Relationship between the lipid solubility of drugs and their oxidation by liver microsomes

BiochemicalPharmacology.1959, Vol. 2, pp. 89-96. PergamonPressLtd. Printedin Great Britain.

RELATIONSHIP BETWEEN THE LIPID DRUGS AND THEIR OXIDATION MICROSOMES L. E.

and B. B.

GAUDETTE*~

Laboratory of Chemical Pharmacology, Public Health Service, U.S. Department

SOLUBILITY BY LIVER

OF

BRODIE

National Heart Institute, National Institutes of Health, of Health, Education and Welfare, Bethesda, Maryland

(Received 23 December

1958)

Abstract-The oxidative dealkylation of foreign N-alkylamines by rabbit liver microsomes appears to be limited to compounds which are lipid soluble, as shown by high chloroform to water partition coefficients at physiologic pH. Since the microsomal hydroxylation of aromatic compounds also appears to be limited to lipid soluble substances, it is suggested that an intracellular fat-like boundary separates normally occurring polar substances from the highly non-specific microsomal enzymes. The dealkylation of foreign alkylamines is catalysed by at least two enzyme systems in liver microsomes.

recent years, enzyme systems that catalyse the dealkylation of foreign alkylamines have been the subject of a number of studies. Mueller and Miller1 reported that 4dimethylaminoazobenzene and related dyes are demethylated by a rat enzyme system which requires TPN, DPN and oxygen, and that the reaction does not involve transfer of the methyl group, but its oxidation to yield formaldehyde. La Du et aL2 demonstrated that N-dealkylation is accomplished in rabbit liver microsomes by a mechanism that is TPNH-dependent and requires oxygen. The system removes methyl, ethyl and butyl groups. For example, monomethyl-4-aminoantipyrine (MMAP) and its ethyl and butyl analogues are converted to 4-aminoantipyrine, the methyl group appearing as formaldehyde and the ethyl groups as acetaldehyde. The microsomal system dealkylates a variety of N-alkylamines, including methylamphetamine, methadone, methylaniline, ethylaniline, pethidine, mepacrine,2 diacetylmorphine and codeine.3 Normally occurring alkylamines are also dealkylated in the body. Mackenzie et al4 have demonstrated that sarcosine and dimethylglycine are demethylated by highly specific enzyme systems present in mitochondria. In addition, a number of other methylated amino acids are demethylated by an acetone extract of rabbit liver or kidney.= The present report is concerned with the dealkylation of a diversity of alkylamines by rabbit liver microsomes. Despite its non-specificity, the microsomal system does not demethylate sarcosine and dimethylglycine. The evidence suggests that microsomal IN

* In partial fulfilment of the requirements for the degree of Doctor of Philosophy, Dept. of Biochemistry, Georgetown University, Washington, D.C. t Present address: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 89

L. E. GAUDETTE

90

and B. B. BRODIE

dealkylation is limited to compounds which are lipid-soluble and that at least two different enzyme systems are involved. METHODS

AND

MATERIALS

Materials fi-Diethylaminoethyl diphenylpropylacetate HCl (SKF 525-A) was donated by Smith, Kline and French Laboratories. Triphosphopyridine nucleotide (TPN) was purchased from the Sigma Chemical Company. Reduced triphosphopyridine nucleotide (TPNH) was made ch~mically6. A preparation of glucose dehydrogenase was made from hog liver.’ Isolation

of tissue microsomes for enzyme studies Tissue samples were prepared at 0-3°C. Male albino rabbits (New Zealand White) were killed by a blow on the head and the livers were immediately exposed and perfused with l-15 per cent (isotonic) KC1 solution. Livers were removed and homogenized in a Waring blender with 10 ~01s. of isotonic sucrose, containing O-2 per cent nicotinamide. The homogenate was strained through cheese cloth and centrifuged at 9000 x g for 20 min to remove unbroken cells, nuclei and mitochondria. The supernatant fraction, containing microsomes and soluble fraction of the cell, was carefully decanted and then centrifuged at 78,000 x g for I hr. Microsomes were separated, washed once with isotonic sucrose solution and recentrifuged. Enzyme activity was assayed in freshly prepared microsomes or in microsomes that had been dried by lyophilization after suspension in water. Microsomes were suspended in O-1 M phosphate buffer, pH 7.4 in a volume equivalent to that of the original liver. With the lyophilized preparation this corresponded to a quantity of microsomes having a protein content of about 40 mg as measured by the method of Warburg and Christians. Lyophilized micromes were as active as freshly prepared microsomes and were stable for several months.

Analytical methods 4-Aminoantipyrine= and aniline9 were estimated by methods previously described. Formaldehyde and acetaldehyde were trapped by @OI M semicarbazide in the incubation mixture and assayed as previously described.2 The distribution of various substances between chloroform and water was determined by shaking solutions of each substance in 0.1 M phosphate buffer (pH 7.4) with an equal volume of chloroform* for 20 min. The distribution of each substance is represented as the percentage in the organic phase. Measurement of dealkylation activity Previous results have shown that alkylated derivatives of 4-aminoantipyrine and aniline yield amine and aldehyde in equivalent amounts2. Accordingly, the dealkylation of these compounds was measured by the amount of primary amine formed. The dealky~ation of the other alkylamines was estimated from the amount of aldehyde produced. One millilitre of the microsomal preparation was incubated with 5 Fmoles of substrate, 100 pmoles of nicotinamide, 75 pmoles of MgCl,, 100 pmoles of neutralized * Chloroform was purified by successive washes with 1 N NaOH, HCI and water.

Lipid solubility of drugs and their oxidation by liver microsomes

91

semicarbazide hydrochloride, 0.2 pmole of TPN, and a TPNH-generating system. The TPNH-generating system consisted of either 30 pmofes of glucose-6-phosphate plus 1-O ml of a liver supernatant fraction prepared at 78,000 x g and used as a source of glucosed-phosphate dehydrogenase, or 40 pmoles of glucose plus O-4 ml (720 units) of a glucose dehydrogenase preparation’ and water to make a final volume of 5-Oml. The reaction mixture was incubated for 1 hr at 37°C in a Dubnoff metabolic shaking incubator in an atmosphere of air. The reaction was stopped by the addition of trichloroacetic acid to a final concentration of 5 per cent. The proteins were removed by ~entrifugation and the filtrate was analysed for aldehyde or for primary amine. Enzyme activity was expressed in pmoies of amine or aldehyde formed in 1 hr. With from 1 to 5 pmoles of MMAP as substrate, dealkylation followed a first-order reaction. EXPERIMENTAL

Dea~ky~a~~5~ of var~5~ ~lk~~arn~ne~by liver rni~r5~5rne~ In Table I are tabulated the dealkylation rates of a variety of alkylamines, normal

body substrates as well as drugs. The alkylamines are arbitrarily divided into two TABLE 1. LIPID SOLUBILITYAND DEALKYLATION OF A NUMBER OF ALKYLAMINES BY LIVER MICROSOMES % Compound extracted into CI-ICI,

Relative* activity

Compound

>95 >95 >95 >95

Monomethyl-4-aminoantipyrine Ephedrine Pethidine (demerol) ~-Diethyl~in~thyl diphenylpropylacetate Caramiphen (parpanit) I -Phenyl-2-ethoxydiethylamino-3 :5dichlorobenzene N-Methyl aniline N-Ethyl aniline Mepacrine (atabrine) N-Butylaniline Methadone Dimethyl~a~noantip~ine (amidopyrine) Levorphan (levo-dromoran) Diphenhydramine (benadryl) ;F;e;;tgmme

>95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 12 11

Theophylline Theobromine Dimethyl~rotonin Pethidine acid Trimethylphenyl ammonium chloride Adrenaline Sarcosine (monomethylglycine) Dimethylglycine Monomethylaminoethanol Dimethylaminoethanol Choline Creatine


-

A..

Lyophilized microsomes equivalent to 1 g of rabbit liver were incubated with 5 pmoles of substrate as described in “Methods”. Glucose and glucose dehydrogenase were used as the TPNH-generating system. * An activity of 100 is equivalent to the dealkylation of 1 mole of substrate per hour.

L. E. GAUDETTE and B. B. BRODIE

92

groups, those extracted 95 per cent or more into chloroform, and those extracted 15 per cent or less. It is evident that the foreign compounds which underwent measurable dealkylation were highly lipid-soluble at physiologic pH, as evidenced by their high chloroform to water partition ratios. In contrast, poorly lipid soluble alkylamines, whether drugs or normally occurring substrates, were not dealkylated by microsomes. Evidence for more than one de~ikyl~tion system in microsomes

A comparison was made of the dealkylation rates of various N-alkyl derivatives of 4-aminoantipyrine and aniline. MMAP was dealkylated much more rapidly than the other N-alkyl derivatives of 4-aminoantipyrine. In contrast, monomethylaniline was dealkylated at about the same rate as its dimethyl, monoethyl and monobutyl homologues (Table 2). This difference between the two series suggests that more than one dealkylation system may be present in liver microsomes. TABLE 2. ~EALKYLATI~N OF VARIOUSN-ALKYL derivatives AND ANILINE 4-Aminoantipyrine derivatives

033 4-AMlNoA~TlPYRlNE

Aniline derivatives

4-An$oa~nJpyrin Alkyl groups

Alkyl groups

Aniline formed (pmoles)

@moles) CH,--

(CH,),-

’

Ct!HV-(W&Z(&J&k

1.26 0‘34 0.32 0.19 0.34 0.10

‘i

CH,~C~l’--

0.98 090 O-96

$$+-i /I

0;6

-

(~*Hgg)Z-

Lyophilized microsomes equivalent to I g of rabbit liver were incubated with 5 pmoles substrate, as described in “Methods”. Glucose and glucose dehydrogenase were used as the TPNH-generating system.

TABLE 3. INHIBITIONOF THE MICROSOMAL DEALKYLATION BY SKF 525-A Substrate Amidopyrine (dimethyl-4-aminoantipyrine) Monomethyl-4aminoantipyrine Ephedrine Pethidine N-Methylaniline N-Ethylaniline N-Butylaniline N : N-Dimethylaniline Monoethyl+aminoantipyrine MonobutyI~-aminoantip~rine Mepacrine

i

lnhibition (%I :!I 45

;

5i 8 : 0

Microsomes prepared from 1 g liver were incubated with 5 ymoles substrate as described under “Methods” with and without the addition of 4 x lo--* M SKF 525-A*. Glucose-dphosphate and the soluble fraction of the cell used as the TPNH-~nerating system. * SKF 525-A at a concentration

of 5 x 10-s M induces partial denaturation

of proteins.

Lipid solubility of drugs and their oxidation by liver microsomes

93

Experiments with the non-competitive inhibitory agent, SKF 525-A,1° provided more definite evidence for more than one dealkylation system in microsomes. This compound, at a concentration of 1 x IO-* M, inhibited the dealkylation of some Nalkylamines but not of others. For example, it inhibited the demethylation of dimethyl4-aminoantipyrine (amidopyrine), MMAP, ephedrine and pethidine but did not affect the demethylation of methylaniline. Furthermore, SKF 525-A did not inhibit the dealkylation of a number of ethyl- and butylalkylamines, including homologues of 4aminoantipyrine (Table 3). Other results, indicative of more than one dealkylation system, were obtained by comparing dealkylation rates of various drugs by rabbit and guinea pig microsomes. Ratios of dealkylation rates for various compounds in the two species are presented in Table 4. The ratios for MMAP and pethidine were the same, whereas the ratio for TABLE

4. DEALKYLATION

OF VARIOUS SUBSTRATES BY MICROSOMES OF RABBIT AND AND GUINEA PIGS

Ratio of dealkylation rates Species MMAP*/Pethidine Rabbit Guinea pig

MMAP/MEAPt

MMAP/Methylaniline

5:l 1 :I

:j;

411 1:l

Microsomes prepared from 1 g liver were incubated with 5 pmoles substrate as described under “Methods”. Glucose-6-phosphate and the soluble fraction were used as the TPNH-generating system. * MMAP, Monomethyl-4aminoantipyrine 7 MEAP, Monoethyl-4aminoantipyrine.

MMAP and its ethyl homologue was higher in rabbits than guinea pig. These results suggest that the enzyme systems involved in the dealkylation of the methyl and ethyl derivatives of 4-aminoantipyrine may be different. Similar studies indicate that methylaniline and MMAP also may be dealkylated by different enzymes.

TABLE

5. DEALKYLATION OF SARCOSINE BY RABBIT LIVER MITOCHONDRIA Form;m;yde Incubation

system -

Mitochondria Mitochondria + 1 x 1O-3 M CNDialysed mitochondria Dialysed mitochondria + TPNH-generating system* Dialysed mitochondria + DPNH-generating system* Dialysed mitochondria -t 1 x 1O-4 M cytochrome c * Glucose dehydrogenase

(pmolesihr per g tissue)

I.50 0.70 0.10 0.19 0.13 0.62

with 0.2 pmole of coenzyme was used as the generating system.

Mitochondria from 25 g of liver were dialysed for 24 hr against 0.1 M phosphate buffer (pH 7.4) and then incubated for 1 hr at 37°C with 5 rmoles of sarcosine and additions as indicated, in a final volume of 5 ml of 0.1 M phosphate buffer.

94

L.

E.

GAUDE‘TTE

and 3. B.

ERODE

Mackenzie4 has reported that dimethylglycine and monomethylgly~ine (sarcosine) are demethylated to glycine and formaldehyde by two different enzyme systems localized in liver mitochondria, Since mi~rosomal systems do not dealkylate these methylamino acids {Table 1), it was of interest to compare the properties of the microsomal and the mitochondrial enzyme systems. Rabbit liver mitochondria, prepared as described by Mackenzie4, readily demethylated sarcosine. Activity was lost on dialysis against phosphate buffer for 24 hr. The addition of flavine adenine dinu~leotide (FAD) (50 pg), TPN (200 pg), DPN (200 pg), DPNH (0.2 pmoie) and TPNH (0.2 pmoles) failed to restore the activity, but considerable activity was restored on the addition of cytochrome-c (lo-& M) (Table 5). This observation is of particular interest since cytochrome-c inhibits the d~aIkylation of foreign compounds by microsomes. In contrast, cyanide which does not affect microsomal dealkylation,ll markedly inhibited the demethylation of sarcosine. When amidopyrine or MMAP was incubated with the mito~hondrial preparation, no demethylation was observed under conditions in which sarcosine was readily demethylated. Moritani et aI.” have demonstrated that an acetone powder extract of rabbit liver or kidney demethylates N-methyl-I-histidine and other methylamino acids, but does not act on sarcosine or dimethylglycine. These workers have postulated that the methylamino acids undergo dehydrogenation by the action of a FAD-flavoprotein. On incubation of amidopyrine and MMAP with acetone powder extracts of rabbit liver and kidney, these ~ornpoun~ were not deme~hylated under conditions in which N-methylhistidine was readily demethylated. DISCUSSION A diversity of foreign alkyIami~es are oxidatively dealkylated by enzyme systems in liver microsomes. These enzymes seem to have an extraordinary degree of nonspecificity. However, more than one dealkylation system may be present in rabbit microsomes. For example, the enzyme system that removes methyl groups from monomethyl-4-aminoantipyrine (MMAP) may differ from that which removes higher alkyl groups from homologues of 4-aminoantipyrine. /3-Dimethylaminoethyl diphenylpropylacetate (SKF 525-A), a non-competitive inhibitor, antagonizes the demethylation of MMAP, pethidine and a number of other methylated amines, but does not interfere with the dealkylation of the ethyl and butyl homologues of MMAP. Moreover, the relative rates of dealkylation in rabbit and guinea pig microsomes are the same for MMAP and pethidine, but different for MMAP and its ethyl homologue. Of particular interest is the evidence that the enzyme system that demethylates methylaniline may differ from that which removes the methyl groups from a number of other methylamines. Axelrod3 has also reported evidence for more than one demethylation system present in microsomes. Despite the probability that more than one dealkylatio~ system is present in microsomes, it is likely that the dealkylation of a host of foreign alkylamine compounds is achieved by relatively few enzymes. A traditional belief holds that drugs undergo chemical transformation because their structures are similar to those of substrates in internlediary mettibolism. It is noteworthy, therefore, that normally occurring ~-alkyl~ines, such as sarcosine, dime~hylglycine, dimethylaminoethanol, choline, creatine and epinephrine are not

Lipid solubility of drugs and their oxidation by liver microsomes

95

demethylated by liver microsomes, though the demethylation of sarcosine and dimethylglycine is accomplished by highly specific systems in mitochondria.4 A similar situation exists for the hydroxylation of aromatic compounds. Despite the nonspecificity of the microsomal hydroxylating system with respect to foreign compounds, it does not promote the hydroxylation of L-phenylalanine, L-tryptophane, kynurenine and anthranilic acid, naturally occurring compounds which are hydroxylated by quite specific systems localized in other parts of the liver ce11.12 The failure of liver microsomes to act on these normally occurring substrates is surprising in view of the unusually broad range of foreign compounds which they metabolize. Some insight into this apparent paradox is provided by a comparison of the metabolism of a series of foreign N-alkylamines. Only those compounds that have a high chloroform to water partition ratio are oxidized by rabbit microsomes in vitro (Table 1). This suggests that the microsomal oxidative systems are protected by a lipoid barrier which only fat soluble substances can penetrate. The evidence for this hypothesis is strengthened by the results with the series, caffeine, theophylline and theobromine; only caffeine, which has a relatively high lipid solubility, is demethylated by microsomes. An alternative explanation may be that the active sites on the microsomal enzymes can interact only with non-polar substances. It should be also noted that the afore-mentioned normal substrates which are not hydroxylated by liver microsomes, are also poorly lipid soluble. Other examples of the importance of lipid solubility are found in substrates of the deamination enzyme present in rabbit microsomes, but absent in a number of other mammalian species.13 This enzyme deaminates the lipid soluble substances amphetamine, methamphetamine and ephedrine, but does not act on substances like tyramine or serotonin,13 which are substrates of monoamine oxidase. Older studies have indicated that monoamine oxidase in rabbits, but not in other species, deaminates mescaline. l4 This observation can be explained if mescaline, a highly lipid soluble compound, is metabolized by rabbit liver microsomes. It is of particular interest that the dealkylation of foreign compounds is achieved by quite a different mechanism than that which acts on sarcosine. Dealkylation by the microsomal system requires TPNH and involves activation of oxygen; similar requirements have been noted for the oxidative mechanisms in microsomes that hydroxylate aromatic rings and aliphatic side chains, cleave ethers, deaminate alkylamines and oxidize sulphur-containing compounds to sulphoxides.15 It has been suggested that a common step in microsomal oxidation may be the formation of an intermediate “hydroxyl” donor, which, in conjunction with a number of non-specific enzymes, can transfer an hydroxyl group to an appropriate acceptor substrate.15 Thus, dealkylation of a foreign N-alkylamine may be written as the direct substitution of a hydroxyl grouping for a hydrogen.

R-NHCH

3

__-:oH1

-+ [R-NHCH 2OH] unstable

------3

RNH, + HCHO

The mitochondrial enzyme system which acts on sarcosine does not require TPNH and unlike the microsomal systems it is blocked by cyanide. It appears to be a de-

L. E.

96

GAUDETTE and

hydrogenase linked with the cytochrome may be written as follows:

I

R-N-CH3

-2H+-

R-N=CH,

B. B.

BRODIE

system. Thus, the dealkylation

HoH+

I

[R-N-CH,OH]

F-3

of sarcosine

R-NH, -I+ HCHO

unstable REFERENCES 1. G. C. MUELLER and

2. 3. 4. 5. 6. 1. 8. 9. 10. 1I. 12. 13. 14. 15.

.I. A.

MILLER, J.

Biol. Chem. 202, 579 (1953). B. N. LA Du, L. E. GAUDETTE, N. TROUSOF and B. B. BRODIP, J. Biol. Chem. 214, 741 (1955). J. AXELROD, J. Pharmacol. 117, 322 (1956). C. G. MACKENZIE and W. R. FRISELL,J. Biol. Chem. 232,417 (1958). M. MORITANI, T. TUNG, S. FUIII, H. MITI, N. IZUMAZI, K. KENMOCHI and R. HIROHATO, J. Biol. Chem. 209,485 (1954). N. 0. KAPLAN, S. P. COLOWICK and E. F. NEUFELD, J. Biol. Chem. 195, 107 (1952). H. J. STRECKERand S. KORKES, J. Biol. Chem. 196, 769 (1952). 0. WARBURG and W. CHRISTIAN, Biochem. Z. 310, 384 (1941). B. B. BRODIE and J. AXELROD, J. Pharmacol. 94, 22 (1948). B. N. LA Du, E. C. HORNING, H. B. WOOD,N. TROUSOF and B. B. BRODIE, Fed. Proc. 13, 377 (1954). J. R. GILLETTE,B. B. BRODIEand B. N. LA Du, J. PharmacoL 119,532 (1957). C. MITOMA, H. S. POSNER, A. C. REITZ and S. UDENFRIEND,Arch. Biochem. and Biophys. 61,43 1 (1956). J. AXELROD, J. Pharmacol. 110, 315 (1954). H. BLASCHKO, Pharmacol. Rev. 4, 415 (1952). B. B. BRODIE, J. R. GILLETTEand B. N. LA Du, Ann. Rev. Biochem. 27,427 (1958).