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332. Fifteen metabolites of thalidomide
TERATOGENESIS
325
332. Fifteen metabolites of thalidomide Previous work on the fate of thalidomide (I) showed that dogs excreted 6 metabolites of I labelled with t4C together with unchanged I in the urine. The major metabolite was ~-(ocarboxybenzoylamino)-glutarimide (see p. 323 of this issue). Also phthaloylisoglutamine, unchanged I, and an unidentified derivative of 3-hydroxyphthalic acid were excreted in the urine by man, rabbits and rats given I (Cited in F.C.T. 1963, 1, 153). In response to the report by Boylen et al. (Lancet 1963, i, 552) discussed in the previous abstract, the author of T
co CO0 ~-.H
Tholidomide
"%0\
(I) \
i
;" ~ COIN__C:H~ -C.H~-..... 2 CHz
~r'~'~""~"C-'Q CI'~2 L~. L ,CHz ~ ~C-~) COOHCONHz
..--
~
%CO
CONH2
N--C"
COOH
Phthaloybsoglulomine (2)
Phthaloylglutamine (3)
/t
° a - ( o - Corboxybenzoylomino)glutarimide(4)
/"
..d. co "~
~
COOH
( 6 ) (7)
COOHt i
Phtholoylglulamic acid (5)
~>~.~ / COOH +
;L oo.
[•C•
NH_c~CH~"~CH COOH ~ !^2,, ~)Ul'l ~,UI~I=l2
L ~ . L..COOH C~ONHz COOH
^.
o - Corboxybenzoylglutomine(7)
ii
o - C o r b o xybenzoyltL~oglulomine (6)
\
f/2~/C~0
~-C H~-~. 9
(IL)
U2)
',,,/ (13)
o - Corboxybenzoylgluta mic ~id(8) /-CH~.... NH~ CH CHz CONHz COOH Zsoglutomine(ll)
/CH~ NHz~H ?Hz~ COOH COOH Glutamic acid(13)
/CHz\ NHz~H ~Hz COOH CONH2 Glulamine (12)
Fig.I.Hydrolytic breakdown of thalidomide.
0~.~NH~
o
Phthallc ocid(9)
F2"~.," C~ONH_CH/" CH~'-~CHz
NH2
c=-Aminogluto rimide(lO)
326
TERATOGENESIS
the present paper presented more recent findings on the metabolism of I, some of which had been previously reported by him (Cited in F.C.T. 1963, 1, 153). Following the administration of unspecified doses of the racemate of I to rabbits, 16 compounds including unchanged I were found in the urine. Twelve of these, isolated in crystalline form, proved to have the following structures: phthaloylisoglutamine, phthaloylglutamine, phthaloylglutamic acid, ct-(o-carboxybenzoylamino)-glutarimide, o-carboxybenzoylisoglutamine, o-carboxybenzoylglutamine, o-carboxybenzoylglutamic acid, ~aminoglutarimide, phthalic acid, glutamine, isoglutamine and glutamic acid. Each of these 12 compounds could arise by simple hydrolysis of I. Indeed it was shown that at 37 ° in phosphate buffer, pH 7.4, 80~o of I decomposed in 24 hr to the 12 products previously found in the urine. All four amide linkages of I undergo hydrolysis, which appears to proceed in three stages as shown in Fig. 1" (Williams, personal communication). Also, I was unstable at pH values above 6.5. The lability of I at physiological pH suggests that, by administering I, one is in effect giving a mixture of the hydrolytic products of I. The urine also contained three hydroxy derivatives of I which have so far been isolated but await identification. An examination of the faeces revealed the presence of ~-(o-carboxybenzoylamino)-glutarimide and unchanged I. In order to see whether I or its metabolites interfered with glutamic acid metabolism, two enzymes of rat brain, glutamine synthetase and glutamate dehydrogenase were selected. One of the hydroxy derivatives was found to be a potent inhibitor of glutamine synthetase --less effective inhibitions were obtained with o-carboxybenzoylglutamic acid and another hydroxy derivative. [The complexity of this problem and the magnitude of work involved in elucidating the fate of I in the body is realized, when one considers that in theory there are over I00 possible metabolites based on the application of the two detoxication mechanisms hydroxylation and hydrolysis to I and on the possible existence of optically active or inactive derivatives.] Williams, R. T. (1963). Teratogenic effects of thalidomide and related substances. Lancet i, 723.
333. Thalidomide and derivatives inhibit leucocyte development One of the major questions still unsolved concerning thalidomide (I) is the way in which it produces teratogenic effects. Since the drug is known to inhibit leucocyte development in man (Cited in F.C.T. 1963, 1, 153) the authors decided that a further investigation into the biological mechanisms involved in the inhibitory effect may shed some light on the mode of teratogenic action. They set out to determine the concentration at which I is most inhibitory; to compare the effectiveness of D and L forms of I and finally to study the effect of several derivatives of I on leucocyte development. The concentration of each compound incubated with cultures of human leucocytes was either 15 or 20 tzg/ml. The compounds used, together with the observed ~ inhibitions of leucocyte development in parentheses are as follows: D-thalidomide (51), L-thalidomide (43), phthaloylglutamic acid (27), phthaloylglutamine (0), phthaloylisoglutamine (0), o-carboxybenzoylglutamine (48), o-carboxybenzoylisoglutamine (54), o-carboxybenzoylglutamic acid (17), ~-(o-carboxybenzoylamino)-glutarimide (24) and glutamic acid (0). A 50 inhibition resulted at a concentration of 3/zg I/ml, which simulates plasma levels of I found in animals and consequently has a bearing on the effects of I in vivo. It is possible on the grounds of structural similarities that I or its active derivatives act as antagonists of glutamic acid, glutamine, folic acid or less likely one of the other vitamins in the B group. Roath, S., Elves, M. W. & Isra/fls, M. C. G. (1963). Effects of thalidomide and its derivatives on human leucocytes cultured in vitro. Lancet i, 249. °We wish to thank Professor R. T. Williams for granting us permission to reproduce Fig. 1.
325
332. Fifteen metabolites of thalidomide Previous work on the fate of thalidomide (I) showed that dogs excreted 6 metabolites of I labelled with t4C together with unchanged I in the urine. The major metabolite was ~-(ocarboxybenzoylamino)-glutarimide (see p. 323 of this issue). Also phthaloylisoglutamine, unchanged I, and an unidentified derivative of 3-hydroxyphthalic acid were excreted in the urine by man, rabbits and rats given I (Cited in F.C.T. 1963, 1, 153). In response to the report by Boylen et al. (Lancet 1963, i, 552) discussed in the previous abstract, the author of T
co CO0 ~-.H
Tholidomide
"%0\
(I) \
i
;" ~ COIN__C:H~ -C.H~-..... 2 CHz
~r'~'~""~"C-'Q CI'~2 L~. L ,CHz ~ ~C-~) COOHCONHz
..--
~
%CO
CONH2
N--C"
COOH
Phthaloybsoglulomine (2)
Phthaloylglutamine (3)
/t
° a - ( o - Corboxybenzoylomino)glutarimide(4)
/"
..d. co "~
~
COOH
( 6 ) (7)
COOHt i
Phtholoylglulamic acid (5)
~>~.~ / COOH +
;L oo.
[•C•
NH_c~CH~"~CH COOH ~ !^2,, ~)Ul'l ~,UI~I=l2
L ~ . L..COOH C~ONHz COOH
^.
o - Corboxybenzoylglutomine(7)
ii
o - C o r b o xybenzoyltL~oglulomine (6)
\
f/2~/C~0
~-C H~-~. 9
(IL)
U2)
',,,/ (13)
o - Corboxybenzoylgluta mic ~id(8) /-CH~.... NH~ CH CHz CONHz COOH Zsoglutomine(ll)
/CH~ NHz~H ?Hz~ COOH COOH Glutamic acid(13)
/CHz\ NHz~H ~Hz COOH CONH2 Glulamine (12)
Fig.I.Hydrolytic breakdown of thalidomide.
0~.~NH~
o
Phthallc ocid(9)
F2"~.," C~ONH_CH/" CH~'-~CHz
NH2
c=-Aminogluto rimide(lO)
326
TERATOGENESIS
the present paper presented more recent findings on the metabolism of I, some of which had been previously reported by him (Cited in F.C.T. 1963, 1, 153). Following the administration of unspecified doses of the racemate of I to rabbits, 16 compounds including unchanged I were found in the urine. Twelve of these, isolated in crystalline form, proved to have the following structures: phthaloylisoglutamine, phthaloylglutamine, phthaloylglutamic acid, ct-(o-carboxybenzoylamino)-glutarimide, o-carboxybenzoylisoglutamine, o-carboxybenzoylglutamine, o-carboxybenzoylglutamic acid, ~aminoglutarimide, phthalic acid, glutamine, isoglutamine and glutamic acid. Each of these 12 compounds could arise by simple hydrolysis of I. Indeed it was shown that at 37 ° in phosphate buffer, pH 7.4, 80~o of I decomposed in 24 hr to the 12 products previously found in the urine. All four amide linkages of I undergo hydrolysis, which appears to proceed in three stages as shown in Fig. 1" (Williams, personal communication). Also, I was unstable at pH values above 6.5. The lability of I at physiological pH suggests that, by administering I, one is in effect giving a mixture of the hydrolytic products of I. The urine also contained three hydroxy derivatives of I which have so far been isolated but await identification. An examination of the faeces revealed the presence of ~-(o-carboxybenzoylamino)-glutarimide and unchanged I. In order to see whether I or its metabolites interfered with glutamic acid metabolism, two enzymes of rat brain, glutamine synthetase and glutamate dehydrogenase were selected. One of the hydroxy derivatives was found to be a potent inhibitor of glutamine synthetase --less effective inhibitions were obtained with o-carboxybenzoylglutamic acid and another hydroxy derivative. [The complexity of this problem and the magnitude of work involved in elucidating the fate of I in the body is realized, when one considers that in theory there are over I00 possible metabolites based on the application of the two detoxication mechanisms hydroxylation and hydrolysis to I and on the possible existence of optically active or inactive derivatives.] Williams, R. T. (1963). Teratogenic effects of thalidomide and related substances. Lancet i, 723.
333. Thalidomide and derivatives inhibit leucocyte development One of the major questions still unsolved concerning thalidomide (I) is the way in which it produces teratogenic effects. Since the drug is known to inhibit leucocyte development in man (Cited in F.C.T. 1963, 1, 153) the authors decided that a further investigation into the biological mechanisms involved in the inhibitory effect may shed some light on the mode of teratogenic action. They set out to determine the concentration at which I is most inhibitory; to compare the effectiveness of D and L forms of I and finally to study the effect of several derivatives of I on leucocyte development. The concentration of each compound incubated with cultures of human leucocytes was either 15 or 20 tzg/ml. The compounds used, together with the observed ~ inhibitions of leucocyte development in parentheses are as follows: D-thalidomide (51), L-thalidomide (43), phthaloylglutamic acid (27), phthaloylglutamine (0), phthaloylisoglutamine (0), o-carboxybenzoylglutamine (48), o-carboxybenzoylisoglutamine (54), o-carboxybenzoylglutamic acid (17), ~-(o-carboxybenzoylamino)-glutarimide (24) and glutamic acid (0). A 50 inhibition resulted at a concentration of 3/zg I/ml, which simulates plasma levels of I found in animals and consequently has a bearing on the effects of I in vivo. It is possible on the grounds of structural similarities that I or its active derivatives act as antagonists of glutamic acid, glutamine, folic acid or less likely one of the other vitamins in the B group. Roath, S., Elves, M. W. & Isra/fls, M. C. G. (1963). Effects of thalidomide and its derivatives on human leucocytes cultured in vitro. Lancet i, 249. °We wish to thank Professor R. T. Williams for granting us permission to reproduce Fig. 1.