Acta pharmacol. et toxicol. 1975, 36, 328-334.
From the Department of Pharmacology, University of Turku, Kiinamyllynk. 10, Turku 52 and the Department of Neurology, University of Turku Central Hospital, Turku, Finland
Cerebrospinal-Fluid Concentrations of Diazepam and its Metabolites in Man BY J. Kanto, L. Kangas and T. Siirtola (Received August 26, 1974; Accepted November 12, 1974)
Abstract: Plasma and cerebrospinal-fluid (CSF) concentrations of diazepam, its
main metabolite, N-demethyldiazepam, and free oxazepam were studied gas chromatographically in 49 patients suffering from neurological diseases, after a 10 mg intramuscular dose of diazepam. These determinations were made in three other patients after repeated intramuscular doses of 10 mg diazepam. In seven of the patients both free and plasma protein-bound diazepam, and N-demethyldiazepam were determined by ultracentrifugation after a single dose of 10 mg intramuscular diazepam. After a single dose of diazepam there was only 2-3 % of the total plasma concentration of diazepam and 1-4 % of that of N-demethyldiazepam in the CSF. After repeated doses of diazepam the same values were 3 % and 4 %, respectively. The plasma protein binding capacity of diazepam was 98 % and that of N-demethyldiazepam 97 %. Free diazepam and N-demethyldiazepam concentrations in the plasma were in equilibrium with their CSF concentrations. No accumulation of diazepam and N-demethyldiazepam could be found in the CSF. No free oxazepam was found. Key-words: Diazepam
- cerebrospinal-fluid
- protein binding - metabolism.
The exchange of drugs between blood, cerebrospinal fluid (CSF), and the brain is determined by many variable (RALL & ZUBROD1962). The plasma protein binding, degree of lipid solubility, extent of ionization and size and shape of the drug molecule play a major role in the pharmacokinetics of drug transfer between blood and the tissue and fluids of the central nervous system. The blood CSF barrier appears to behave as a lipid membrane toward most drugs (BRODIEet al. 1960). Diazepam is a long-acting, slowly metabolized, lipophilic and undissociated drug with a low molecular weight (285), and is highly bound to plasma proteins (96 %) (VAN DER KLEIJNet al. 1971). Several studies have been published on its
CEREBROSPINAL FLUID DIAZEPAM
329
pharmacokinetics in the central nervous system of various animals (IDANPXAN-HEIKKILA et al. 1971a; VAN DER KLEIJN& WIJFFELS 1971; MARCUCCI et al. 1971; UNTO & PIHLAJAMAKI 1973), although human studies are scanty. Foetal human brain concentrations of diazepam have been determined (IDANPAWN-HEIKKILA et al. 1971b; ERKKOLA et al. 1973a; UNTO & ERKKOLA 1974), and it was decided to study the entry of diazepam and its metabolites into the human CSF. To elucidate this mechanism the plasma protein binding of diazepam and its main metabolite, N-demethyldiazepam, were studied.
Methods Forty two patients at the Neurological Department of Turku University Central Hospital received 10 mg of diazepam (tensopam@,Laake Oy) intramuscularly before lumbar puncture (Group I). The patient material consisted of 25 males and 17 females from 19 to 72 years of age (mean age 47). As far as possible the material was chosen from new patients who were examined at the clinic without medication. Eleven of the patients suffered from epilepsy or undefined convulsions, including one patient who had been receiving phenytoin and carbamazepine. Ten patients were diagnosed as suffering from cerebral arterial insufficiency, of whom two received papaverine, one an anticoagulant (phenindione) and one a long-acting glycerylnitrate. Seven patients had had hemiparetic strokes, two of whom received papaverine only and one papaverine, phenindione, and hydrochlorothiazide. Four patients not on medication were examined because of cephalalgia and vertigo. The remaining 10 patients were given different neurological diagnoses. One of these patients received dexamethasone, another methotrimeprazine and chlorprotixene. Each patient in Group I had only one lumbar puncture from 0.5 to 24 hours after diazepam administration, as seen in fig. 1 and 2. No lumbar punctures were done at zero time. None of the patients received diazepam before this experiment. The blood and CSF samples were taken at the same time. Group I1 consisted of three epileptic patients all taking phenytoin. The concentrations of diazepam and its metabolites in their plasma and CSF were measured after repeated intramuscular doses of 10 mg diazepam (2-4 doses). The time intervals between drug administrations were 6-12 hours and the last dose was given 7-10 hours before lumbar puncture. The blood and CSF samples were taken again at the same time. Group 111 consisted of one female and six male patients with ages ranging from 26 to 74 years (mean age 45). Two of these seven patients were examined in the clinic without medication. Five other patients suffered from cerebral arterial insufficiency, two of whom received no medication, one received doxepine and papaverine, one methyl dopa and hydrochlorothiazide and one received digoxin, clonidine, haloperidol, and methotrimeprazine. The plasma and CSF concentrations of diazepam and its metabolites in this group were determined a t 2 hours after a 10 mg i.m. dose of diazepam, and in addition plasma protein-bound and free diazepam and N-demethyldiazepam were determined. The plasma and CSF concentrations of both diazepam and its main metabolite, Ndemethyldiazepam, as well as the concentrations of free oxazepam were determined by gas chromatography (KANGAS et al. 1974), using a 63Ni-electroncapture detector. The volume
330
J. U N T O , L. KANGAS AND T. SIIRTOLA
of plasma or CSF required for drug estimation was 0.5 ml. External standards were used. The plasma percentage of recovery of diazepam, plasma N-derethyldiazepam and plasma oxazepam were 96+2, 92+3 and 96+3,respectively (n = 10); those of CSF diazepam, CSF N-demethyldiazepam, and CSF oxazepam were 99 0.5,95 3, and 91 +4, respectively (n = 11). The lower limit of the method was 0.2 ng/ml in CSF and in the proteinfree fraction of the plasma ultracentrifugate and 0.5 ng/ml in plasma. To differentiate between the protein-bound and free diazepam and N-demethyldiazepam in plasma, ultracentrifugation was done with a MSE Superspeed 65 Mk 2 ultracentrifuge with a titanium angle rotor (cat. No. 59592) and with a capacity of 10 x 10 ml. Adapters fitting 2 ml tubes were used to reduce the plasma volume. Runs were made at 37" for 5 hours at maximum speed (65000 r.p.m. = 407000 x g). At least 1 ml of protein free supernatant was made available from each sample. The initial 200 pl at the top of the tube contained lipid material and other undesirable impurities. The next 500 p1 fraction was taken for gas chromatographic analyses. No protein volume rectifications were made (VANDER KLEIJN1969) since these would not have changed the results.
+
+
Results
The concentrations of diazepam in plasma and CSF after a single 10 mg intramuscular dose of diazepam (Group I) can be seen in fig. 1 . The CSF concentrations of diazepam as per cent of plasma concentration were as follows : at 0.5 hours 2 %, at 1 hour 3 %, at 2 hours 3 %, at 3 hours 3 %, at 6 hours 2 %, and at 24 hours 2 YO(mean 2 %).
Fig. 1. Plasma (solid line) and cerebrospinal-fluid (broken line) concentrations of diazepam after a 10 mg intramuscular dose of diazepam to 42 patients (mean+S.E.M.). At each point determinations on seven patients are given.
CEREBROSPINAL FLUID DIAZEPAM
331
E
'12 1 2 3 6 24 Hou r s Fig. 2 Plasma (solid enil) and cerebrospinal-fluid (broken line) concentrations of N-demethyldiazepam after a 10 mg intramuscular dose of diazepam. Mean values k S.E.M. of those patients in Group I who had N-demethyldiazepam in their plasma.
N-demethyldiazepam was found in 25 plasma samples and only in 21 CSF samples of the 42 patients in Group I, as seen in fig. 2. At 0.5 and 1 hour there was no detectable amount of N-demethyldiazepam in the CSF of any of the patients. The CSF concentrations of N-demethyldiazepam as per cent of plasma concentration were as follows: at 2 hours 4%, at 3 hours 3 YO,at 6 hours 1 YO, and at 24 hours 2 YO(mean 2 YO). No free oxazepam could be found in any of the plasma or CSF samples in Group I. In Group I1 after repeated intramuscular doses of diazepam the CSF concentrations of diazepam and N-demethyldiazepam 7-10 hours after the last dose were 3% and 4%, respectively of the plasma concentration. No free oxazepam was found.
Table I. Results of Group 111 (n = 7). Free plasma diazepam and N-demethyldiazepam concentrations are in equilibrium with those of the cerebrospinal fluid. Total plasma diazepam (nglml) 126.6& 57.4
Free plasma diazepam (nglml) 2.1k1.1
CSF diazepam (nglml) 2.2 f 0.7
Total plasma N-demethyldiazepam (nglml) 41.7 5 14.6
Free plasma N-demethyldiazepam (ng/ml) 1.3k0.4
CSF N-demethyldiazepam (nglml) 1.3A0.8
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J. KANTO, L. KANGAS AND T. SIIRTOLA
In table 1 the results of Group 111 are shown. It is apparent that only the free plasma concentrations of diazepam and N-demethyldiazepam are in equilibrium with the CSF.In Group 111 at 2 hours the CSF concentrations of diazepam and N-demethyldiazepam as per cent of plasma concentration were 2 % and 3 %, respectively. No free oxazepam was found. The plasma protein binding capacity of diazepam was 98 YOand that of N-demethyldiazepam 97 %. In the 49 patients of this study who received a single dose of diazepam, the CSF concentrations of diazepam and N-demethyldiazepam were 2 % and 3 %, respectively of the plasma concentration.
Discussion The exchange mechanism of drugs between blood and cerebrospinal fluid is usually passive diffusion (RALL& ZUBROD1962), although an active transport process in the choroid plexus has been known to take place as well (BONTING et al. 1964). The entry of diazepam and N-demethyldiazepam into the CSF seems to be by passive diffusion, since the concentrations of diazepam and N-demethyldiazepam in the CSF follow the plasma concentrations. Because diazepam and its main metabolite enter into the CSF so quickly, it is possible that they equilibrate with the CSF both across the choroid plexus and the ependyma and pia-glia. Vessels within the subarachnoid space may allow diffusion into the subarachnoid CSF as well. There is an exchange of free plasma diazepam and N-demethyldiazepam between the blood and the CSF. No accumulation of diazepam or its main metabolite, N-demethyldiazepam, could be observed in the CSF. The lipid solubility and small molecular size of diazepam and its main metabolite facilitate their entry into the CSF.The protein concentration of the CSF is only 1/500 that of the plasma (BOWMAN et al. 1968). Because there is no protein for binding, the rapid turn-over rate of the CSF decreases its concentrations of diazepam and N-demethyldiazepam (RALL& ZUBROD1962). Diazepam has been shown to accumulate rapidly in animal brains (IDANel al. 1971a; MARCUCCI et al. 1971; KANTO& PIHLAJAMAKI PUN-HEIKKILA 1973), and in human foetal brain (IDANPAAN-HEIKKILA et al. 1971b; KANTO & ERKKOLA 1974). During labour there is a rapid placental transfer and it has been shown to accumulate in foetal plasma (ERKKOLA et al. 1973b; KANTO et al. 1973). Under these conditions diazepam is bound to foetal brain proteins and foetal plasma proteins. The protein binding seems to influence the distribution of diazepam and N-demethyldiazepam across membranes. In human and animal studies diazepam has also been shown to accumulate in adipose tissue (MARCUCCI et al. 1968; UNTO et al. 1974). However, the
CEREBROSPINAL FLUID DIAZEPAM
333
CSF has no lipid component and this mechanism of accumulation in the CSF is not possible (RALL& ZUBROD 1962). In the patients of this study receiving a single dose of diazepam the CSF concentrations of diazepam and N-demethyldiazepam were 2 YO and 3 YO, respectively, of the plasma concentration. Drugs such as digoxin may reduce the CSF production in man by inhibiting the CSF-producing system in the choroid plexus (NEBLETT er al. 1972). Drug interaction of this kind may have an effect on the CSF level of other drugs. However, in this study no drug therapy or neurological disease had any clear effect on the percentage CSF concentrations of diazepam or N-demethyldiazepam. In earlier studies a protein binding capacity of 96 % (VAN DER KLEIJN1969; UNTO et al. 1973) and 90% (MULLER& WOLLERT 1973) were calculated for diazepam, whereas in this study it was 98 %. However, it should be remembered that all the patients had one or more neurological diseases, and some were on multi-drug therapy. These factors could have had some effect on the protein binding of diazepam and N-demethyldiazepam. To our knowledge, the protein binding of N-demethyldiazepam has not as yet been measured. After the small doses of diazepam given in this study, no free oxazepam was found in the plasma or in the CSF. After large doses of diazepam in a case of suicidal intoxication it was possible to find measurable amounts of free plasma oxazepam (KANTO,unpublished results). It seems possible that the serum protein binding capacity of a drug could be determined by measuring the concentration of the drug in the CSF. Such determinations have been made with, for instance, carbamazepine (JOHANNESSEN & STRANDJORD 1971).
REFERENCES Bonting, S. L., N. M. Hawkins & M. R. Canady: Studies of sodium potassium activated adenosine triphosphatase. VII-inhibition by erythrophleum alkaloids. Biochem. Pharmacol. 1964, 13, 13-22. Bowman, W. C., M. J. Rand & G. B. West: Textbook ofpharmacology. Blackwell Scientific Publications, Oxford and Edinburgh, 1968, pp. 501-502. Brodie, B. B., H. Kurz & L. S. Schanker: The importance of dissociation constant and lipid-solubility in influencing the passage of drugs into the cerebrospinal fluid. J . Pharmacol. 1960, 130, 20-25. Erkkola, R., J. Kanto & R. Sellman: Diazepam concentrations in mother and foetus in early pregnancy. Excerpta Medica International Congress Series 1973a, No. 279, pp. 321. Erkkola, R., L. Kangas & A. Pekkarinen: The transfer of diazepam across the placenta during labour. Acta obstet. gynec. scand. 1973b, 52, 167-170. Idanpaan-Heikkila, J. E., R. J. Taska, H. A. Allen & J. E. Scholaar: Autoradiographic
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study on the fate of diazepam-C" in the monkey brain. Arch. Int. Pharmacodyn. 1971a, 194, 68-77. Idanpaan-Heikkila, J. E., P. E. Jouppila, J. 0. Puolakka & M. S. Vorne: Placental transfer and fetal metabolism of diazepam in early human pregnancy. Amer. Obsret. Gynec. 1971b, 109, 1011-1013. Johannessen, S. & R. Strandjord: Koncentrationen av karbamazapin i serum och cerebrospinalvatska (CSF) hos 9 patienter med epilepsi. Plasma koncentrationsbestlmningar av antiepileptica: metodologiska och kliniska aspekter. Geigy Lakemedel Fack, 43120 Molndal 1, Lidingo 1971. Kangas, L., A. Pekkarinen, C. Sourander & E. Raijola: A comparative gaschromatographic study on absorption of diazepam tablets in man. Ann. Clin. Res. 1974, 5, suppl. 1 1 , 12-20. Kanto, J. & K. Pihlajamaki: Interactions of diazepam and halothane in rats. Ann. Chir. Cynaec. Fenn. 1973,62,247-250. Kanto, J., R. Erkkola & R. Sellman: Accumulation of diazepam and N-demethyldiazepam in the fetal blood during labour. Ann. Clin. Res. 1973, 5 , 375-379. Kanto, J. & R. Erkkola: Distribution of diazepam in early pregnancy. Ann. Chir. Gynaec. Fenn. 1974,63, 489-491. Kanto, J., K. Pihlajamaki & E. U. M. Iisalo: Concentrations of diazepam in adipose tissue of children. Brit. J. Anaesth, 1974. 46, 168. van der Kleijn, E.: Protein binding and lipophilic nature of ataractics of the meprobamateand diazepine-group. Arc. Int. Pharmacodyn. Therap. 1969, 179, 225-250. van der Kleijn, E. & C. C. G. Wijffels: Whole-body and regional brain distribution of diazepam in newborn rhesus monkey. Arch. Int. Pharmacodyn. Therap. 1971, 192, 25 5-264. van der Kleijn, E., J. M. van Rossum, E. T. J. M. Muskens & N. V. M. Rijntjes: Pharmacokinetics of diazepam in dogs, mice, and man. Acta pharmacol. et toxicol. 1971, 29, 109-127. Marcucci, F., R. Fanelli, M. Frova & P. L. Morselli: Levels of diazepam in adipose tissue of rats, mice, and man. Eur. J . Pharmacol. 1968,4,464466. Marcucci, F., E. Mussini, A. Guaitani, R. Fanelli & S. Garattini: Anticonvulsant activity and brain levels of diazepam and its metabolites in mice. Eur. J . Pharmacol. 1971, 16, 311-314. Muller, W. & U. Wollert: Characterization of the binding of benzodiazepines to human serum albumin. Naunyn-Schmiedeberg's Arch. Pharmacol. 1973, 280, 229-237. Neblett, C. R., D. P. Mc Neel, T. A. Waltz & G. M. Harrison: Effect of cardiac glycosides on human cerebrospinal-fluid production. Lancet 1972,2, 1008-1009. Rall, D. P. & C. G. Zubrod: Mechanism of drug absorption and excretion. Passage of drugs in and out of the central nervous system. Amer. Rev. Pharmacol. 1962, 2, 109128.
From the Department of Pharmacology, University of Turku, Kiinamyllynk. 10, Turku 52 and the Department of Neurology, University of Turku Central Hospital, Turku, Finland
Cerebrospinal-Fluid Concentrations of Diazepam and its Metabolites in Man BY J. Kanto, L. Kangas and T. Siirtola (Received August 26, 1974; Accepted November 12, 1974)
Abstract: Plasma and cerebrospinal-fluid (CSF) concentrations of diazepam, its
main metabolite, N-demethyldiazepam, and free oxazepam were studied gas chromatographically in 49 patients suffering from neurological diseases, after a 10 mg intramuscular dose of diazepam. These determinations were made in three other patients after repeated intramuscular doses of 10 mg diazepam. In seven of the patients both free and plasma protein-bound diazepam, and N-demethyldiazepam were determined by ultracentrifugation after a single dose of 10 mg intramuscular diazepam. After a single dose of diazepam there was only 2-3 % of the total plasma concentration of diazepam and 1-4 % of that of N-demethyldiazepam in the CSF. After repeated doses of diazepam the same values were 3 % and 4 %, respectively. The plasma protein binding capacity of diazepam was 98 % and that of N-demethyldiazepam 97 %. Free diazepam and N-demethyldiazepam concentrations in the plasma were in equilibrium with their CSF concentrations. No accumulation of diazepam and N-demethyldiazepam could be found in the CSF. No free oxazepam was found. Key-words: Diazepam
- cerebrospinal-fluid
- protein binding - metabolism.
The exchange of drugs between blood, cerebrospinal fluid (CSF), and the brain is determined by many variable (RALL & ZUBROD1962). The plasma protein binding, degree of lipid solubility, extent of ionization and size and shape of the drug molecule play a major role in the pharmacokinetics of drug transfer between blood and the tissue and fluids of the central nervous system. The blood CSF barrier appears to behave as a lipid membrane toward most drugs (BRODIEet al. 1960). Diazepam is a long-acting, slowly metabolized, lipophilic and undissociated drug with a low molecular weight (285), and is highly bound to plasma proteins (96 %) (VAN DER KLEIJNet al. 1971). Several studies have been published on its
CEREBROSPINAL FLUID DIAZEPAM
329
pharmacokinetics in the central nervous system of various animals (IDANPXAN-HEIKKILA et al. 1971a; VAN DER KLEIJN& WIJFFELS 1971; MARCUCCI et al. 1971; UNTO & PIHLAJAMAKI 1973), although human studies are scanty. Foetal human brain concentrations of diazepam have been determined (IDANPAWN-HEIKKILA et al. 1971b; ERKKOLA et al. 1973a; UNTO & ERKKOLA 1974), and it was decided to study the entry of diazepam and its metabolites into the human CSF. To elucidate this mechanism the plasma protein binding of diazepam and its main metabolite, N-demethyldiazepam, were studied.
Methods Forty two patients at the Neurological Department of Turku University Central Hospital received 10 mg of diazepam (tensopam@,Laake Oy) intramuscularly before lumbar puncture (Group I). The patient material consisted of 25 males and 17 females from 19 to 72 years of age (mean age 47). As far as possible the material was chosen from new patients who were examined at the clinic without medication. Eleven of the patients suffered from epilepsy or undefined convulsions, including one patient who had been receiving phenytoin and carbamazepine. Ten patients were diagnosed as suffering from cerebral arterial insufficiency, of whom two received papaverine, one an anticoagulant (phenindione) and one a long-acting glycerylnitrate. Seven patients had had hemiparetic strokes, two of whom received papaverine only and one papaverine, phenindione, and hydrochlorothiazide. Four patients not on medication were examined because of cephalalgia and vertigo. The remaining 10 patients were given different neurological diagnoses. One of these patients received dexamethasone, another methotrimeprazine and chlorprotixene. Each patient in Group I had only one lumbar puncture from 0.5 to 24 hours after diazepam administration, as seen in fig. 1 and 2. No lumbar punctures were done at zero time. None of the patients received diazepam before this experiment. The blood and CSF samples were taken at the same time. Group I1 consisted of three epileptic patients all taking phenytoin. The concentrations of diazepam and its metabolites in their plasma and CSF were measured after repeated intramuscular doses of 10 mg diazepam (2-4 doses). The time intervals between drug administrations were 6-12 hours and the last dose was given 7-10 hours before lumbar puncture. The blood and CSF samples were taken again at the same time. Group 111 consisted of one female and six male patients with ages ranging from 26 to 74 years (mean age 45). Two of these seven patients were examined in the clinic without medication. Five other patients suffered from cerebral arterial insufficiency, two of whom received no medication, one received doxepine and papaverine, one methyl dopa and hydrochlorothiazide and one received digoxin, clonidine, haloperidol, and methotrimeprazine. The plasma and CSF concentrations of diazepam and its metabolites in this group were determined a t 2 hours after a 10 mg i.m. dose of diazepam, and in addition plasma protein-bound and free diazepam and N-demethyldiazepam were determined. The plasma and CSF concentrations of both diazepam and its main metabolite, Ndemethyldiazepam, as well as the concentrations of free oxazepam were determined by gas chromatography (KANGAS et al. 1974), using a 63Ni-electroncapture detector. The volume
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of plasma or CSF required for drug estimation was 0.5 ml. External standards were used. The plasma percentage of recovery of diazepam, plasma N-derethyldiazepam and plasma oxazepam were 96+2, 92+3 and 96+3,respectively (n = 10); those of CSF diazepam, CSF N-demethyldiazepam, and CSF oxazepam were 99 0.5,95 3, and 91 +4, respectively (n = 11). The lower limit of the method was 0.2 ng/ml in CSF and in the proteinfree fraction of the plasma ultracentrifugate and 0.5 ng/ml in plasma. To differentiate between the protein-bound and free diazepam and N-demethyldiazepam in plasma, ultracentrifugation was done with a MSE Superspeed 65 Mk 2 ultracentrifuge with a titanium angle rotor (cat. No. 59592) and with a capacity of 10 x 10 ml. Adapters fitting 2 ml tubes were used to reduce the plasma volume. Runs were made at 37" for 5 hours at maximum speed (65000 r.p.m. = 407000 x g). At least 1 ml of protein free supernatant was made available from each sample. The initial 200 pl at the top of the tube contained lipid material and other undesirable impurities. The next 500 p1 fraction was taken for gas chromatographic analyses. No protein volume rectifications were made (VANDER KLEIJN1969) since these would not have changed the results.
+
+
Results
The concentrations of diazepam in plasma and CSF after a single 10 mg intramuscular dose of diazepam (Group I) can be seen in fig. 1 . The CSF concentrations of diazepam as per cent of plasma concentration were as follows : at 0.5 hours 2 %, at 1 hour 3 %, at 2 hours 3 %, at 3 hours 3 %, at 6 hours 2 %, and at 24 hours 2 YO(mean 2 %).
Fig. 1. Plasma (solid line) and cerebrospinal-fluid (broken line) concentrations of diazepam after a 10 mg intramuscular dose of diazepam to 42 patients (mean+S.E.M.). At each point determinations on seven patients are given.
CEREBROSPINAL FLUID DIAZEPAM
331
E
'12 1 2 3 6 24 Hou r s Fig. 2 Plasma (solid enil) and cerebrospinal-fluid (broken line) concentrations of N-demethyldiazepam after a 10 mg intramuscular dose of diazepam. Mean values k S.E.M. of those patients in Group I who had N-demethyldiazepam in their plasma.
N-demethyldiazepam was found in 25 plasma samples and only in 21 CSF samples of the 42 patients in Group I, as seen in fig. 2. At 0.5 and 1 hour there was no detectable amount of N-demethyldiazepam in the CSF of any of the patients. The CSF concentrations of N-demethyldiazepam as per cent of plasma concentration were as follows: at 2 hours 4%, at 3 hours 3 YO,at 6 hours 1 YO, and at 24 hours 2 YO(mean 2 YO). No free oxazepam could be found in any of the plasma or CSF samples in Group I. In Group I1 after repeated intramuscular doses of diazepam the CSF concentrations of diazepam and N-demethyldiazepam 7-10 hours after the last dose were 3% and 4%, respectively of the plasma concentration. No free oxazepam was found.
Table I. Results of Group 111 (n = 7). Free plasma diazepam and N-demethyldiazepam concentrations are in equilibrium with those of the cerebrospinal fluid. Total plasma diazepam (nglml) 126.6& 57.4
Free plasma diazepam (nglml) 2.1k1.1
CSF diazepam (nglml) 2.2 f 0.7
Total plasma N-demethyldiazepam (nglml) 41.7 5 14.6
Free plasma N-demethyldiazepam (ng/ml) 1.3k0.4
CSF N-demethyldiazepam (nglml) 1.3A0.8
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J. KANTO, L. KANGAS AND T. SIIRTOLA
In table 1 the results of Group 111 are shown. It is apparent that only the free plasma concentrations of diazepam and N-demethyldiazepam are in equilibrium with the CSF.In Group 111 at 2 hours the CSF concentrations of diazepam and N-demethyldiazepam as per cent of plasma concentration were 2 % and 3 %, respectively. No free oxazepam was found. The plasma protein binding capacity of diazepam was 98 YOand that of N-demethyldiazepam 97 %. In the 49 patients of this study who received a single dose of diazepam, the CSF concentrations of diazepam and N-demethyldiazepam were 2 % and 3 %, respectively of the plasma concentration.
Discussion The exchange mechanism of drugs between blood and cerebrospinal fluid is usually passive diffusion (RALL& ZUBROD1962), although an active transport process in the choroid plexus has been known to take place as well (BONTING et al. 1964). The entry of diazepam and N-demethyldiazepam into the CSF seems to be by passive diffusion, since the concentrations of diazepam and N-demethyldiazepam in the CSF follow the plasma concentrations. Because diazepam and its main metabolite enter into the CSF so quickly, it is possible that they equilibrate with the CSF both across the choroid plexus and the ependyma and pia-glia. Vessels within the subarachnoid space may allow diffusion into the subarachnoid CSF as well. There is an exchange of free plasma diazepam and N-demethyldiazepam between the blood and the CSF. No accumulation of diazepam or its main metabolite, N-demethyldiazepam, could be observed in the CSF. The lipid solubility and small molecular size of diazepam and its main metabolite facilitate their entry into the CSF.The protein concentration of the CSF is only 1/500 that of the plasma (BOWMAN et al. 1968). Because there is no protein for binding, the rapid turn-over rate of the CSF decreases its concentrations of diazepam and N-demethyldiazepam (RALL& ZUBROD1962). Diazepam has been shown to accumulate rapidly in animal brains (IDANel al. 1971a; MARCUCCI et al. 1971; KANTO& PIHLAJAMAKI PUN-HEIKKILA 1973), and in human foetal brain (IDANPAAN-HEIKKILA et al. 1971b; KANTO & ERKKOLA 1974). During labour there is a rapid placental transfer and it has been shown to accumulate in foetal plasma (ERKKOLA et al. 1973b; KANTO et al. 1973). Under these conditions diazepam is bound to foetal brain proteins and foetal plasma proteins. The protein binding seems to influence the distribution of diazepam and N-demethyldiazepam across membranes. In human and animal studies diazepam has also been shown to accumulate in adipose tissue (MARCUCCI et al. 1968; UNTO et al. 1974). However, the
CEREBROSPINAL FLUID DIAZEPAM
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CSF has no lipid component and this mechanism of accumulation in the CSF is not possible (RALL& ZUBROD 1962). In the patients of this study receiving a single dose of diazepam the CSF concentrations of diazepam and N-demethyldiazepam were 2 YO and 3 YO, respectively, of the plasma concentration. Drugs such as digoxin may reduce the CSF production in man by inhibiting the CSF-producing system in the choroid plexus (NEBLETT er al. 1972). Drug interaction of this kind may have an effect on the CSF level of other drugs. However, in this study no drug therapy or neurological disease had any clear effect on the percentage CSF concentrations of diazepam or N-demethyldiazepam. In earlier studies a protein binding capacity of 96 % (VAN DER KLEIJN1969; UNTO et al. 1973) and 90% (MULLER& WOLLERT 1973) were calculated for diazepam, whereas in this study it was 98 %. However, it should be remembered that all the patients had one or more neurological diseases, and some were on multi-drug therapy. These factors could have had some effect on the protein binding of diazepam and N-demethyldiazepam. To our knowledge, the protein binding of N-demethyldiazepam has not as yet been measured. After the small doses of diazepam given in this study, no free oxazepam was found in the plasma or in the CSF. After large doses of diazepam in a case of suicidal intoxication it was possible to find measurable amounts of free plasma oxazepam (KANTO,unpublished results). It seems possible that the serum protein binding capacity of a drug could be determined by measuring the concentration of the drug in the CSF. Such determinations have been made with, for instance, carbamazepine (JOHANNESSEN & STRANDJORD 1971).
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