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STUDIES ON THE CONJUGATION OF GLUCURONIC ACID WITH MORPHINE
STUDIES
ON THE
CONJUGATION WITH
OF GLUCURONIC
ACID
MORPHINE
PART 1 : EFFECTS OF EXOGENOUS GLUCURONIC ACID UPON THE ANALGESIA DUE TO MORPHINE IN MICE SEIJI OTOBE Department of Pharmacology, School of Medicine, KeioUnucrsity, Tokyo Receivedfor publication July 7, 1959
INTRODUCTION Though it is a %,-ellknown fact that some drugs or substances brought into animal body conjugate with glucuronic acid in z'izvand lose their action on the animal by the conjugation with it, it remains to be doubt as yet whether exogenous glucuronic acid can be utilized for the conjugation or not. L. A. Woods (1) has already reported that the most parts of morphine brought into the animal body conjugate with glucuronic acid and form a compounds so-called "bound morphine" which does not cause an analgesia as morphine does. So, by the examination of analgesia due to morphine the author studied on the prob lem whether or not exogenous glucuronic acid or glucuronolactone can be utilized for the conjugation with morphine brought into the animal body. MATERIALS Male periments. the other
albino
mice
And at least seven for the same animal.
Unless
otherwise
ly as 0.1?o solution Sodium soon after plate
of S. M. strain,
indicated, at the rate
glucuronate dissolving
(55°C) method
days
AND METHODS
weighing intervals
morphine
in water.
improved
were
The
by Sanuki
Analgesia
due to morphine in mice
Though
there
the hotplate, to it exceeded
were
no mouse over
some
mice which
was found
20 seconds.
And
placed
among
between
throughout
the ex
one experiment
was administered
and
intrape ritoneal
as free base. was administered sensitivity
of mice
intraperitoneally
or orally
was measured
by the
hot
(2).
EXPERIMENTAL 1.
20 g, were used
hydrochloride
of 50 mg/kg
or glucuronolactone them
about
sprang
RESULTS
up at the moment
more
most of them
than
two
showed
hundreds an attitude
of putting whose
them
reaction
of avoidance
on time from
the hotplate in 7-12 seconds and the reaction time became 9±2 seconds on the average (thin line in Fig. 1). However, the average reaction time of the animal to it was prolonged significantly after peritoneal injection of morphine in dose of 50 mg/kg . And at 30-60 minutes after the morphine administration almost all of the mice did not show any attitude of avoid ance from the hotplate for 120 seconds, as shown by the thick full line in Fig . 1. ' Accurately speaking, the average reaction times of such animal to the hotplate in the figures do not reflect the true values exactly and they are expressed shorter than real , because the animal was not placed on the hotplate over 120 seconds in order to avoid burning the soles and therefore maximal reaction time was cut at 120seconds. The frac tion 20'20, noticed at the peak of the full line in the figure means that twenty mice out of twenty which had received morphine 60 minutes earlier before putting them on the hotplate did not show any attitude of avoidance from it for 120 seconds. In other words, the numerator shows the numbers of mice which showed no attitude of avoidance for 120 seconds and the denominator shows the numbers of the mice used for the experi ment at the same time. Now it is clear that morphine should induct also an analgesia in mice. ?.
E11ectsof scthuin,Jlufuronateon the analgesiadue t-, morphine After confirming by the experiments as shown in Table 1 that the toxicity of 400 mg/kg morphine to mica was the least when 825 mg/ kg sodium glucuronate was mixed with it, the same amount of the glucuronate or glucuronolactone were used for the follow ing experiments. TABLE.
In average
Fig.
reaction
at various
nistration
1, the
times
dotted
line
times of mice after
as above
their true values exactly.
shows
the
to the hotplate
intraperitoneal
of the mixture
the same reason
1
admi
of 50 mg/kg morphine
--v ~--o--~~
Fin. 1 50 mg/kg Morphine (20 Mice) ~(50 825mg/kg Morphine (26 Mice) mg/kg Sod. Glu. Dist. Water (30 Mice)
and 825 mg/kg sodium glucuronate . By mentioned , these average reaction times do not also reflect And the fraction 10/26, noticed at. the peak of the dotted line
means had
that ten mice out of twenty received
sodium fore
the
mixture
glucuronate
putting
show any
15 minutes
them attitude
six which
of morphine
and
earlier
on the hotplate of avoidance
be
did not
from it for
120 seconds. Then, times
the
of mice
prolongation to the hotplate
by morphine
was
simultaneous
administration
and
the
tion
tered
But
the glucuronate
of the
weakly
reduced
glucuronate.
effects of
reaction
when
of the
times
reaction
to be caused apparently of the
the
by drug
inhibitory Fl(-.. 2
on the prolonga appeared
*-~
much
825 mg; kg of it were
16.2 mg/kg Sod. Glu. 3?0 mg/kg Sod . Glu. 825 mg; kg Sod . Glu. 4950 mg!kg Sod. Glu.
-
adminis
60 minutes earlier to morphine. In Fig. 2. it can be seen that
ministered of the
with
morphine
glucuronate
to morphine
simultaneously prolongation
more
the
amount
is increased, of the
reaction
of sodium the
glucuronate
stronger
times
to be ad
the inhibitory
of mice
to the hotplate
effects due
become.
Therefore, ministered
on the
the
with
the analgesia
due
to morphine
should
be reduced
by the
glucuronate
ad
it simultaneously.
3. Ejects of glucuronolactone on the analgesia due to morphine The prolongation of the reaction times of mice to the hotplate to be caused by administration of 50 mg.kg morphine ap peared also much weakly when 825 mg/kg glucuronolactone was administered to the animal 60 minutes in advance to morphine. Especially in the case of oral administra tion of the lactone the effect was significant, Fic. 3 as shown in Fig. 3, while such a strong effect _T H,O (p.o. 60 min in advance) was not recognized by the simultaneous ad 825 mg/kg Glu. lactone (p.o. 60 min in advance) ministration of morphine and the lactone. Then, the analgesia due to morphine should be also reduced by glucuronolactone ad ministered in advance to it. 4. Influenceof o-aminophenol upon the effectsof '.glucuronolactone which reduce the analgesia due to morphine o-Aminophenol is a compound well known by its strong affinity to glucuronic acid and it has no analgetic action.
As shown in Fig. 4, the prolongation of the reaction times in
mice to the hotplate to be caused by mor phine was intensified by the simultaneous intraperitoneal administration of 50 mg/kg morphine and 250 mg/kg o-aminophenol. And also in mice which had received 825 mg/kg glucuronolactone orally 60 minutes in advance, the prolongation of the reaction times to be caused by morphine appeared
Ft(,-. 4 825 mg/kg Glu.-lactone
(p.o. 60 min
iin advance)
__ 150mg/kg Morphine 250 mg/kg o (Aminophenol (i.p.) 1825 i n advance) mg/kg Glu.-lactone (p.o. 60 min '50 mg/kg Morphine -H2O (i .p.)
quite well by the administration of the same amount of morphine and o-aminophenol as used in the cantrol experiment. Then, o-aminophenol might strengthen the analgesia due to morphine and inhibit the effect of glucuro-iolactone by robbing the glucuronic acid to be conjugated with the morphine. .~. Influenceof p-glucuronidase ulronthe effectof glucuronolactone which reduces the analgesia due to morphine It is needless to say that fl-glucuroni dase is an enzyme which catalyses the dis sociation cf glucuronide into glucuronic acid and its aglycon. As shown in Fig. 5, the
Fu:. 5 ~~ -~ 4-.
50 mg'kg Morphine only 1825 mg/kg Sod . Glu. Morphine )66 mg/kg 1;-Glucuronidase 825 mg/kg Sod. G1u.
Morphine
prolongation of the reaction times of mice to the hotplate to be caused by morphine appeared very well by the simultaneous intraperitoneal administration of 50 mg,!kg morphine, 825 mg/kg sodium glucuronate and 66 mg/kg $-glucuronidase. Then, /9-glucuronidase might inhibit the effect of glucuronic acid which reduces the analgesia due to morphine by dissociat ing the conjugation of morphine with glu curonic acid dissociating hindering.
SUMMARYAND DISCUSSION According to the author's experimental results above mentioned : 1. The reaction times of mice to the hotplate were prolonged significantly by the intraperitoneal injection of morphine in the animal . Then, the drug should cause surely an analgesia in mice.
2. The prolongation of the react ion times of the animal to the hotplate to be caused by morphine was reduced apparently by the intraperitoneal injection of sodium glucuro nate simultaneously or by oral administration of glucuronolac.one 60 minutes in advance before morphine. And the more the amount of sodium glucuronate is increased, the stronger the inhibi.ion becomes. Then, exogenous glucuronic acid should reduce evident ly the analgesia due to morphine in mice. 3. On the contrary, the prolongation of the reac ion times of the animal to the hot plate due to morphine was intensified by the simultaneous intraperitoneal injection of o aminophenol which is a compound well known by it's strong affinity to glucuronic acid and has no analgesic acion. And the inhibitory effect:of previously administered glucuronolactone upon the analge sia due to morphine in mice was also reduced by the peritoneal injection of o-aminophenol. Then, o-aminophenol might streng.hen the a'ialgesia due to morphine by robbing glucu ronic acid which conjugates with morphine in iizv. And the previously administered glucu ronolacione might inhibit the analgesia due to morphine by its alteration to glucuronic acid. 4. Moreover, the inhibitory effect of the administered sodium glucuronate upon the analgesia due to morphine was also reduced by simultaneously administered p-glucuroni dase, which dissociates morphine glucuronide into free morphine and glucuronic acid. Then, exogenous glucuronic acid should be also u ilized for the conjugation with mor phine in mice. But it is not yet clear whether the exogenous glucuronic acid can direct ly conjugate with morphine or it only accelerates the co'ijuga,ion of endogenous glucu ronic acid with morphine by some mechanisms. M. Ogawa (3) studied quite independently on the same problem and his results agreed with the author's report which had been presented two months earlier than him at the annual meeting of Japanese Pharmacological S::cie(y in 1957, in principles though there were some differences in detail. CONCLUSION 1. The prolongation of the reaction time of mice to the hotplate due to morphine is reduced by the simultaneous injec ion of sodium glucuronate with it or by the oral ad ministration of glucuronolac`one 60 minutes in advance to the drug, while it is intensified by the simultaneous administration of morphine and o-aminophenol. 2. The inhibitory effect of sodium glucuronate or-glucuronolac=one upon the anal gesia due to morphine is reduced by o-aminophenol or by 13-glucuronidase. 3. Then, exogenous glucuronic acid should be utilized for the conjugation with mor phine within the bodies of mice. REFERENCES 1) Woons, L.A.: J. Pharmacol. 112, 158 (1954) 2) SANUKI, K. ANDOHNO,H. : Arta Med.Okayama 19, 89 (1956) 3) OGAWA, M.: Foliapharmacol. japon.54, 533 (1958) (inJapanese)
ON THE
CONJUGATION WITH
OF GLUCURONIC
ACID
MORPHINE
PART 1 : EFFECTS OF EXOGENOUS GLUCURONIC ACID UPON THE ANALGESIA DUE TO MORPHINE IN MICE SEIJI OTOBE Department of Pharmacology, School of Medicine, KeioUnucrsity, Tokyo Receivedfor publication July 7, 1959
INTRODUCTION Though it is a %,-ellknown fact that some drugs or substances brought into animal body conjugate with glucuronic acid in z'izvand lose their action on the animal by the conjugation with it, it remains to be doubt as yet whether exogenous glucuronic acid can be utilized for the conjugation or not. L. A. Woods (1) has already reported that the most parts of morphine brought into the animal body conjugate with glucuronic acid and form a compounds so-called "bound morphine" which does not cause an analgesia as morphine does. So, by the examination of analgesia due to morphine the author studied on the prob lem whether or not exogenous glucuronic acid or glucuronolactone can be utilized for the conjugation with morphine brought into the animal body. MATERIALS Male periments. the other
albino
mice
And at least seven for the same animal.
Unless
otherwise
ly as 0.1?o solution Sodium soon after plate
of S. M. strain,
indicated, at the rate
glucuronate dissolving
(55°C) method
days
AND METHODS
weighing intervals
morphine
in water.
improved
were
The
by Sanuki
Analgesia
due to morphine in mice
Though
there
the hotplate, to it exceeded
were
no mouse over
some
mice which
was found
20 seconds.
And
placed
among
between
throughout
the ex
one experiment
was administered
and
intrape ritoneal
as free base. was administered sensitivity
of mice
intraperitoneally
or orally
was measured
by the
hot
(2).
EXPERIMENTAL 1.
20 g, were used
hydrochloride
of 50 mg/kg
or glucuronolactone them
about
sprang
RESULTS
up at the moment
more
most of them
than
two
showed
hundreds an attitude
of putting whose
them
reaction
of avoidance
on time from
the hotplate in 7-12 seconds and the reaction time became 9±2 seconds on the average (thin line in Fig. 1). However, the average reaction time of the animal to it was prolonged significantly after peritoneal injection of morphine in dose of 50 mg/kg . And at 30-60 minutes after the morphine administration almost all of the mice did not show any attitude of avoid ance from the hotplate for 120 seconds, as shown by the thick full line in Fig . 1. ' Accurately speaking, the average reaction times of such animal to the hotplate in the figures do not reflect the true values exactly and they are expressed shorter than real , because the animal was not placed on the hotplate over 120 seconds in order to avoid burning the soles and therefore maximal reaction time was cut at 120seconds. The frac tion 20'20, noticed at the peak of the full line in the figure means that twenty mice out of twenty which had received morphine 60 minutes earlier before putting them on the hotplate did not show any attitude of avoidance from it for 120 seconds. In other words, the numerator shows the numbers of mice which showed no attitude of avoidance for 120 seconds and the denominator shows the numbers of the mice used for the experi ment at the same time. Now it is clear that morphine should induct also an analgesia in mice. ?.
E11ectsof scthuin,Jlufuronateon the analgesiadue t-, morphine After confirming by the experiments as shown in Table 1 that the toxicity of 400 mg/kg morphine to mica was the least when 825 mg/ kg sodium glucuronate was mixed with it, the same amount of the glucuronate or glucuronolactone were used for the follow ing experiments. TABLE.
In average
Fig.
reaction
at various
nistration
1, the
times
dotted
line
times of mice after
as above
their true values exactly.
shows
the
to the hotplate
intraperitoneal
of the mixture
the same reason
1
admi
of 50 mg/kg morphine
--v ~--o--~~
Fin. 1 50 mg/kg Morphine (20 Mice) ~(50 825mg/kg Morphine (26 Mice) mg/kg Sod. Glu. Dist. Water (30 Mice)
and 825 mg/kg sodium glucuronate . By mentioned , these average reaction times do not also reflect And the fraction 10/26, noticed at. the peak of the dotted line
means had
that ten mice out of twenty received
sodium fore
the
mixture
glucuronate
putting
show any
15 minutes
them attitude
six which
of morphine
and
earlier
on the hotplate of avoidance
be
did not
from it for
120 seconds. Then, times
the
of mice
prolongation to the hotplate
by morphine
was
simultaneous
administration
and
the
tion
tered
But
the glucuronate
of the
weakly
reduced
glucuronate.
effects of
reaction
when
of the
times
reaction
to be caused apparently of the
the
by drug
inhibitory Fl(-.. 2
on the prolonga appeared
*-~
much
825 mg; kg of it were
16.2 mg/kg Sod. Glu. 3?0 mg/kg Sod . Glu. 825 mg; kg Sod . Glu. 4950 mg!kg Sod. Glu.
-
adminis
60 minutes earlier to morphine. In Fig. 2. it can be seen that
ministered of the
with
morphine
glucuronate
to morphine
simultaneously prolongation
more
the
amount
is increased, of the
reaction
of sodium the
glucuronate
stronger
times
to be ad
the inhibitory
of mice
to the hotplate
effects due
become.
Therefore, ministered
on the
the
with
the analgesia
due
to morphine
should
be reduced
by the
glucuronate
ad
it simultaneously.
3. Ejects of glucuronolactone on the analgesia due to morphine The prolongation of the reaction times of mice to the hotplate to be caused by administration of 50 mg.kg morphine ap peared also much weakly when 825 mg/kg glucuronolactone was administered to the animal 60 minutes in advance to morphine. Especially in the case of oral administra tion of the lactone the effect was significant, Fic. 3 as shown in Fig. 3, while such a strong effect _T H,O (p.o. 60 min in advance) was not recognized by the simultaneous ad 825 mg/kg Glu. lactone (p.o. 60 min in advance) ministration of morphine and the lactone. Then, the analgesia due to morphine should be also reduced by glucuronolactone ad ministered in advance to it. 4. Influenceof o-aminophenol upon the effectsof '.glucuronolactone which reduce the analgesia due to morphine o-Aminophenol is a compound well known by its strong affinity to glucuronic acid and it has no analgetic action.
As shown in Fig. 4, the prolongation of the reaction times in
mice to the hotplate to be caused by mor phine was intensified by the simultaneous intraperitoneal administration of 50 mg/kg morphine and 250 mg/kg o-aminophenol. And also in mice which had received 825 mg/kg glucuronolactone orally 60 minutes in advance, the prolongation of the reaction times to be caused by morphine appeared
Ft(,-. 4 825 mg/kg Glu.-lactone
(p.o. 60 min
iin advance)
__ 150mg/kg Morphine 250 mg/kg o (Aminophenol (i.p.) 1825 i n advance) mg/kg Glu.-lactone (p.o. 60 min '50 mg/kg Morphine -H2O (i .p.)
quite well by the administration of the same amount of morphine and o-aminophenol as used in the cantrol experiment. Then, o-aminophenol might strengthen the analgesia due to morphine and inhibit the effect of glucuro-iolactone by robbing the glucuronic acid to be conjugated with the morphine. .~. Influenceof p-glucuronidase ulronthe effectof glucuronolactone which reduces the analgesia due to morphine It is needless to say that fl-glucuroni dase is an enzyme which catalyses the dis sociation cf glucuronide into glucuronic acid and its aglycon. As shown in Fig. 5, the
Fu:. 5 ~~ -~ 4-.
50 mg'kg Morphine only 1825 mg/kg Sod . Glu. Morphine )66 mg/kg 1;-Glucuronidase 825 mg/kg Sod. G1u.
Morphine
prolongation of the reaction times of mice to the hotplate to be caused by morphine appeared very well by the simultaneous intraperitoneal administration of 50 mg,!kg morphine, 825 mg/kg sodium glucuronate and 66 mg/kg $-glucuronidase. Then, /9-glucuronidase might inhibit the effect of glucuronic acid which reduces the analgesia due to morphine by dissociat ing the conjugation of morphine with glu curonic acid dissociating hindering.
SUMMARYAND DISCUSSION According to the author's experimental results above mentioned : 1. The reaction times of mice to the hotplate were prolonged significantly by the intraperitoneal injection of morphine in the animal . Then, the drug should cause surely an analgesia in mice.
2. The prolongation of the react ion times of the animal to the hotplate to be caused by morphine was reduced apparently by the intraperitoneal injection of sodium glucuro nate simultaneously or by oral administration of glucuronolac.one 60 minutes in advance before morphine. And the more the amount of sodium glucuronate is increased, the stronger the inhibi.ion becomes. Then, exogenous glucuronic acid should reduce evident ly the analgesia due to morphine in mice. 3. On the contrary, the prolongation of the reac ion times of the animal to the hot plate due to morphine was intensified by the simultaneous intraperitoneal injection of o aminophenol which is a compound well known by it's strong affinity to glucuronic acid and has no analgesic acion. And the inhibitory effect:of previously administered glucuronolactone upon the analge sia due to morphine in mice was also reduced by the peritoneal injection of o-aminophenol. Then, o-aminophenol might streng.hen the a'ialgesia due to morphine by robbing glucu ronic acid which conjugates with morphine in iizv. And the previously administered glucu ronolacione might inhibit the analgesia due to morphine by its alteration to glucuronic acid. 4. Moreover, the inhibitory effect of the administered sodium glucuronate upon the analgesia due to morphine was also reduced by simultaneously administered p-glucuroni dase, which dissociates morphine glucuronide into free morphine and glucuronic acid. Then, exogenous glucuronic acid should be also u ilized for the conjugation with mor phine in mice. But it is not yet clear whether the exogenous glucuronic acid can direct ly conjugate with morphine or it only accelerates the co'ijuga,ion of endogenous glucu ronic acid with morphine by some mechanisms. M. Ogawa (3) studied quite independently on the same problem and his results agreed with the author's report which had been presented two months earlier than him at the annual meeting of Japanese Pharmacological S::cie(y in 1957, in principles though there were some differences in detail. CONCLUSION 1. The prolongation of the reaction time of mice to the hotplate due to morphine is reduced by the simultaneous injec ion of sodium glucuronate with it or by the oral ad ministration of glucuronolac`one 60 minutes in advance to the drug, while it is intensified by the simultaneous administration of morphine and o-aminophenol. 2. The inhibitory effect of sodium glucuronate or-glucuronolac=one upon the anal gesia due to morphine is reduced by o-aminophenol or by 13-glucuronidase. 3. Then, exogenous glucuronic acid should be utilized for the conjugation with mor phine within the bodies of mice. REFERENCES 1) Woons, L.A.: J. Pharmacol. 112, 158 (1954) 2) SANUKI, K. ANDOHNO,H. : Arta Med.Okayama 19, 89 (1956) 3) OGAWA, M.: Foliapharmacol. japon.54, 533 (1958) (inJapanese)