i.v.

FiG. 10. Contraction response of nictitating membrane. Injection of 100 jig of ACh into blood supply of the acute ganglionecto mized nictitating membrane every 15 minutes.

by C6 even in a dose of 6 mg/kg. After the intravenous injection of 2 mg/kg of atro pine the nictitating membrane failed to respond to ACh (Fig. 10). g) Effects on contractionresponsedue to the injectionof 100 fig of ACh into the bloodsupply of the denervatednictitating membrane The contraction response of the denervated nictitating membrane to ACh was usually more potent than that of a normal nictitating membrane; the potentiation was repre sented by an increased in duration and contraction height. Thiamine, in a dose of 30 mg/kg, depressed the response slightly. h)

Comparison of ganglion blocking potency of each test compound

An attempt

was made to evaluate

the ganglion

blocking potencies of test compounds.

Fig;. 11. Dose-response curves of C6 and a test compound for the inhibition of contraction response of the nictitating membrane to preganglionic maximum electric stimulation of the cervical sympathetic nerve in a single preparation. (A) C6 and thiamine. (B) C6 and TM. (C) C6 and TPD. Each compound was injected intraarterially.

Dose-response curves of Cs and a test compound for the inhibition of the contraction response to preganglionic maximum stimulation in intraarterial graded doses were ob tained in a single preparation (Fig. 11). At least 1 hour after serial injections of graded doses of Cs, a test compound was injected at graded doses. Then 50% blocking doses (BD50) were estimated from both dose-response curves and the ratio of BD50 of each test compound to that of C6 was calculated as follows : BD50 of a test compound/BD50 of C6. As shown in Fig. 11, A and B, dose-response curves were obtained with less than 20 mg of thiamine and TM in a dose. While with TPD and TTFD usually oc curred the inhibition in less than 50% in a dose more than 20 mg. However, in the preparation which was sensitive to a low dose of Cs, a clear-cut dose-response curve was obtained with TPD in a dose less than 20 mg (Fig. 11, C). As shown in Fig . 11, A to C, the ratio of each test compound was as follows : thiamine 17, TM 20 and TPD 64. DISCUSSION In the experiments on blood pressure, a single dose of thiamine caused a pressure lowering effect in dogs and cats, and these effects were observed in parallel with block ing effects on the pressure response to preganglionic stimulation of the vagal and splan chnic nerves. In addition, the pressure lowering effect of thiamine was diminished following a pretreatment with C6. Thus, it seems most reasonable to conclude that the pressure lowering effect of thiamine is mainly attributable to its peripheral ganglion blocking action, as stressed by Tamori (4) and Yamamoto (6). This conclusion is also supported by the present experimental result in which thia mine hardly acts on peripheral region away from the ganglion. That is, the hypoten sive response to ACh was not affected by thiamine, and thiamine hardly depress a con traction response of the nictitating membrane due to postganglionic stimuli of the cer vical sympathetic nerve and depress slightly a contraction response to ACh which had been injected into the blood supply of the nictitating membrane. This contraction response to ACh was abolished by intravenous atropine. Therefore, it is obvious that thiamine has a weaker effect on the muscarinic "receptor of the nictitating membrane (10) than the effect on the ACh receptor of the superior cervical ganglion. On the contrary, thiamine potentiated a contraction response of the acute ganglionectomized nictitating membrane to intravenous Ad. A hypertensive response to intravenous Ad was also potentiated by thiamine. However, those potentiating effects of thiamine were observed usually at a rather larger dose than a depressive dose to the normal ganglion. The above results also indicate that the most significant site of the action of thia mine on the autonomic nervous system is the ganglion. In the present study, contrac tion of the nictitating membrane elicited by preganglionic sympathetic nerve stimula tion was depressed 40 to 70% by intravenous injection of 30 mg/kg of thiamine as reported by Yamamoto (6). Following intravenous administration of 4 mg/kg of TEA or 1.5 mg/kg of Cs, that contraction response was depressed at almost the same potency and duration as shown by 30 mg/kg of thiamine, Moreover, similar results were ob

tained

by the depression

ACh.

From the report

arterial

ACh is about

fore, it appears identical

On the other

of TEA or C6 of which

by Perry

and

It led to the conclusion

Reinart

while

thiamine

cervical

blocking

ganglion.

action

thiamine

There

is essentially results

or TEA

from a

depressed

C6 augmented

It was also observed

acted

of 100 iag of

100 ,cag of intra

cell.

ganglion,

was analogous

that

the

the response

that a direct

effect of

to that of TEA rather

than C6.

on the peripheral

site of the autonomic

system as in the case of TEA.

Thiamine

as well as TEA

has a quaternary

and it seems to be of interest zole ring structure

with three

ammonium

but not in TPD and TTFD.

peripheral

carbones

In the experiment

ever, even in a intravenous

differing

of thiamine

structure in its thia

from that of TEA or C6.

of the nictitating

membrane

response to the preganglionic

dose of 60 mg/kg,

depression.

both TPD and

blocking

by intravenous

potency

both thia

stimulation.

TTFD

It was also observed that pressure

nerve stimuli were not inhibited of the ganglion

in its chemical

ammonium

just at the same position as in the case of thiamine

mine and TM depressed a contraction slight and transient

ammonium

that the quaternary

is combined

TM has a quaternary

parison

ganglion

that,

(12).

membrane

that

injection

on the ganglion

site of the ganglion

hand, it was observed

on the nictitating

nervous

dose to the superior

of action of thiamine

response to ACh on the decentralized

as reported thiamine

a 90% responsive

with ACh at the receptor

contraction

in case of intraarterial

et al. (11) it may be estimated

that the mechanism

with that

competition

of the response of Mclsaac

only a

responses to various

TPD and TTFD.

of each test compound

How

caused

A com

evidenced

that

the

ganglion blocking activity of TPD was 1/4 to 1/3 as potent as that of thiamine and TM. TTFD showed the potency similar to TPD which would be attributed to the failure

of obtaining

As a result

a clear-cut

it is assumed

dose-response

that

curve

the ganglion

mainly

attributable

to a quaternary

TTFD,

which showed a weak ganglion

at graded

blocking

ammonium blocking

action

in thiazole

doses less than of thiamine ring

20 mg.

and

structure.

TM is TPD or

potency as described above, has a pyri

midine ring in its chemical structure and this part of structure is common to the test compounds. Therefore, it is also suggested that the ganglion blocking activity of thia mine and its related derived

compounds

from the pyrimidine

involves

the weak

ring structure

sure lowering hypertensive

ganglionic

actions.

TPD and TTFD

effect which was not augmented

response of TPD and

tolazoline TTFD

in cats. in different

which would be

responses of TPD and TTFD

effect which was not antagonized

by preadministered

activity

of these agents.

It is also very likely that the pressure able to factor other than

blocking

It may

with atropine

species resulted

blood vessel wall or, may be, from an indirect

showed a transient

action

pres

in dogs, and a transient

by a pretreatment be considered,

were attribut

of C6 but abolished therefore,

from a direct

such pressure action

of some histamine-like

to the

substance

released by these compounds. Matsukawa

et al. (13) reported

that serial compounds

of allithiamine,

involving TPD,

show the vitamin

B, activity

after receiving

a reduction

the living body, and they are excreted slowly.

and forming

Therefore,

thiazole ring in

those thiamine

related

com

pounds may cause significant pharmacological effects due to accumulation after the chronic administration. However, from the fact that TM, which has no vitamin acti vity, had the same ganglion able to conclude

blocking potency as that of thiamine,

that the ganglion

pounds has nothing

blocking activity

to do with the vitamin

it seems most reason

of thiamine

and its related

com

activity.

SUMMARY Pharmacological

properties

with those of TEA and It was shown that nervous

system is the ganglion

follows : thiamine

and

its related

Cs, with special reference

the most significant

lion blocking property from 50% blocking

of thiamine

to their

site of action

compounds ganglion

of thiamine

and that there was a significant

between thiamine

and TEA.

dose of a test compound

were compared blocking

similarity

on the gang

Ratios of test compounds,

versus 50% blocking

17, TM 20 and TPD 64. It was evident

from

activity.

on the autonomic calculated

dose of C6, were as these ratios

that

the

ganglion blocking activity of TPD was 1/4 to 1/3 as potent as that of thiamine and TM. It was concluded that the ganglion blocking activity of thiamine was mainly due to the quaternary ammonium of thiazole ring structure and that the ganglion block ing activity of thiamine

and its related compounds

has nothing

to do with the vitamin

activity. REFERENCES 1) YAMAMOTO, I., IWATA,H., TAMORI,Y. ANDHIRAYAMA, M.: Folia pharmacol.japon. 52, 429 (1956) (Japanese) 2) YAMAMOTO, I., IWATA,H., TAMORI,Y. AND HIRAYAMA, M.: Ibid. 53, 307 (1957) (Japanese) 3) YAMAMOTO, I., KUROGOCHI, Y., KITAMURA, T., NISHIO,H. ANDTAMORI,Y. ; J. Nara Med. Assoc. 9, 36 (1958) (Japanese) 4) TAMORI,Y.: Folia pharmacol.japon. 54, 571 (1958) (Japanese) 5) YAMAMOTO, I., INOKi,R. ANDTsuJIMOTO,A. : Japan.J. Pharm. Chem.33, 731 (1961) (Japanese) 6) YAMAMOTO, I. : THISJOURNAL13, 240 (1963) 7) MAZZELLA, H. ANDFERRERO, N. : Arch. int. Pharmacodyn. 82, 220 (1950) 8) TRENDELENBURG, U. : Brit. J. Pharmacol.11, 74 (1956) 9) JONES,A. : J. Pharmacol.141, 195 (1963) 10) TRENDELENBURG, U. : Ibid. 135, 39 (1962) 11) McIsAAC,R.J. ANDMILLERSCHOEN, N.R.: Ibid. 139, 18 (1963) 12) PERRY,W.L.M. ANDREINERT,H. : J. Physiol.126, 101 (1954) 13) MATSUKAWA, T., YURUGI,S., KAWASAKI, H., ARAMAKI, Y. AND SUZUOKI, J. : Ann. Rep. Takeda Res. Lab. 12, 1 (1953) (Japanese)