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Aspects of the interaction of thyroxine and its binding serum proteins with 1,4-benzodiazepine tranquillizers
LUOCHEMICAL
MEDICINE
26, 191-198
(1981)
Aspects of the Interaction of Thyroxine and its Binding Serum Proteins with 1,dBenzodiazepine Tranquillizers M. A. F. EL-HAZMI Department
of Biochemistry, College of Medicine, P. 0. Box University of Riyadh, Riyadh, Saudi Arabia
2925,
ReceivedDecember2, 1980
Tranquilizers, like many cations, anions, metabolites, and drugs, are transported in the bloodstream and stored mainly by albumin. Binding of these molecules onto human serum albumin (HSA) is not random, but is a defined phenomenon subject to the requirement of noncovalent bonds between these molecules and albumin. Using various methods, Miiller and Wallert (6, 7) and Sjodin and co-workers (10) have shown that chlordiazepoxide and diazepam are mainly bound to one site on HSA. A limited information is available in the literature concerning the binding of drugs to blood globulins. In general, several drugs, particularly lipid-soluble, can bind to globulins (2). Our previous work showed that diazepam and chlordiazepoxide interfere with thyroid function tests as they compete with L-Tq-‘251for binding sites (1). The present studies were carried out in order to investigate the specificity of the interactions between thyroxine binding protein (TBP) and chlordiazepoxide and diazepam and its metabolites. The electrophoretic behavior of the interaction was studied to illustrate the drugs’ effects on the distribution of T, among its binding proteins. It was hoped that more information about the specificity of the TBP binding sites might be gained. MATERIALS
AND METHODS
Normal male sera were obtained from the local blood transfusion center, pooled, and kept at 4°C for at least 24 hr before the experiments were performed. Chlordiazepoxide-HCl solution (Librium, 50 mg) was prepared by dissolving 1 ml saline solution. Increments of this solution were added to the serum to yield the desired concentrations. Diazepam (Valium, 50 191 ooo6-2944/81/050191-08$02.0010 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
192
M. A. F. EL-HAZMI
mg) was dissolved in 1 ml of 96% (v/v) methyl alcohol. Increments of the drug solution were dried under nitrogen stream and serum was added to the dried drug which was then mixed and incubated at 37°C for 6 hr before electrophoresis. A serum control, without added drug, was run in parallel with drug-containing sera in each experiment. Electrophoreses were carried on 30 ~1 of serum using specially designed apparatus (Fig. 1) following the addition of 1.4 ~1 L-T,-‘25I (0.2 mWm1) to 100 l.~l serum. Preliminary attempts revealed poor separation of thyroxine binding prealbumin (TBPA) from albumin on paper electrophoresis and of thyroxine binding globulin (TBG) from albumin on starch gel electrophoresis. There was a need for control of both temperature and pH in view of the marked effects of pH and temperature variations on the distribution of T, among serum proteins. These findings necessitate design of an apparatus (Fig. 1) for preparative purpose that allows material manipulation for radioactive counting,
FIG. 1. Basic outfit for vertical slab gel electrophoresis apparatus: (a) Higher buffer reservoir; (a’) lower buffer reservoir with platinum electrodes and a maximum capacity of 5 liters buffer: (b) base of cooling plate that is in contact with outer gel plate. The water runs straight to the bottom of the plate and rises through openings in a transverse tube to the top of the plate and runs into smaller tubes to a cooling circulating water bath: (c) screw to hold cooling plates with gel sandwiched in between: (d) side cover: (e) upper safety cover, with (f) an electricity input plug; (f) inflow of water. The large arrows indicate direction of buffer flow that is circulated by a pump.
THYROXINE-TRANQUILLIZER
INTERACTIONS
193
staining, and autoradiography with provision for slicing of the gel and maintaining constant pH and temperature. The variables were overcome by using a large volume of circulating buffer and cooling water through cooling plates on both sides of the gel. Variation of buffer, voltage, thickness of the gel, and length of run established that the optimum system was 8% (w/v) cyanogum 41 polyacrylamide gel, 0. I M phosphate buffer, pH 7.4, over 15 hr electrophoresis at 100 V during which the temperature can be held constant at 4°C. A 6-mm-thick gel (100 x 120 mm) with 13 slots was prepared from 20 g cyanogum 41 in 0. I M phosphate buffer, pH 7.4. The gel was aliowed to cool to 4°C for 30 min before preelectrophoresis was carried out at 100 V (about 300 mA) for 1 hr. Serum (30 ~1) with or without drug or hormone, was applied in duplicate and electrophoresis was carried out for 15 hr at 100 V with buffer circulating maintaining the temperature at 4°C. At the end of electrophoresis, the gel was sliced into two parts, one of which was stored at 4°C until the other was stained. The unstained gel was aligned with the stained half of the gel with a glass plate in between and the unstained gel was cut longitudinally between electrophoretic paths and each path cut out laterally into eight fractions with fractions l-4 corresponding to protein run between origin and Pre-TBG region. Fractions 5, 6, 7 and 8 correspond to TBG, albumin, prealbumin1, and prealbumin-2, respectively. The protein-bound radioactive thyroxine in each fraction was counted for 1 min and the result was corrected for background. The amount of radioactive T, bound to specific protein was expressed as a percentage of the total counts per sample. For autoradiography, the gel was exposed to X-ray film, sandwiched between two glass plates, and stored at -70°C for an appropriate period of time (l-3 days). The X-ray film was developed using Exprol Rapid Developer, diluted with an equal volume of water, and Ilford Hypam Rapid Fixer. RESULTS
Electrophoresis of drug-free (control) and drug-containing sera with added L-T,-“‘1 revealed a remarkable differences in the affinity of the drug for the T, binding sites on TBG. Assessed as a function of the drug ability to displace L-Tq-‘251, where the percentage of bound L-T,-‘*‘1 was used to measure the degree of displacement, chlordiazepoxide and diazepam exhibited different dissociation patterns for L-T,-“‘I. Moreover, chlordiazepoxide and diazepam differ somewhat in the T, binding proteins on which they exert their effect. While chlordiazepoxide effects TBPA, diazepam has it displacing effect on L-T,‘*‘1 binding to TBG (Fig. 2). With chlordiazepoxide albumin seems to be the main
194
M. A. F. EL-HAZMI
A 1
a
+
234567123456
FIG. 2. Autoradiogram of TBP-L-T,-‘? separated by polyacrylamide gel electrophoresis. Al, prealbumin marker; A2, serum-L-T,-‘*‘1 control: A3-A7. serum-L-T4-‘2SI in the presence of increasing chlordiazepoxide concentration (0.02-I .O g/liter). B I-B6, serum-lT,-“‘1 in the presence of increasing concentration of diazepam (0.01-I .O g/liter).
recipient of the displaced hormone and with diazepam the displaced LT,-“‘I was shown to be bound to TBPA. Diazepam metabolites showed similar displacing pattern for L-T4-‘25I to that of diazepam but of lesser effect (Fig. 3). The three metabolites differ in that desmethyldiazepam has a stronger effect on L-T,-“‘1 binding to TBG than 3-hydroxydiazepam and oxazepam. TBPA is the common recipient of the displaced L-T4-12’I from TBG by these drugs (Fig. 4). DISCUSSION The variation in the binding affinity to TBP for thyroid hormones and the benzodiazepines in human sera indicated that these proteins, in spite of their diversity and complexity, have certain reactive groups in common and that there is a unique sequence involved in this highly specific binding site. Nevertheless, the detailed organization of each binding site on a specific binding protein is different. The structural and conformational requirement for optimal binding of T, appears to be best met on the TBG molecule hence this protein has the highest affinity for T,. The hypothesis of Schussler (9) and Oppenheimer and Tavernitti (8) proposed that the T, binding site on TBG is complimentary to the diphenyl ether structure of T4, as was predicted from structural similarities. However, the decreasing affinity of T, for binding to TBG suggests that anionic substituents on the benzene rings are necessary for strong binding. Based on the similarities in the benzene structure of chlordiazepoxide, diazepam, and its metabolites (Fig. 5) the percentage of increasing or
THYROXINE-TRANQUILLIZER
01 0.00
.? 2 5 a
0.20
0.40 Drug
concentration
195
INTERACTIONS
0.60
0.60
1.0 0
(s/l)
FIG. 3. Effect of the presence of diazepam and its metabolites in the TBP solution and TBG binding of L-T,-‘% A, Diazepam; 0, 3-hydroxydiazepam; A, desmethyldiazepam; n , oxazepam.
decreasing binding of L-T,- “? to a serum protein can be used to elaborate on the effect of functional groups on the affinity of protein binding. As is shown by the ability of the drugs to displace L-T,-“‘1 from its specific binding sites on TBG and TBPA, the C-diazepine ring appears to play an additional role in the binding to T, sites on TBP. Furthermore, variation in the displacing effect of the benzodiazepines revealed the importance of the substituents on the diazepine ring for the binding onto TBP. It is possible that benzodiazepine binding onto TBG may involve the whole molecule. The remarkable ability of diazepam to displace LT,-12’I from TBG compared with chlordiazepoxide indicates the importance of the methyl group at N, for strong binding. Having a less binding affinity, 3-hydroxydiazepam and oxazepam revealed the effect of the substitution and the extent of the involvement of the positions 2 and 3 of the diazepine ring of the drug molecules. To elaborate on the differential binding properties of diazepam and chlordiazeposide it could be worthwhile to consider special arrangements of thyroxine. Leonard and Sutton (5) proposed that a valency angle of about 120” is assumed for the diphenyl ether oxygen, and Jorgensen (4) reported that the two phenyl rings are not in the same plane but lie at
196
M. A. F. EL-HAZMl
300 I 260
4 a m I”
,,....“’ ..4
260_:’
,:’
..
,.A..”
. ..’ 240-
,:’
L
Drug concentration(Q/l) FIG. 4. Effect of diazepam and its metabolites on TBPA binding of L-T,-‘% as for Fig. 3.
Symbols
almost right angles to each other. As 1,Cbenzodiazepines are assymetric and their B-benzene rings are not planar, it is conceivable that the B-benzene ring has a valency angle with the C-benzodiazepine ring similar to that of diphenyl ether oxygen. Schussler (9) postulated that the N, of the diazepine ring is involved in the binding to TBG. If this is true, the N-O of chlordiazepoxide, which exhibits a low affinity for TBG, may affect the spatial orientation of the benzene ring. In a similar way, the B-benzene ring of the benzodiazepine molecule is not situated in the same plane as the C-ring owing to steric hinderance (IO). It is possible that N-O bond can conceivably affect the free rotation of the
THYROXINE-TRANQUILLIZER
197
INTERACTIONS
Cl
--RI Ii
R2
3
NH-Cl-l3
H
- R4 -0
Chlordiarcpoxide
(librium(*))
CH3
0
H
Diazepam
H
0
H
Desmethyldiazepam
CH3
0
OH
3-Hydroxydiarepam
H
0
OH
Oxazepam
FIG.
(ValiumcR)
)
(SetaxfR))
5. Chemical structure of 1,4-benzodiazepines.
B-benzene ring for optimal binding, or alternatively by altering the electron distribution, may affect its hydrophobic interactions for the binding onto TBG. The hydrogen substitution for oxygen at N, of the diazepam ring would increase the lipophilic properties of the chlordiazepoxide molecule, but this might be suppressed by the restriction of spatial orientation of the B-rings by the NHCH, group at C2. This may explain the low affinity of chlordiazepoxide for TBG binding site. Of interest is the finding that diazepam is a more potent tranquilizer and is also a more effective binding competitor of T, than chlordiazepoxide. To relate the chemical structure to the pharmacological effectiveness, these findings may indicate a common receptor for thyroid hormones and benzodiazepines, the organization of which is optimal for thyroid hormones and diazepam. In light of these reports of previous work and our studies, it can be concluded that chlordiazepoxide and diazepam, and possibly also chemically related compounds, compete for the T, binding sites on TBP in vitro. Greenberg and co-workers (3) showed that in vim, neither drug exerts a constant effect on the binding of thyroid hormones or their concentration in the circulation. However, these observations do not preclude that under abnormal conditions, for example in case of a combination of albumin deficiency and multiple drug treatment, some disturbance might result. This is particularly relevant for the low albumin
198
level in the elderly therapy.
M. A. F. EL-HAZMI
who are also quite frequently
on a multiple-drug
REFERENCES 1. El-Hazmi, M. A. F., C/in. Chirn. Acta 63, 211 (1975). 2. Goldstein, A., Pharmacol. Rev. 1, 102 (1949). 3. Greenberg, A. H., Gzernichow, P., and Blizzard, R. M.. Johns Hopkins Med. J. 126, 134 (1970). 4. Jorgensen, E. C., Mayo Clinic Proc. 39, 560 (1964). 5. Leonard, J. N., and Sutton, L. E., J. Amer. Chern. Sot. 70, 1564 (1948). 6. Miiller, W.. and Wallert, U.. Narrnvn-Schiedr~berg’s Arch. Pharmukol. 283, 67 (1974). 7. Miiller, W., and Wallert, U.. Naun~n-Schtniedeberg*s Arch. Pharmakol. 280, 229 (1973). 8. Oppenheimer, H. J., and Tavernitti. R. P., J. CIirl. Invest. 41, 2213 (1962). 9. Schussler. G. C.. J. Pharmacol. Exp. Ther. 178, 204 (1971). 10. Sjodin, T.. Roosdrop, N., and Sjoholm, I.. Biochern. fhnrmucoi. 25, 2131 (1976).
MEDICINE
26, 191-198
(1981)
Aspects of the Interaction of Thyroxine and its Binding Serum Proteins with 1,dBenzodiazepine Tranquillizers M. A. F. EL-HAZMI Department
of Biochemistry, College of Medicine, P. 0. Box University of Riyadh, Riyadh, Saudi Arabia
2925,
ReceivedDecember2, 1980
Tranquilizers, like many cations, anions, metabolites, and drugs, are transported in the bloodstream and stored mainly by albumin. Binding of these molecules onto human serum albumin (HSA) is not random, but is a defined phenomenon subject to the requirement of noncovalent bonds between these molecules and albumin. Using various methods, Miiller and Wallert (6, 7) and Sjodin and co-workers (10) have shown that chlordiazepoxide and diazepam are mainly bound to one site on HSA. A limited information is available in the literature concerning the binding of drugs to blood globulins. In general, several drugs, particularly lipid-soluble, can bind to globulins (2). Our previous work showed that diazepam and chlordiazepoxide interfere with thyroid function tests as they compete with L-Tq-‘251for binding sites (1). The present studies were carried out in order to investigate the specificity of the interactions between thyroxine binding protein (TBP) and chlordiazepoxide and diazepam and its metabolites. The electrophoretic behavior of the interaction was studied to illustrate the drugs’ effects on the distribution of T, among its binding proteins. It was hoped that more information about the specificity of the TBP binding sites might be gained. MATERIALS
AND METHODS
Normal male sera were obtained from the local blood transfusion center, pooled, and kept at 4°C for at least 24 hr before the experiments were performed. Chlordiazepoxide-HCl solution (Librium, 50 mg) was prepared by dissolving 1 ml saline solution. Increments of this solution were added to the serum to yield the desired concentrations. Diazepam (Valium, 50 191 ooo6-2944/81/050191-08$02.0010 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
192
M. A. F. EL-HAZMI
mg) was dissolved in 1 ml of 96% (v/v) methyl alcohol. Increments of the drug solution were dried under nitrogen stream and serum was added to the dried drug which was then mixed and incubated at 37°C for 6 hr before electrophoresis. A serum control, without added drug, was run in parallel with drug-containing sera in each experiment. Electrophoreses were carried on 30 ~1 of serum using specially designed apparatus (Fig. 1) following the addition of 1.4 ~1 L-T,-‘25I (0.2 mWm1) to 100 l.~l serum. Preliminary attempts revealed poor separation of thyroxine binding prealbumin (TBPA) from albumin on paper electrophoresis and of thyroxine binding globulin (TBG) from albumin on starch gel electrophoresis. There was a need for control of both temperature and pH in view of the marked effects of pH and temperature variations on the distribution of T, among serum proteins. These findings necessitate design of an apparatus (Fig. 1) for preparative purpose that allows material manipulation for radioactive counting,
FIG. 1. Basic outfit for vertical slab gel electrophoresis apparatus: (a) Higher buffer reservoir; (a’) lower buffer reservoir with platinum electrodes and a maximum capacity of 5 liters buffer: (b) base of cooling plate that is in contact with outer gel plate. The water runs straight to the bottom of the plate and rises through openings in a transverse tube to the top of the plate and runs into smaller tubes to a cooling circulating water bath: (c) screw to hold cooling plates with gel sandwiched in between: (d) side cover: (e) upper safety cover, with (f) an electricity input plug; (f) inflow of water. The large arrows indicate direction of buffer flow that is circulated by a pump.
THYROXINE-TRANQUILLIZER
INTERACTIONS
193
staining, and autoradiography with provision for slicing of the gel and maintaining constant pH and temperature. The variables were overcome by using a large volume of circulating buffer and cooling water through cooling plates on both sides of the gel. Variation of buffer, voltage, thickness of the gel, and length of run established that the optimum system was 8% (w/v) cyanogum 41 polyacrylamide gel, 0. I M phosphate buffer, pH 7.4, over 15 hr electrophoresis at 100 V during which the temperature can be held constant at 4°C. A 6-mm-thick gel (100 x 120 mm) with 13 slots was prepared from 20 g cyanogum 41 in 0. I M phosphate buffer, pH 7.4. The gel was aliowed to cool to 4°C for 30 min before preelectrophoresis was carried out at 100 V (about 300 mA) for 1 hr. Serum (30 ~1) with or without drug or hormone, was applied in duplicate and electrophoresis was carried out for 15 hr at 100 V with buffer circulating maintaining the temperature at 4°C. At the end of electrophoresis, the gel was sliced into two parts, one of which was stored at 4°C until the other was stained. The unstained gel was aligned with the stained half of the gel with a glass plate in between and the unstained gel was cut longitudinally between electrophoretic paths and each path cut out laterally into eight fractions with fractions l-4 corresponding to protein run between origin and Pre-TBG region. Fractions 5, 6, 7 and 8 correspond to TBG, albumin, prealbumin1, and prealbumin-2, respectively. The protein-bound radioactive thyroxine in each fraction was counted for 1 min and the result was corrected for background. The amount of radioactive T, bound to specific protein was expressed as a percentage of the total counts per sample. For autoradiography, the gel was exposed to X-ray film, sandwiched between two glass plates, and stored at -70°C for an appropriate period of time (l-3 days). The X-ray film was developed using Exprol Rapid Developer, diluted with an equal volume of water, and Ilford Hypam Rapid Fixer. RESULTS
Electrophoresis of drug-free (control) and drug-containing sera with added L-T,-“‘1 revealed a remarkable differences in the affinity of the drug for the T, binding sites on TBG. Assessed as a function of the drug ability to displace L-Tq-‘251, where the percentage of bound L-T,-‘*‘1 was used to measure the degree of displacement, chlordiazepoxide and diazepam exhibited different dissociation patterns for L-T,-“‘I. Moreover, chlordiazepoxide and diazepam differ somewhat in the T, binding proteins on which they exert their effect. While chlordiazepoxide effects TBPA, diazepam has it displacing effect on L-T,‘*‘1 binding to TBG (Fig. 2). With chlordiazepoxide albumin seems to be the main
194
M. A. F. EL-HAZMI
A 1
a
+
234567123456
FIG. 2. Autoradiogram of TBP-L-T,-‘? separated by polyacrylamide gel electrophoresis. Al, prealbumin marker; A2, serum-L-T,-‘*‘1 control: A3-A7. serum-L-T4-‘2SI in the presence of increasing chlordiazepoxide concentration (0.02-I .O g/liter). B I-B6, serum-lT,-“‘1 in the presence of increasing concentration of diazepam (0.01-I .O g/liter).
recipient of the displaced hormone and with diazepam the displaced LT,-“‘I was shown to be bound to TBPA. Diazepam metabolites showed similar displacing pattern for L-T4-‘25I to that of diazepam but of lesser effect (Fig. 3). The three metabolites differ in that desmethyldiazepam has a stronger effect on L-T,-“‘1 binding to TBG than 3-hydroxydiazepam and oxazepam. TBPA is the common recipient of the displaced L-T4-12’I from TBG by these drugs (Fig. 4). DISCUSSION The variation in the binding affinity to TBP for thyroid hormones and the benzodiazepines in human sera indicated that these proteins, in spite of their diversity and complexity, have certain reactive groups in common and that there is a unique sequence involved in this highly specific binding site. Nevertheless, the detailed organization of each binding site on a specific binding protein is different. The structural and conformational requirement for optimal binding of T, appears to be best met on the TBG molecule hence this protein has the highest affinity for T,. The hypothesis of Schussler (9) and Oppenheimer and Tavernitti (8) proposed that the T, binding site on TBG is complimentary to the diphenyl ether structure of T4, as was predicted from structural similarities. However, the decreasing affinity of T, for binding to TBG suggests that anionic substituents on the benzene rings are necessary for strong binding. Based on the similarities in the benzene structure of chlordiazepoxide, diazepam, and its metabolites (Fig. 5) the percentage of increasing or
THYROXINE-TRANQUILLIZER
01 0.00
.? 2 5 a
0.20
0.40 Drug
concentration
195
INTERACTIONS
0.60
0.60
1.0 0
(s/l)
FIG. 3. Effect of the presence of diazepam and its metabolites in the TBP solution and TBG binding of L-T,-‘% A, Diazepam; 0, 3-hydroxydiazepam; A, desmethyldiazepam; n , oxazepam.
decreasing binding of L-T,- “? to a serum protein can be used to elaborate on the effect of functional groups on the affinity of protein binding. As is shown by the ability of the drugs to displace L-T,-“‘1 from its specific binding sites on TBG and TBPA, the C-diazepine ring appears to play an additional role in the binding to T, sites on TBP. Furthermore, variation in the displacing effect of the benzodiazepines revealed the importance of the substituents on the diazepine ring for the binding onto TBP. It is possible that benzodiazepine binding onto TBG may involve the whole molecule. The remarkable ability of diazepam to displace LT,-12’I from TBG compared with chlordiazepoxide indicates the importance of the methyl group at N, for strong binding. Having a less binding affinity, 3-hydroxydiazepam and oxazepam revealed the effect of the substitution and the extent of the involvement of the positions 2 and 3 of the diazepine ring of the drug molecules. To elaborate on the differential binding properties of diazepam and chlordiazeposide it could be worthwhile to consider special arrangements of thyroxine. Leonard and Sutton (5) proposed that a valency angle of about 120” is assumed for the diphenyl ether oxygen, and Jorgensen (4) reported that the two phenyl rings are not in the same plane but lie at
196
M. A. F. EL-HAZMl
300 I 260
4 a m I”
,,....“’ ..4
260_:’
,:’
..
,.A..”
. ..’ 240-
,:’
L
Drug concentration(Q/l) FIG. 4. Effect of diazepam and its metabolites on TBPA binding of L-T,-‘% as for Fig. 3.
Symbols
almost right angles to each other. As 1,Cbenzodiazepines are assymetric and their B-benzene rings are not planar, it is conceivable that the B-benzene ring has a valency angle with the C-benzodiazepine ring similar to that of diphenyl ether oxygen. Schussler (9) postulated that the N, of the diazepine ring is involved in the binding to TBG. If this is true, the N-O of chlordiazepoxide, which exhibits a low affinity for TBG, may affect the spatial orientation of the benzene ring. In a similar way, the B-benzene ring of the benzodiazepine molecule is not situated in the same plane as the C-ring owing to steric hinderance (IO). It is possible that N-O bond can conceivably affect the free rotation of the
THYROXINE-TRANQUILLIZER
197
INTERACTIONS
Cl
--RI Ii
R2
3
NH-Cl-l3
H
- R4 -0
Chlordiarcpoxide
(librium(*))
CH3
0
H
Diazepam
H
0
H
Desmethyldiazepam
CH3
0
OH
3-Hydroxydiarepam
H
0
OH
Oxazepam
FIG.
(ValiumcR)
)
(SetaxfR))
5. Chemical structure of 1,4-benzodiazepines.
B-benzene ring for optimal binding, or alternatively by altering the electron distribution, may affect its hydrophobic interactions for the binding onto TBG. The hydrogen substitution for oxygen at N, of the diazepam ring would increase the lipophilic properties of the chlordiazepoxide molecule, but this might be suppressed by the restriction of spatial orientation of the B-rings by the NHCH, group at C2. This may explain the low affinity of chlordiazepoxide for TBG binding site. Of interest is the finding that diazepam is a more potent tranquilizer and is also a more effective binding competitor of T, than chlordiazepoxide. To relate the chemical structure to the pharmacological effectiveness, these findings may indicate a common receptor for thyroid hormones and benzodiazepines, the organization of which is optimal for thyroid hormones and diazepam. In light of these reports of previous work and our studies, it can be concluded that chlordiazepoxide and diazepam, and possibly also chemically related compounds, compete for the T, binding sites on TBP in vitro. Greenberg and co-workers (3) showed that in vim, neither drug exerts a constant effect on the binding of thyroid hormones or their concentration in the circulation. However, these observations do not preclude that under abnormal conditions, for example in case of a combination of albumin deficiency and multiple drug treatment, some disturbance might result. This is particularly relevant for the low albumin
198
level in the elderly therapy.
M. A. F. EL-HAZMI
who are also quite frequently
on a multiple-drug
REFERENCES 1. El-Hazmi, M. A. F., C/in. Chirn. Acta 63, 211 (1975). 2. Goldstein, A., Pharmacol. Rev. 1, 102 (1949). 3. Greenberg, A. H., Gzernichow, P., and Blizzard, R. M.. Johns Hopkins Med. J. 126, 134 (1970). 4. Jorgensen, E. C., Mayo Clinic Proc. 39, 560 (1964). 5. Leonard, J. N., and Sutton, L. E., J. Amer. Chern. Sot. 70, 1564 (1948). 6. Miiller, W.. and Wallert, U.. Narrnvn-Schiedr~berg’s Arch. Pharmukol. 283, 67 (1974). 7. Miiller, W., and Wallert, U.. Naun~n-Schtniedeberg*s Arch. Pharmakol. 280, 229 (1973). 8. Oppenheimer, H. J., and Tavernitti. R. P., J. CIirl. Invest. 41, 2213 (1962). 9. Schussler. G. C.. J. Pharmacol. Exp. Ther. 178, 204 (1971). 10. Sjodin, T.. Roosdrop, N., and Sjoholm, I.. Biochern. fhnrmucoi. 25, 2131 (1976).