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Demethylation of N-methyl derivatives of barbituric acid, hydantoin, and 2,4-oxazolidinedione by rat liver microsomes
Lite Sciences 1(0 . 7s PP " ~6-~92r 1963 " United States .
Preea, Inc . Printed in the
DEMETHYLATION OF N-METHYL DERIVATIVES OF BARBITURIC ACID, HYDANTOIN, AND 2,4-OXAZOLIDINEDIONE BY RAT LIVER MICROSOMES * Jerry A . Smith, William J . Waddell, and Thomas C . Butler Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina (Received l9 June 1963) Dealkylation of N-alkyl derivatives of a variety of chemical structures has been found to be carried out by enzymatic systems localized entirely in the microsomal fraction of liver homogenates . for the reaction,
In N-demethylation one mole of formaldehyde is produced for
each mole of demethylated product, field,
NADPH 2 and oxygen are required
Axelrod (1) has recently reviewed this
Pretreatment of animals with a number of different drugs has been found
to cause large increases of microsomal enzyme activity . reviewed by Remmer (2) .
This subject has been
Electron micrographa of livers of rats pretreated with
phenobarbital, one of the potent inducers of microsomal enzymes,
show extensive
proliferation of the smooth-surfaced endoplasmic reticulum (3) . The present study concerne the N-demethylation by rat liver microsome preparationa of several N-methyl derivatives of barbituric acid, hydantoin, and 2,4-oxazolidinedione .
Demethylation in vivo has previously been demonstrated
for all of these compounds in the dog and for some in the rat ; and for those that are used as antiepileptic drugs, (4-14),
it has also been demonstrated in man
Furthermore, experiments in partially hepatectomized rate with three
drugs representative of the N-methyl derivatives of these three heterocylea indicated that the liver is the principal, if not the only, site of the
* This investigation was supported in part by a grant (NB 00384) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, U. S, Public Health Service,
rna,~rvra~rr~ pir 1(-1~7CL D~tIYATIVSB
Xo . 7 demethylation (5,11) .
487
The purpose of the present investigation was to ascer-
tain whether the N-methyl derivatives of these structures resemble the N-methyl compounds studied by other workers in that the enzymes effecting demethylation are located in the hepatic microsomes, whether the demethylation is accompanied by formaldehyde production, and whether increased enzymatic activity toward these substrates can be produced by pretreatment with some of the drugs that have been reported to induce enzymes metabolizing other types of substrates . Materials and Methods Substrates .
The following compounds were studied as substrates :
5,5-
diethyl-l-methyl barbituric acid (metharbital, N-methylbarbital), 5-ethyl-lmethyl-5-phenyl barbituric acid (mephobarbital, N-methylphenobarbital), 1,3 dimethyl-5,5-diethyl barbituric acid (dimethylbarbital), d-,
1-, and d1-5-
ethyl-3-methyl-5-phenyl hydantoin (mephenytoin, Mesantoin, 3-methylnirvanol), 5-ethyl-l-methyl-5-phenyl hydantoin (1-methylnirvanol),
1,3-dimethyl-5-ethyl-
5-phenyl hydantoin (dimethylnirvanol), and 3,5,5-trimethyl-2,4-oxazolidinedione (trimethadione, N-methyl DMO) . Mic rosome preparations . were decapitated .
Male Carworth Farms CFN rata weighing about 250 g
The liver was removed immediately and homogenized with a
glass homogenizer .
For each gram of liver 4 ml of a cold 0 .2 M potassium
phosphate buffer of pH 7 .4 was used for homogenizing .
The homogenate was
centrifuged at 9000 X g for 30 min in a refrigerated centrifuge .
The super-
natant containing microsomea and the soluble fraction was refrigerated and used within a few hours . Incubations .
Of the microsome preparation 2 .5 ml was added to 2 .5 ml of
a solution containing 100 pmole of nicotinamide, 1 .4
mole of NADP, 10 p,mole of
glucose-6-phosphate, 68 pmole of semicarbazide, 50 p,mole of MgC1 2 , 140 pmole of K 2 HP04 , 35 pmole of KH 2P04 , and 2 mg of substrate .
The flask containing this
mixture was flushed with oxygen and shaken at 37 ° C for 3 hr .
Proteins were
precipitated by addition of 2 ml of 20~ ZnS04 followed by 2 ml of a saturated solution of Ba(OH) 2 .
After centrifugation the supernatant was used for form-
D~IATIQ;f 0~ lf-1~YL D~iIVATIVS3
k88
lâ . 7
aldehyde determination and paper chromatography . Formaldehyde was determined by the method of Cochin and Axelrod (15), a 3 ml sample of the supernatant being used, Paper chromatography was used to identify the demethylated products in the supernatant from the protein precipitation . changed substrates was essential .
Preliminary separation of the un-
By extraction and countercurrent distri
bution schemes with solvents appropriate for the particular compounds involved, final extracts were prepared containing the products but little of the original substrates .
These extracts were spotted on Whatman No . 2 paper .
ecending systems were used .
Three de-
For the hydantoins the solvent was 2,2,4-tri-
methylpentane-butanol-ethanol-0 .1 N HC1 (100 :5 :2 :2) .
For the oxazolidinedione
the solvent was 1,2-dichloroethane saturated with water .
For the barbituric
acids, the paper had previously been dipped in 0 .5 N NaOH and dried . solvent was chloroform saturated with water .
The
The barbituric acids and hy-
daatoina could be visualized by illuminating the paper with a mercury vapor lamp transmitting the 254 nm line .
S,5-Damethyl-2,4-oxazolidinedione
(DMO) was
located by cutting the paper in strips, eluting with a buffer of pH 9, and measuring the absorption spectrum between 205 and 225 nm .
An abaorbancy maxi-
mum of DMO is at 208 nm, End i nduction .
With the exception of 3-methylcholanthrene, the drugs
tested for enzyme inducing activity were given daily for three days . were killed on the fourth day,
The daily doses were :
The rats
phenobarbital 20 mg,
nikethamide 50 mg, diphenhydramine 2 mg, meprobamate 5 mg .
3-Methylcholan-
threne was given in a single dose of 5 mg one day before the rats were killed, Results The yields of formaldehyde from the various substrates are shown in Table 1 .
Although there were considerable variations among individual rats
with any one substrate, there were consistent differences between substrates when they were incubated with the microsomea of a single liver .
Notable are
the greater yield from 1-mephenytoin than from its optical isomer and the much
LA'l'I011 0~ !~ -1~18YL I85RIVIITIVSB
greater yields from the dimethyl derivatives of Nirvanol and barbital than from their monomethyl derivativen,
With most of the preparations from rata without
pretreatment, the yields of formaldehyde from metharbital, mephobarbital, and trimethadione were too small to be definitely determinable by the analytical method used, TABLE 1 Formaldehyde Production (~molee/g liver in 3 hr) In each tabulation, the first number in parentheses ie the nwnber of animals . The number underlined is the mean formaldehyde production, It is followed by the range in parentheses, Pretreatment
Substrate
Phenobarbital
None
Nikethamide
Metharbital
(6) 0 .11 (0 .02-0 .17)
(4)
5 .66 (3 .36- 7 .56)
(1)
5 .00
Mephobarbital
(9) 0 .12 (0 .04-0 .24)
(3)
2 .24 (2 .12- 2 .30)
(1)
2.30
Dimethylbarbital
(6) 0 .72 (0 .64-0 .93)
(4) 13 .30 (8 .10-15 .80)
(1) 15 .00
(5)
1,76 (0 .97- 2 .28)
(1)
2 .24
dl-Mephenytoin
(10) 0 .54 (0 .35-0 .83)
d-Mephenytoin
(3) 0 .31 (0 .22-0 .40)
(1)
1,38
1-Mephenytoin
(4) 0 .59 (0 .37-0 .71)
(1)
3 .90
1-Methylnirvanol
(10) 0,31 (0 .20-0 .56)
(5)
2 .95 (1,25- 4 .16)
(1)
3 .52
Dimethylnirvanol
(10)
2 .10 (1 .17-3 .10)
(5)
9 .92 (8,40-14 .90)
(1)
15 .30
Trimethadione
(11) 0,07
(5)
0,68 (0,38- 1 .05)
(1)
0 .61
(0 .01-0 .11)
Of the compounds tented for enzyme inducing activity, 3rmethylcholanthrene, meprobamate, and diphenhydramine showed no significant effect .
Pretreatment
with phenobarbital and nikethamide caused a marked increase is formaldehyde production from all substrates . different substrates .
However, the increases were quite unequal for
For some substrates the increase was only 3 or 4 fold,
whereas for others it was 20 to 50 fold .
Some of the greatest proportional
increases occurred with those substrates that without pretreatment had the lowest yields . Products of demethylation of all substrates were identified by paper chromatography .
With all except trimethadione thin could be done even in
preparations from rata without pretreatment,
Only with pretreatment with an
D~LATI03f OF lf-1~1SYL D~tIYATIVBB
No . 7
inducing agent was the product of demethylation of trimethadione formed in sufficient amounts to identify,
Barbital was identified as a product from methar
bital and phenobarbital from mephobarbital, arbital and barbital were identified,
From dimethylbarbital, both meth-
Nirvanol (5-ethyl-5-phenyl hydantoin)
was found as a product of the metabolism of both isomers of mephenytoin as well as of 1-methylnirvanol,
From mephenytoin another product, hitherto unrecog-
nized, was identified as 5-ethyl-5-p-hydroxyphenyl-3-methyl hydantoin .
Both
monomethyl derivatives of Nirvanol as well as the completely demethylated compound were .ideatified as products from dimethylnirvanol, tive, mephenytoin, was present in much the largest amount,
The 3-methyl derivaDMO was found as a
product from trimethadione in preparations from pretreated rats . Discussion Like the N-methyl compounds of different structures previously studied by other workers, the N-methyl derivatives of the three heterocycles studied here are demethylated by liver microsomes with the production of formaldehyde .
How
ever, they are demethylated much more slowly than are some other types of compounds .
Meperidine,
for instance, is demethylated about ten times as rapidly
by rat liver microsomea as is mephenytoin .
Demethylation of the monomethyl
compounds of this study also proceeds slowly in vivo , many hours being required for the demethylation of single doses to come to completion in the dog (4,6, 7,9) .
The pronounced differences among the substrates in rates of demethyla-
tion by the microsomal system have not all been in evidence in vivo , but some correlations between rates in vitro and in vivo can be noted,
1-Mephenytoin
is demethylated more rapidly than is its optical isomer in the living rat (10) as well as in vitro .
In the dog, the first methyl group is removed from di-
methylbarbital at a far more rapid rate than is the second (8) .
This corre-
later with the high rate of formaldehyde production from dimethylbarbital in vitro .
It was also concluded from indirect evidence that in the dog the methyl
group in the 1-position in dimethylnirvanol is removed more rapidly than that in the 3-position and much more rapidly than the methyl group of mephenytoin
Ao .
7
(14),
D~~tSYLATIOdf ~ ~-i~THYL D~iIVATIVSB
~91
This correlates with the high rate of formaldehyde production from di-
methylnirvanol in the microsomal preparation and the presence of greater amounts of mephenytoin than of 1-methylnirvanol, No explanation of the large differences among the substrates in their rates of demethylation is apparent,
No correlation with physical properties
such as lipid solubility or degree of ionization is obvious,
That spatial
configuration of the substrate molecule aside from physical properties is an important factor is evident from the difference between the optical isomers of mephenytoin . Drugs capable of inducing enzyme activity evidently act selectively, inducing only certain enzymes and inducing those in unequal degree .
Some of
the drugs highly effective in inducing enzymes attacking other substrates are ineffective in inducing enzymes demethylating the substrates of this study, 3-Methylcholanthrene, diphenhydramiae, and meprobamate, which we found ineffective as enzyme inducers, had been found by other workers to be effective in inducing enzymes against other substrates (16,17) .
The differences between the
closely related compounds of the present study in the degree to which activity was induced toward them by phenobarbital and nikethamide are notable,
The
simplest explanation is that a multiplicity of enzymes are involved and that they are induced to unequal extents,
It seems likely that each substrate is
attacked by a number of enzymes, each of which is active toward a number of substrates,
If the members of a series of substrates have unequal suscepti-
bilities to an enzyme that attacks them all and if the order of susceptibility in the aeries is different toward another enzyme that also attacks them, then induction of the two enzymes in unequal amounts would be expected to give results such as were obtained in these experiments, Summary Several N-methyl derivatives of barbituric acid, hydantoin, and 2,4oxazolidinedione were demethylated by a preparation of the microsomal and soluble fractions of a rat liver homogenate with added NADP, glucose-6-
492
Ro . Î
D~THYIATIOI( Q~ 11-l~18YL D~tIVAi'IVSS
phosphate, and nicotinamide and under an atmosphere of pure oxygen . tion was accompanied by production of formaldehyde,
Demethyla-
There were large differ-
entes among the substrates in their rates of demethylation .
Livers of rats
pretreated with Phenobarbital or nikethamide had higher demethylating activity toward all the substrates than did livers of untreated rata, but the degree of augmentation of activity was quite different for the different substrates . References 1.
J . AXELROD, Proceedings of First International Pharmacological Meeting . Vol . 6, p, 97 . Pergamon Press, London (1962) .
2.
H . REMMER, Proceedings _of First International Pharmacological Meeting , Pergamon Press, London (1962) . Vol . 6, p . 235 .
3.
H . REMMER and H,-J, MERKER, Klin, tischr . 41, 276 (1963) .
4.
T . C . BUTLER, J . Pharmacol . ~, Ther ,
5.
T . C . BUTLER, D, MAHAFFEE, and C, MAHAFFEE, J . Pharmacol . ~. Ther , 106, 364 (1952) .
6.
T . C . BUTLER, J . Pharmacol , ex~ . Ther . 108, 11 (1953) .
7.
T . C, BUTLER, J . Pharmacol , ~ . Ther . 108, 474 (1953) .
8,
T . C . BUTLER, Proc , Soc . ~. Biol ., N, Y . 84, 105 (1953) .
9.
T . C, BUTLER, J . Pharmacol , ex~ . Ther , 109, 340 (1953) .
106, 235 (1952) .
10 .
T, C . BUTLER and W, J . WADDELL, J . Pharmacol , exp . Ther . 110, 120 (1954) .
11,
T, C, BUTLER and W, J . WADDELL, J . Pharmacol . exP . Ther , 110, 241 (1954) .
12 .
T . C . BUTLER and W, J . WADDELL, NeuroloSy 8, Suppl, 1, 106 (1958),
13 .
T . C, BUTLER and W, J . WADDELL, J . Pharmacol , ex~. Ther . 127, 171 (1959) .
14,
W, J, WADDELL and T . C . BUTLER, J . Pharmacol ,
15,
J . COCHIN and J . AXELROD, .J, Pharmacol , ex~, Ther . 125, 105 (1959),
16 .
A, H, CONNEY, C . DAMSON, R . GASTEL, and J . J . BURNS, J . Pharmacol , Ther . 130, 1 (1960) .
17 .
R . KATO and E . CHIESARA, Brit , J . Phazmacol , 18, 29 (1962) .
ex~. Ther . 132,
291 (1961) .
Preea, Inc . Printed in the
DEMETHYLATION OF N-METHYL DERIVATIVES OF BARBITURIC ACID, HYDANTOIN, AND 2,4-OXAZOLIDINEDIONE BY RAT LIVER MICROSOMES * Jerry A . Smith, William J . Waddell, and Thomas C . Butler Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina (Received l9 June 1963) Dealkylation of N-alkyl derivatives of a variety of chemical structures has been found to be carried out by enzymatic systems localized entirely in the microsomal fraction of liver homogenates . for the reaction,
In N-demethylation one mole of formaldehyde is produced for
each mole of demethylated product, field,
NADPH 2 and oxygen are required
Axelrod (1) has recently reviewed this
Pretreatment of animals with a number of different drugs has been found
to cause large increases of microsomal enzyme activity . reviewed by Remmer (2) .
This subject has been
Electron micrographa of livers of rats pretreated with
phenobarbital, one of the potent inducers of microsomal enzymes,
show extensive
proliferation of the smooth-surfaced endoplasmic reticulum (3) . The present study concerne the N-demethylation by rat liver microsome preparationa of several N-methyl derivatives of barbituric acid, hydantoin, and 2,4-oxazolidinedione .
Demethylation in vivo has previously been demonstrated
for all of these compounds in the dog and for some in the rat ; and for those that are used as antiepileptic drugs, (4-14),
it has also been demonstrated in man
Furthermore, experiments in partially hepatectomized rate with three
drugs representative of the N-methyl derivatives of these three heterocylea indicated that the liver is the principal, if not the only, site of the
* This investigation was supported in part by a grant (NB 00384) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, U. S, Public Health Service,
rna,~rvra~rr~ pir 1(-1~7CL D~tIYATIVSB
Xo . 7 demethylation (5,11) .
487
The purpose of the present investigation was to ascer-
tain whether the N-methyl derivatives of these structures resemble the N-methyl compounds studied by other workers in that the enzymes effecting demethylation are located in the hepatic microsomes, whether the demethylation is accompanied by formaldehyde production, and whether increased enzymatic activity toward these substrates can be produced by pretreatment with some of the drugs that have been reported to induce enzymes metabolizing other types of substrates . Materials and Methods Substrates .
The following compounds were studied as substrates :
5,5-
diethyl-l-methyl barbituric acid (metharbital, N-methylbarbital), 5-ethyl-lmethyl-5-phenyl barbituric acid (mephobarbital, N-methylphenobarbital), 1,3 dimethyl-5,5-diethyl barbituric acid (dimethylbarbital), d-,
1-, and d1-5-
ethyl-3-methyl-5-phenyl hydantoin (mephenytoin, Mesantoin, 3-methylnirvanol), 5-ethyl-l-methyl-5-phenyl hydantoin (1-methylnirvanol),
1,3-dimethyl-5-ethyl-
5-phenyl hydantoin (dimethylnirvanol), and 3,5,5-trimethyl-2,4-oxazolidinedione (trimethadione, N-methyl DMO) . Mic rosome preparations . were decapitated .
Male Carworth Farms CFN rata weighing about 250 g
The liver was removed immediately and homogenized with a
glass homogenizer .
For each gram of liver 4 ml of a cold 0 .2 M potassium
phosphate buffer of pH 7 .4 was used for homogenizing .
The homogenate was
centrifuged at 9000 X g for 30 min in a refrigerated centrifuge .
The super-
natant containing microsomea and the soluble fraction was refrigerated and used within a few hours . Incubations .
Of the microsome preparation 2 .5 ml was added to 2 .5 ml of
a solution containing 100 pmole of nicotinamide, 1 .4
mole of NADP, 10 p,mole of
glucose-6-phosphate, 68 pmole of semicarbazide, 50 p,mole of MgC1 2 , 140 pmole of K 2 HP04 , 35 pmole of KH 2P04 , and 2 mg of substrate .
The flask containing this
mixture was flushed with oxygen and shaken at 37 ° C for 3 hr .
Proteins were
precipitated by addition of 2 ml of 20~ ZnS04 followed by 2 ml of a saturated solution of Ba(OH) 2 .
After centrifugation the supernatant was used for form-
D~IATIQ;f 0~ lf-1~YL D~iIVATIVS3
k88
lâ . 7
aldehyde determination and paper chromatography . Formaldehyde was determined by the method of Cochin and Axelrod (15), a 3 ml sample of the supernatant being used, Paper chromatography was used to identify the demethylated products in the supernatant from the protein precipitation . changed substrates was essential .
Preliminary separation of the un-
By extraction and countercurrent distri
bution schemes with solvents appropriate for the particular compounds involved, final extracts were prepared containing the products but little of the original substrates .
These extracts were spotted on Whatman No . 2 paper .
ecending systems were used .
Three de-
For the hydantoins the solvent was 2,2,4-tri-
methylpentane-butanol-ethanol-0 .1 N HC1 (100 :5 :2 :2) .
For the oxazolidinedione
the solvent was 1,2-dichloroethane saturated with water .
For the barbituric
acids, the paper had previously been dipped in 0 .5 N NaOH and dried . solvent was chloroform saturated with water .
The
The barbituric acids and hy-
daatoina could be visualized by illuminating the paper with a mercury vapor lamp transmitting the 254 nm line .
S,5-Damethyl-2,4-oxazolidinedione
(DMO) was
located by cutting the paper in strips, eluting with a buffer of pH 9, and measuring the absorption spectrum between 205 and 225 nm .
An abaorbancy maxi-
mum of DMO is at 208 nm, End i nduction .
With the exception of 3-methylcholanthrene, the drugs
tested for enzyme inducing activity were given daily for three days . were killed on the fourth day,
The daily doses were :
The rats
phenobarbital 20 mg,
nikethamide 50 mg, diphenhydramine 2 mg, meprobamate 5 mg .
3-Methylcholan-
threne was given in a single dose of 5 mg one day before the rats were killed, Results The yields of formaldehyde from the various substrates are shown in Table 1 .
Although there were considerable variations among individual rats
with any one substrate, there were consistent differences between substrates when they were incubated with the microsomea of a single liver .
Notable are
the greater yield from 1-mephenytoin than from its optical isomer and the much
LA'l'I011 0~ !~ -1~18YL I85RIVIITIVSB
greater yields from the dimethyl derivatives of Nirvanol and barbital than from their monomethyl derivativen,
With most of the preparations from rata without
pretreatment, the yields of formaldehyde from metharbital, mephobarbital, and trimethadione were too small to be definitely determinable by the analytical method used, TABLE 1 Formaldehyde Production (~molee/g liver in 3 hr) In each tabulation, the first number in parentheses ie the nwnber of animals . The number underlined is the mean formaldehyde production, It is followed by the range in parentheses, Pretreatment
Substrate
Phenobarbital
None
Nikethamide
Metharbital
(6) 0 .11 (0 .02-0 .17)
(4)
5 .66 (3 .36- 7 .56)
(1)
5 .00
Mephobarbital
(9) 0 .12 (0 .04-0 .24)
(3)
2 .24 (2 .12- 2 .30)
(1)
2.30
Dimethylbarbital
(6) 0 .72 (0 .64-0 .93)
(4) 13 .30 (8 .10-15 .80)
(1) 15 .00
(5)
1,76 (0 .97- 2 .28)
(1)
2 .24
dl-Mephenytoin
(10) 0 .54 (0 .35-0 .83)
d-Mephenytoin
(3) 0 .31 (0 .22-0 .40)
(1)
1,38
1-Mephenytoin
(4) 0 .59 (0 .37-0 .71)
(1)
3 .90
1-Methylnirvanol
(10) 0,31 (0 .20-0 .56)
(5)
2 .95 (1,25- 4 .16)
(1)
3 .52
Dimethylnirvanol
(10)
2 .10 (1 .17-3 .10)
(5)
9 .92 (8,40-14 .90)
(1)
15 .30
Trimethadione
(11) 0,07
(5)
0,68 (0,38- 1 .05)
(1)
0 .61
(0 .01-0 .11)
Of the compounds tented for enzyme inducing activity, 3rmethylcholanthrene, meprobamate, and diphenhydramine showed no significant effect .
Pretreatment
with phenobarbital and nikethamide caused a marked increase is formaldehyde production from all substrates . different substrates .
However, the increases were quite unequal for
For some substrates the increase was only 3 or 4 fold,
whereas for others it was 20 to 50 fold .
Some of the greatest proportional
increases occurred with those substrates that without pretreatment had the lowest yields . Products of demethylation of all substrates were identified by paper chromatography .
With all except trimethadione thin could be done even in
preparations from rata without pretreatment,
Only with pretreatment with an
D~LATI03f OF lf-1~1SYL D~tIYATIVBB
No . 7
inducing agent was the product of demethylation of trimethadione formed in sufficient amounts to identify,
Barbital was identified as a product from methar
bital and phenobarbital from mephobarbital, arbital and barbital were identified,
From dimethylbarbital, both meth-
Nirvanol (5-ethyl-5-phenyl hydantoin)
was found as a product of the metabolism of both isomers of mephenytoin as well as of 1-methylnirvanol,
From mephenytoin another product, hitherto unrecog-
nized, was identified as 5-ethyl-5-p-hydroxyphenyl-3-methyl hydantoin .
Both
monomethyl derivatives of Nirvanol as well as the completely demethylated compound were .ideatified as products from dimethylnirvanol, tive, mephenytoin, was present in much the largest amount,
The 3-methyl derivaDMO was found as a
product from trimethadione in preparations from pretreated rats . Discussion Like the N-methyl compounds of different structures previously studied by other workers, the N-methyl derivatives of the three heterocycles studied here are demethylated by liver microsomes with the production of formaldehyde .
How
ever, they are demethylated much more slowly than are some other types of compounds .
Meperidine,
for instance, is demethylated about ten times as rapidly
by rat liver microsomea as is mephenytoin .
Demethylation of the monomethyl
compounds of this study also proceeds slowly in vivo , many hours being required for the demethylation of single doses to come to completion in the dog (4,6, 7,9) .
The pronounced differences among the substrates in rates of demethyla-
tion by the microsomal system have not all been in evidence in vivo , but some correlations between rates in vitro and in vivo can be noted,
1-Mephenytoin
is demethylated more rapidly than is its optical isomer in the living rat (10) as well as in vitro .
In the dog, the first methyl group is removed from di-
methylbarbital at a far more rapid rate than is the second (8) .
This corre-
later with the high rate of formaldehyde production from dimethylbarbital in vitro .
It was also concluded from indirect evidence that in the dog the methyl
group in the 1-position in dimethylnirvanol is removed more rapidly than that in the 3-position and much more rapidly than the methyl group of mephenytoin
Ao .
7
(14),
D~~tSYLATIOdf ~ ~-i~THYL D~iIVATIVSB
~91
This correlates with the high rate of formaldehyde production from di-
methylnirvanol in the microsomal preparation and the presence of greater amounts of mephenytoin than of 1-methylnirvanol, No explanation of the large differences among the substrates in their rates of demethylation is apparent,
No correlation with physical properties
such as lipid solubility or degree of ionization is obvious,
That spatial
configuration of the substrate molecule aside from physical properties is an important factor is evident from the difference between the optical isomers of mephenytoin . Drugs capable of inducing enzyme activity evidently act selectively, inducing only certain enzymes and inducing those in unequal degree .
Some of
the drugs highly effective in inducing enzymes attacking other substrates are ineffective in inducing enzymes demethylating the substrates of this study, 3-Methylcholanthrene, diphenhydramiae, and meprobamate, which we found ineffective as enzyme inducers, had been found by other workers to be effective in inducing enzymes against other substrates (16,17) .
The differences between the
closely related compounds of the present study in the degree to which activity was induced toward them by phenobarbital and nikethamide are notable,
The
simplest explanation is that a multiplicity of enzymes are involved and that they are induced to unequal extents,
It seems likely that each substrate is
attacked by a number of enzymes, each of which is active toward a number of substrates,
If the members of a series of substrates have unequal suscepti-
bilities to an enzyme that attacks them all and if the order of susceptibility in the aeries is different toward another enzyme that also attacks them, then induction of the two enzymes in unequal amounts would be expected to give results such as were obtained in these experiments, Summary Several N-methyl derivatives of barbituric acid, hydantoin, and 2,4oxazolidinedione were demethylated by a preparation of the microsomal and soluble fractions of a rat liver homogenate with added NADP, glucose-6-
492
Ro . Î
D~THYIATIOI( Q~ 11-l~18YL D~tIVAi'IVSS
phosphate, and nicotinamide and under an atmosphere of pure oxygen . tion was accompanied by production of formaldehyde,
Demethyla-
There were large differ-
entes among the substrates in their rates of demethylation .
Livers of rats
pretreated with Phenobarbital or nikethamide had higher demethylating activity toward all the substrates than did livers of untreated rata, but the degree of augmentation of activity was quite different for the different substrates . References 1.
J . AXELROD, Proceedings of First International Pharmacological Meeting . Vol . 6, p, 97 . Pergamon Press, London (1962) .
2.
H . REMMER, Proceedings _of First International Pharmacological Meeting , Pergamon Press, London (1962) . Vol . 6, p . 235 .
3.
H . REMMER and H,-J, MERKER, Klin, tischr . 41, 276 (1963) .
4.
T . C . BUTLER, J . Pharmacol . ~, Ther ,
5.
T . C . BUTLER, D, MAHAFFEE, and C, MAHAFFEE, J . Pharmacol . ~. Ther , 106, 364 (1952) .
6.
T . C . BUTLER, J . Pharmacol , ex~ . Ther . 108, 11 (1953) .
7.
T . C, BUTLER, J . Pharmacol , ~ . Ther . 108, 474 (1953) .
8,
T . C . BUTLER, Proc , Soc . ~. Biol ., N, Y . 84, 105 (1953) .
9.
T . C, BUTLER, J . Pharmacol , ex~ . Ther , 109, 340 (1953) .
106, 235 (1952) .
10 .
T, C . BUTLER and W, J . WADDELL, J . Pharmacol , exp . Ther . 110, 120 (1954) .
11,
T, C, BUTLER and W, J . WADDELL, J . Pharmacol . exP . Ther , 110, 241 (1954) .
12 .
T . C . BUTLER and W, J . WADDELL, NeuroloSy 8, Suppl, 1, 106 (1958),
13 .
T . C, BUTLER and W, J . WADDELL, J . Pharmacol , ex~. Ther . 127, 171 (1959) .
14,
W, J, WADDELL and T . C . BUTLER, J . Pharmacol ,
15,
J . COCHIN and J . AXELROD, .J, Pharmacol , ex~, Ther . 125, 105 (1959),
16 .
A, H, CONNEY, C . DAMSON, R . GASTEL, and J . J . BURNS, J . Pharmacol , Ther . 130, 1 (1960) .
17 .
R . KATO and E . CHIESARA, Brit , J . Phazmacol , 18, 29 (1962) .
ex~. Ther . 132,
291 (1961) .