Monoamine metabolites in cerebrospinal fluid and serotonin uptake inhibition during treatment with chlorimipramine The effects of chlorimipramine on the concentrations of the main metabolites of serotonin (5-HT) norepinephrine (NE), and dopamine, i.e. 5-hydroxyindoleacetic acid (5-H1AA), 4-hydroxy-3-methoxyphenyl glycol (HMPG), and homovanillic acid (HVA), respectively, were studied in cerebrospinal fluid from 14 depressed patients, and related to the serotonin- and NE uptake inhibiting activity in vitro Qf'plasma drawnfrom the patients. Chlorimipramine inhibited the uptake Qf both transmitter amines in all patients. During treatment, the levels of 5-HIAA and HMPG in cerebrospinal fluid (CSF) were significantly reduced. HV A levels were reduced in 6 patients and increased in 8 patients; there was no mean change. The decrease in 5-HIAA level in CSF was correlated to the uptake inhibition Qf 5-HT but there was no corresponding relationship between NE uptake and HMPG levels. The changes in HVA levels were also correlated to the uptake of 5-HT despite the absence Qf' a unidirectional change of this metabolite.
Marie ASberg, M.D., Vivi-Ann Ringberger, Folke Sjöqvist, M.D., Peter Thoren, M.D., Lil Träskman, M.D., and J. Richard Tuck, M.D. Huddinge and Stockholm, Sweden Departments Qf Psychiatry (Karolinska and Huddinge Hospital) and Clinical Pharmacology (Huddinge Hospital), Karolinska Institute
In attempts to enhance the efficacy of drug treatment in depressive disorders, attention has been focused on the impact of individual differences in drug metabolism, and the subsequent variability in drug plasma levels. 1, 29 The dimethylated tricyclic antidepressants (imipSupported by grants [rom the Swedish Medical Research Council (3902-05A and 4676-01) and the Bank of Sweden Tercentenary Fund (74/110). Presented in abstract format at the Sixth International Congress of Pharmacology, Helsinki, July, 1975. Received for publication July 15, 1976. Accepted for publication Aug. 3, 1976. Reprint requests to: Prof. Folke Sjöqvist, Department of Clinical Pharmacology, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
ramine, amitriptyline, chlorimipramine) have active metabolites, which complicates the interpretation of relationships between drug levels and central effects. The antidepressant effect of these drugs l l is thought to be mediated by inhibition of transmitter reuptake into noradrenergic and serotoninergic neurons. This property can be assessed in vitro, by measuring the uptake of labeled transmitter into small pieces of rat cerebral cortex. 19 When tricyclic antidepressants are added to the incubation medium (buffer or blood plasma), the amine uptake is inhibited in proportion to the concentration of the drug. 8 , 29 The procedure can be used as a bioassay of all active drugs and drug metabolites when applied 201
202
Asberg et al.
on plasma specimens drawn during antidepressant therapy. Antidepressant drugs differ in their relative potency to inhibit uptake of norepinephrine (NE) and serotonin (5_HT).12. 13 In concentrations corresponding to those encountered during treatment of depression, nortriptyline has a pronounced inhibitory effect on NE uptake, while its effect on 5-HT uptake is negligible. 19 Chlorimipramine, on the other hand, is also a potent inhibitor of 5-HT uptake. 12 . 13. 19 When these drugs are given to patients, the levels of the transmitter metabolites, 4hydroxy-3-methoxyphenyl glycol (HMPG) and 5-hydroxyindoleacetic acid (5-HIAA) decrease in the cerebrospinal fluid (CSF) , indicating that the release or synthesis of the transmitters in the central nervous system (CNS) has been decreased. 2. 5. 9. 24. 25 Analogous with the differential effect of the drugs on serotonin (5-HT) and NE uptake, chlorimipramine causes a relatively larger decrease of 5-HIAA than of HMPG, while the opposite holds true for nortriptyline. 5 In the present investigation, the decreases in 5-HIAA and HMPG levels in the CSF of patients treated with chlorimipramine were studied in relation to the degree of inhibition of serotonin and NE uptake exerted in vitro by plasma drawn from the patients. It was thought that a study of this relationship should bring the idea that drug levels in plasma are important for the central effects of antidepressants to a critical test. Patients and methods
Fourteen depressed patients (l0 endogenous and 4 neurotic depressions), diagnosed according to the scales constructed by Gurney and co-workers,18 were studied in a psychiatric research ward setting. A placebo washout period of 4 to 7 days preceded the 3-wk treatment period. Only 3 of the patients had received tricyclic antidepressant drugs prior to hospitalization. The drug-free period in these cases (Nos. 9, 10, and 13) was 7, 15, and 10 days, respectively. Chlorimipramine, 50 mg, was given at 7 A.M., 2 P.M., and 10 P.M., and the patients were routinely observed to ingest the drug. No additional drug treatment was given
Clinical Pharmacology and Therapeutics
except for occasional doses of benzodiazepines and chloralhydrate or barbiturates for night sedation. * Lumbar punctures. Lumbar punctures were performed by a standardized technique at the end of the placebo period and after 3 wk of chlorimipramine treatment. The CSF was collected in the early morning before the patients had risen and after at least 8 hr of fasting and bedrest. About 13 ml of CSF was drawn using a fine, disposable needle. Except for occasional moderate headache, there were no complications to the lumbar punctures. After centrifugation, the sampies were immediately frozen in 2-ml aliquots in silanized glass tubes and stored at -200 C until analyzed. Chemical analyses. The CSF sampies were analyzed for their contents of 5-HIAA, HMPG, and HV A according to the mass fragmentographic methods developed by Bertilsson and associates,6 Bertilsson,4 and Bertilsson and Palmer,1 respectively. Sampies before and during treatment with chlorimipramine were determined at the same time. Each metabolite was analyzed in duplicate. An LKB Model 9000 gas chromatograph-mass spectrometer was used. Deuterium-Iabeled isotopes of the amine metabolites were used as internal standards. Unconjugated as weIl as conjugated HMPG were measured. Conjugated HMPG amounted to 15.2 ± 6.8% of the unconjugated metabolite both before and during drug treatment. The sampies were analyzed within 6 mo. During that period of time the 5-HIAA and HMPG are reasonabl y stable in frozen CSF. 2. 5 Plasma samples. Plasma sampies were drawn immediately prior to the lumbar puncture. About 50 ml of blood was drawn into heparinized tubes and centrifuged immediately at 10,000 X g for 30 min. Plasma was stored frozen at -20 C until analysis (within 1-2 mo). 0
Determination of NE and 5-HT uptake. Uptake of 3H-NE and 3H-5-HT was determined according to the procedure described by Hamberger and Tuck. 19 . 20 Coronal brain slices (0.5 mm thick, 3 mm diameter, weight approximately 5 mg) from untreated Sprague-Dawley 'Clinical outcome in relation to biochemical and pharmacokinetic data will be presented elsewhere when a sufficiently large sampIe of patients has been investigated. These data are available on request.
Valume 21 Number 2
Serotonin inhibition after chlorimipramine
203
Table I. 5-HIAA. HMPG. and HVA in CSF and uptake of W-5-HT and 3H-NE in plasma
Patient No.
1 2 3 4 5 6 7 8 9 10 II
12 13 14
Be/are treatment-metabolite in CSF (nglml)
During chlorimipramine treatment-metabolite in CSF (nglml)
5-HIAA .1 HMPG 1 HVA
5-HlAA 1 HMPG 1 HVA
20.4 31.9 26.0 34.2 23.9 23.7 7.7 18.4 24.9 19.4 21.9 12.4 23.7 13.6
10.6 12.5 10.5 16.7 15.5 13.4 10.8 8.0 18.6 13.7 5.6 12.7 12.7 13.0
23.1 89.9 43.8 60.3 81.1 60.9 28.0 28.3 42.4 16.5 24.0 34.6 40.5 44.9
15.4 10.9 21.3 21.1 7.5 16.9 5.0 8.8 10.8 11.0 14.9 6.9 16.7 7.7
rats were used. The slices were incubated at 37° C in patient plasma. After 15 min incubation of 1 ml plasma, 3H-NE and 3H-5-HT were added to a concentration of 2 x 10- 8 M and 2 X 10- 9 M, respectively. After a 15-min incubation, the tissue was removed and rapidly rinsed in buffer; the radioactivity was determined after solubilization with Soluene and addition of toluene scintiIIation solution. The results are expressed as percent of their own contrals. In addition, model studies with desmethyIchlorimipramine were performed according to the same technique as previous studies with the parent compound. 19 Brain slices were incubated with labeled amine as above in 10 ml Krebs-Ringer solution to which known amounts of the compound were added. Results
Patients. The concentrations of 5-HIAA, HMPG, and HVA in the CSF, before and during treatment with chlorimipramine, are shown in Table I together with the uptake of 3H-5-HT and 3H-NE in plasma. Before treatment. The distribution of 5-HIAA in CSF of depressed patients has previously been found to be bimodal. 2, 3 In the present material, 3 patients (Nos. 7, 12, and 14) had 5-HIAA levels below 15 ng/ml and thus belonged to the lower mode. Since there was no
4.7 7.2 10.5 9.0 10.0 10.4 9.9 5.4 12.2 11.4 4.3 9.0 10.1 8.9
34.1 77.9 64.9 82.3 69.0 72.2 35.5 24.4 27.2 26.0 28.4 33.2 74.5 43.1
Uptake in plasma (% 0/ control) 5-HT
65 41 68 68 35 60 60 47 37 70 58 55 51 55
1
NE
47 30 46 32 29 31 47 48 40 44 32 48 24 30
indication of a different reaction to chlorimipramine in these patients, they are inc1uded in aII caIculations. There was a significant correlation between the levels of 5-HIAA and HV A prior to treatment (r = 0.60, t = 2.57, P = 0.024, Fig. 1). The correlations between 5-HIAA and HMPG, and HVA and HMPG, were not significant (r = 0.33, t = l.22, and r = 0.45, t = 1.74, respectively) . During treatment. TRANSMITTER UPTAKE IN VITRO. Chlorimipramine treatment reduced serotonin and NE uptake in a11 patients (mean 3H-5-HT uptake, 55.0 ± SD 11.5%, mean 3H-NE uptake, 37.7 ± SD 8.7%). There was no significant correlation between the uptake of the two amines during treatment (r = 0.33; t = 1.21). AMINE METABOLITE LEVELS. For aII metabolites, there was a significant correlation between concentrations before and during treatment (Table 11). The changes in metabolite levels are summarized in Table III. During treatment, 5-HIAA concentrations were reduced in a11 patients (mean, 58.6% of pretreatment contral) , and HMPG was reduced in a11 patients but one (mean, 70.4% of pretreatment contraI). HV A levels were increased in 8 patients and reduced in 4, and there was no sig-
204 Asberg et al.
100
Clinical Pharmacology and Therapeutics
Table 11. Correlations between amine metabolite concentrations in CSF before and during chlorimipramine treatment
HVA, ng. per ml.
• •
• 50
• •
•
•
••
•
•• • 30
20
Fig. 1. Concentration of 5-HIAA and HVA in cerebrospinal fluid from untreated depressed patients. There is a correlation (r = 0.60, P = 0.024) between the levels of the metabolites.
5HIAA decrease, per cent
• •
•
,
50
•
Metabolite
Mean
5-HIAA HMPG HVA
-9.1 -3.7 +5.3
3H-NA
3H-5-HT
25
5HTuptake
5
3.30 3.77 4.50
0.006 0.003
Marie ASberg, M.D., Vivi-Ann Ringberger, Folke Sjöqvist, M.D., Peter Thoren, M.D., Lil Träskman, M.D., and J. Richard Tuck, M.D. Huddinge and Stockholm, Sweden Departments Qf Psychiatry (Karolinska and Huddinge Hospital) and Clinical Pharmacology (Huddinge Hospital), Karolinska Institute
In attempts to enhance the efficacy of drug treatment in depressive disorders, attention has been focused on the impact of individual differences in drug metabolism, and the subsequent variability in drug plasma levels. 1, 29 The dimethylated tricyclic antidepressants (imipSupported by grants [rom the Swedish Medical Research Council (3902-05A and 4676-01) and the Bank of Sweden Tercentenary Fund (74/110). Presented in abstract format at the Sixth International Congress of Pharmacology, Helsinki, July, 1975. Received for publication July 15, 1976. Accepted for publication Aug. 3, 1976. Reprint requests to: Prof. Folke Sjöqvist, Department of Clinical Pharmacology, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
ramine, amitriptyline, chlorimipramine) have active metabolites, which complicates the interpretation of relationships between drug levels and central effects. The antidepressant effect of these drugs l l is thought to be mediated by inhibition of transmitter reuptake into noradrenergic and serotoninergic neurons. This property can be assessed in vitro, by measuring the uptake of labeled transmitter into small pieces of rat cerebral cortex. 19 When tricyclic antidepressants are added to the incubation medium (buffer or blood plasma), the amine uptake is inhibited in proportion to the concentration of the drug. 8 , 29 The procedure can be used as a bioassay of all active drugs and drug metabolites when applied 201
202
Asberg et al.
on plasma specimens drawn during antidepressant therapy. Antidepressant drugs differ in their relative potency to inhibit uptake of norepinephrine (NE) and serotonin (5_HT).12. 13 In concentrations corresponding to those encountered during treatment of depression, nortriptyline has a pronounced inhibitory effect on NE uptake, while its effect on 5-HT uptake is negligible. 19 Chlorimipramine, on the other hand, is also a potent inhibitor of 5-HT uptake. 12 . 13. 19 When these drugs are given to patients, the levels of the transmitter metabolites, 4hydroxy-3-methoxyphenyl glycol (HMPG) and 5-hydroxyindoleacetic acid (5-HIAA) decrease in the cerebrospinal fluid (CSF) , indicating that the release or synthesis of the transmitters in the central nervous system (CNS) has been decreased. 2. 5. 9. 24. 25 Analogous with the differential effect of the drugs on serotonin (5-HT) and NE uptake, chlorimipramine causes a relatively larger decrease of 5-HIAA than of HMPG, while the opposite holds true for nortriptyline. 5 In the present investigation, the decreases in 5-HIAA and HMPG levels in the CSF of patients treated with chlorimipramine were studied in relation to the degree of inhibition of serotonin and NE uptake exerted in vitro by plasma drawn from the patients. It was thought that a study of this relationship should bring the idea that drug levels in plasma are important for the central effects of antidepressants to a critical test. Patients and methods
Fourteen depressed patients (l0 endogenous and 4 neurotic depressions), diagnosed according to the scales constructed by Gurney and co-workers,18 were studied in a psychiatric research ward setting. A placebo washout period of 4 to 7 days preceded the 3-wk treatment period. Only 3 of the patients had received tricyclic antidepressant drugs prior to hospitalization. The drug-free period in these cases (Nos. 9, 10, and 13) was 7, 15, and 10 days, respectively. Chlorimipramine, 50 mg, was given at 7 A.M., 2 P.M., and 10 P.M., and the patients were routinely observed to ingest the drug. No additional drug treatment was given
Clinical Pharmacology and Therapeutics
except for occasional doses of benzodiazepines and chloralhydrate or barbiturates for night sedation. * Lumbar punctures. Lumbar punctures were performed by a standardized technique at the end of the placebo period and after 3 wk of chlorimipramine treatment. The CSF was collected in the early morning before the patients had risen and after at least 8 hr of fasting and bedrest. About 13 ml of CSF was drawn using a fine, disposable needle. Except for occasional moderate headache, there were no complications to the lumbar punctures. After centrifugation, the sampies were immediately frozen in 2-ml aliquots in silanized glass tubes and stored at -200 C until analyzed. Chemical analyses. The CSF sampies were analyzed for their contents of 5-HIAA, HMPG, and HV A according to the mass fragmentographic methods developed by Bertilsson and associates,6 Bertilsson,4 and Bertilsson and Palmer,1 respectively. Sampies before and during treatment with chlorimipramine were determined at the same time. Each metabolite was analyzed in duplicate. An LKB Model 9000 gas chromatograph-mass spectrometer was used. Deuterium-Iabeled isotopes of the amine metabolites were used as internal standards. Unconjugated as weIl as conjugated HMPG were measured. Conjugated HMPG amounted to 15.2 ± 6.8% of the unconjugated metabolite both before and during drug treatment. The sampies were analyzed within 6 mo. During that period of time the 5-HIAA and HMPG are reasonabl y stable in frozen CSF. 2. 5 Plasma samples. Plasma sampies were drawn immediately prior to the lumbar puncture. About 50 ml of blood was drawn into heparinized tubes and centrifuged immediately at 10,000 X g for 30 min. Plasma was stored frozen at -20 C until analysis (within 1-2 mo). 0
Determination of NE and 5-HT uptake. Uptake of 3H-NE and 3H-5-HT was determined according to the procedure described by Hamberger and Tuck. 19 . 20 Coronal brain slices (0.5 mm thick, 3 mm diameter, weight approximately 5 mg) from untreated Sprague-Dawley 'Clinical outcome in relation to biochemical and pharmacokinetic data will be presented elsewhere when a sufficiently large sampIe of patients has been investigated. These data are available on request.
Valume 21 Number 2
Serotonin inhibition after chlorimipramine
203
Table I. 5-HIAA. HMPG. and HVA in CSF and uptake of W-5-HT and 3H-NE in plasma
Patient No.
1 2 3 4 5 6 7 8 9 10 II
12 13 14
Be/are treatment-metabolite in CSF (nglml)
During chlorimipramine treatment-metabolite in CSF (nglml)
5-HIAA .1 HMPG 1 HVA
5-HlAA 1 HMPG 1 HVA
20.4 31.9 26.0 34.2 23.9 23.7 7.7 18.4 24.9 19.4 21.9 12.4 23.7 13.6
10.6 12.5 10.5 16.7 15.5 13.4 10.8 8.0 18.6 13.7 5.6 12.7 12.7 13.0
23.1 89.9 43.8 60.3 81.1 60.9 28.0 28.3 42.4 16.5 24.0 34.6 40.5 44.9
15.4 10.9 21.3 21.1 7.5 16.9 5.0 8.8 10.8 11.0 14.9 6.9 16.7 7.7
rats were used. The slices were incubated at 37° C in patient plasma. After 15 min incubation of 1 ml plasma, 3H-NE and 3H-5-HT were added to a concentration of 2 x 10- 8 M and 2 X 10- 9 M, respectively. After a 15-min incubation, the tissue was removed and rapidly rinsed in buffer; the radioactivity was determined after solubilization with Soluene and addition of toluene scintiIIation solution. The results are expressed as percent of their own contrals. In addition, model studies with desmethyIchlorimipramine were performed according to the same technique as previous studies with the parent compound. 19 Brain slices were incubated with labeled amine as above in 10 ml Krebs-Ringer solution to which known amounts of the compound were added. Results
Patients. The concentrations of 5-HIAA, HMPG, and HVA in the CSF, before and during treatment with chlorimipramine, are shown in Table I together with the uptake of 3H-5-HT and 3H-NE in plasma. Before treatment. The distribution of 5-HIAA in CSF of depressed patients has previously been found to be bimodal. 2, 3 In the present material, 3 patients (Nos. 7, 12, and 14) had 5-HIAA levels below 15 ng/ml and thus belonged to the lower mode. Since there was no
4.7 7.2 10.5 9.0 10.0 10.4 9.9 5.4 12.2 11.4 4.3 9.0 10.1 8.9
34.1 77.9 64.9 82.3 69.0 72.2 35.5 24.4 27.2 26.0 28.4 33.2 74.5 43.1
Uptake in plasma (% 0/ control) 5-HT
65 41 68 68 35 60 60 47 37 70 58 55 51 55
1
NE
47 30 46 32 29 31 47 48 40 44 32 48 24 30
indication of a different reaction to chlorimipramine in these patients, they are inc1uded in aII caIculations. There was a significant correlation between the levels of 5-HIAA and HV A prior to treatment (r = 0.60, t = 2.57, P = 0.024, Fig. 1). The correlations between 5-HIAA and HMPG, and HVA and HMPG, were not significant (r = 0.33, t = l.22, and r = 0.45, t = 1.74, respectively) . During treatment. TRANSMITTER UPTAKE IN VITRO. Chlorimipramine treatment reduced serotonin and NE uptake in a11 patients (mean 3H-5-HT uptake, 55.0 ± SD 11.5%, mean 3H-NE uptake, 37.7 ± SD 8.7%). There was no significant correlation between the uptake of the two amines during treatment (r = 0.33; t = 1.21). AMINE METABOLITE LEVELS. For aII metabolites, there was a significant correlation between concentrations before and during treatment (Table 11). The changes in metabolite levels are summarized in Table III. During treatment, 5-HIAA concentrations were reduced in a11 patients (mean, 58.6% of pretreatment contral) , and HMPG was reduced in a11 patients but one (mean, 70.4% of pretreatment contraI). HV A levels were increased in 8 patients and reduced in 4, and there was no sig-
204 Asberg et al.
100
Clinical Pharmacology and Therapeutics
Table 11. Correlations between amine metabolite concentrations in CSF before and during chlorimipramine treatment
HVA, ng. per ml.
• •
• 50
• •
•
•
••
•
•• • 30
20
Fig. 1. Concentration of 5-HIAA and HVA in cerebrospinal fluid from untreated depressed patients. There is a correlation (r = 0.60, P = 0.024) between the levels of the metabolites.
5HIAA decrease, per cent
• •
•
,
50
•
Metabolite
Mean
5-HIAA HMPG HVA
-9.1 -3.7 +5.3
3H-NA
3H-5-HT
25
5HTuptake
5
3.30 3.77 4.50
0.006 0.003
