Clinical Pharmacokinetics 3: 72-91 (1978) © ADIS Press 1978 Clinical Pharmacokinetics of Diazepam Marinella Mandelli, Gianni Tognoni and Silvio Garattini Istituto di Ricerche Farmacologiche ‘Mario Negr’, Milan ‘Summary Diazepam is still one of the most used of the benzodiazepine group of drugs. Extensive studies over 10 years have defined a fairly complete profile of ts kinetics. Minor aspects relat- ing 1 patterns of ts metabolism and excretion in certain age groups and in some disease states remain to be described quantitatively. However, there is more than sufficient kinetic informa- ‘ion available for the requirements of good clinical practice. For optimum clinical benefit with minimum side-effects the following kinetic properties should be borne in mind: (a) there is a large interindividual variation (up to 30-fold) in dose! blood level ratios, especially when treatment is short-term; (6) the elimination half-life is prolonged in the elderly and the newborn and in some cases of liver disease; (c) there is ac- ‘cumulation of the active N-desmethylated metabolite during long-term treatment; and (d) ad~ ‘ministration of diazepam to pregnant women leads to rapid distribution from the maternal io Jetal compartment: accumulation of both diazepam and desmeihyldiazepam could cause rotonged sedation in the newborn, As there does not appear to be any clear relationship bet- ween the conceniration of diazepam in the plasma and clinical effect, measurement of blood levels, other than for research purposes, is unnecessary. Based on kinetic data, a single administration of diazepam at night should be adequate for hypnotic and anxiolytic effects in most patients. There are many excellent articles on the benzodiazepines, their properties, uses and patterns of disposition (Garattini et al., 1973a, 1975; Tyrer, 1974; Greenblatt and Shader, 1974; Costa and Greengard, 1975; Lasagna, 1977). The phar- macokinetics of diazepam, still probably the most widely prescribed conpound in this group, have been extensively investigated. The purpose of this review is, to show how the results of these studies may be ap- plied in general clinical practice. Previous articles in the journal have discussed the kinetic properties of diazepam relevant to its use in epilepsy (Hvidberg and Dam, 1976) and anaesthetic practice (Ghoneim and Kortilla, 1977), There is general agreement that neither chemical manipulation of the basic diazepam molecule, nor use of its active metabolites (fig. 1) has lead to any sub- stantial gain in therapeutic efficacy (Kesson et al., 1976; Shader and Greenblatt, 1977). However, side- effects in groups at risk because of age and/or associ- ated disease states, may be minimised by the applica- tion of kinetic principles: either by adjusting the dosage regimen, or by choosing a benzodiazepine with a shorter half-life (and possibly no after-effects), Such as oxazepam or methyloxazepam (Nicholson and Stone, 1976; Fuccella et al., 1977). ‘The kinetic and metabolic patterns of diazepam have been extensively investigated using sensitive andClinical Pharmacokinetics of Diazepam n Diazepam ee Sal Methyloxazepam demethylation a ‘ x mnethylation, CL Desmethyidiazepam hydroxylation Oxazepam Fig. 1. Biotransformation pathways of major active metabolites of diszepam. specific analytical techniques (Marcucci et al., 1968a; Van der Kleijn et al., 1971; Belvedere et al., 1972; Zingales, 1973; Arnold, 1975). Diazepam may be considered almost a classical model for the study of the various factors known to influence the disposition of a drug in the body. By far the most clinically rele- vant information to emerge has been the absence of any evidence of a correlation between plasma levels and therapeutic effect. Thus, measurement of blood levels is unnecessary except in a patient where knowledge of the concentration of the drug could help in clarifying an unexpected reaction, or where changes in the metabolic pattern of diazepam might be considered as a marker of other biological events, ‘such as enzyme induction (Garattini et al., 1973b; Hvidberg and Dam, 1976; Bond et al., 1977). 1. Fundamental Pharmacokinetic Properties 1.1 Absorption Table I summarises some of the studies dealing specifically with the absorption of diazepam follow-Giinical Pharmacokinetics of Diazepam 74 Table |. Gastrointestinal absorption of diazepam Author Patients Dose Peak time Peak levels (min) (ng/mi) number age ly) ‘Schwartz et al. (1965) 2 - 10mg 120 70° Garattiniet al.(1973b) = 17-59 0.25mg/kg 60 140-2508 60-80 0.25mg/kg 60 80-908 Hillestad et al. (1974a) 9 19-35 20mg 30 492 Kanto and Erkkola (1974) — = _ _ 17-492 Amold (1975) 16 21-87 10mg - 300-400 Gamble et al. (1975) 40 32) 10mg 90 Wea 8 Measured in blood: @ blood/serum or plasma ratio of about 0.6 should be calculated. b- Mean. ‘Note: Both mean and range values are reported whenever available. Table 11. Intramuscular absorption of diazepam ‘Author Patients Dose Peak time Peak level (mg) (min) (og/mi) number age (y) Baird and Hailey (1973) 4 = 10 60 200-300 Hillestad et al. (1974a) 8 19-35, 20 60 283 Gamble et al. (1975) Buttock (nurses)® 31 3 10 90 43278 Buttock (doctors}* 10 33 10 90 1005.1 Thigh (doctors)* 33 28> 10 30 1493.7 2 See text for explanation. b_ Mean, ing various routes of administration. Where no con- traindications exist, the oral is preferable to the in- tramuscular or rectal route of administration. Absorption after oral administration is rapid and ‘complete; peak plasma levels being reached within 30 ‘to 90 minutes. Age is a major factor influencing ab- Sorption; an earlier peak is seen in children, a delayed and lower one is observed in the elderly (fig. 2; Garat- tini et al., 1973b). In chronic alcoholic cirrhosis, a Substantially lower (44% at 2 hours), but not delayed, absorption peak has been observed (Sellman et al., 1975a).Clinical Pharmacokinetics of Diazepam rey st #_<005 in respect to children Plasma level ng/ml) 30-60 180 300 Time (min) Fig, 2, Diazepam blood levels in children, adults and elderly people after a single oral dose (0.25mg/kg), ‘Children (0.3-4 years! ‘e+ Adults (17-59 years) ‘ia Etderiy people (60-80 years) 24680246 8 24680 Patient numbers Fig. 3. Individual variability of diazepam blood levels 180min after oral administration of 15mg,Clinical Pharmacokinetics of Diazepam 78 Solid dosage forms with different dissolution rates (5 cf 48min) yield the same blood levels (Arnold, 1975) but faster absorption is seen following the use of a suspension compared with tablets (Berlin et al. 1972). Therapeutic or symptomatic effects first ap- pear 60min after administration. Interindividual variability in plasma concentration (up to 30-fold) has been noted in patients receiving the same oral dose (fig. 3; Garattini et al., 1973b; Gamble et al., 1973, 1975). A plasma concentration of 400ng/ml is con- sidered necessary to produce a demonstrable and last- ing effect (section 1.5). Suppositories are rarely used. When their use seems to be clinically justified, lower and more erratic absorption can be expected: plasma levels are only 505% of those obtained with the same dose by mouth, and pharmaceutical preparations with different dis- solution rates (6 cf 60min) achieve peak levels at different times (1.8 to 4.4h) [Schwartz et al., 1966; Arnold, 1975] Similarly, poor and irregular absorption is seen after intramuscular administration. Plasma levels are only 60% of those attained with the same oral dose. Possible contributing factors are the site and depth of the injection, the amount of adipose tissue, and perhaps precipitation at the injection site. In routine practice the influence of these variables can be great. Table II shows the differing results attributable to variation in injection site (with higher plasma con- centrations after injection in the thigh than the but- tock) and injection technique (higher concentrations being achieved when a group of doctors, injecting into the buttock, paid deliberate attention to ensuring penetration into muscle, than when injections, at the same site, were given by nurses not thus briefed) [Gamble et al., 1975]. ‘After intravenous injection, no linear increase in plasma levels has been found with increasing doses; boluses of 10 and 20mg result in concentrations after 15min of about 400 and 1,200ng/ml respectively (Hillestad et al., 1974a), After a few minutes all sub- jects are relaxed and drowsy, with slurring of speech 10 to 15 minutes after administration of the drug the Patient is usually asleep. These effects persist for 120 minutes; the sleep can be easily broken at any time, with the subjects being comfortable and alert (Baird and Hailey, 1972; Hillestad et al., 1974a), 1.2 Plasma Protein Binding Diazepam is highly bound to plasma proteins; re- ported values according to different methods ranging from 96.8 to 98.6% (Van der Kleijn et al., 1971; Klotz et al., 1975, 1976a; Thiessen et al., 1976). The percentage of the free drug fraction is independent of the total amount of drug present. Samples loaded with up to 16ng/ml and up to 10yg/ml were tested respectively by Van der Kleijn et al. (1971) and Thiessen et al., (1976). In the fetus and newborn, a lower degree of binding (84%) has been reported by some investigators (Kanto et al., 1974a). Significant differences have been observed for other age groups, buta highly significant (p < 0.001) increase of the free fraction is seen in cirrhotic patients (Klotz et al., 1975). The same binding values as for diazepam have been reported for desmethyldiazepam (Klotz et al., 1976a). 1.3 Distribution in Various Body Tissues 1.3.1 Cerebrospinal Fluid (CSF) Ina study in neurological patients, the distribution from plasma to CSF has been shown to correspond to the free fraction of diazepam (2 to 3%) and of desmethyldiazepam (1 to 4%). The pattern is the same following single and repeated doses (Kanto et al., 1975). A long-term high dose regimen (15 to 30mg/day for many months) does not produce any change in diazepam concentration (8.7% of plasma level), but is accompanied by a significant accumula- tion of desmethyldiazepam (30.9% of plasma level) (Hendel, 1975]. 13.2 Brain A detailed study of the kinetics of diazepam and its, metabolites in the brain has been made in cats (Mor- selli et al., 1973a). A rapid distribution phase in theCinical Pharmacokinetics of Diazepam n Table Il. Diazepam apparent halt-ves after single intravenous administration Author Pationts Dose tye typ th) number age ly) Klotz etal. (1975) 33 15-82 O.1mg/kg - 20-90 ‘Andreasen et al (1976) 4 26-49 10mg - 143-612 (ane Klot et al. (18760) 10 23-96 O.1mg/kg 0.96-1.1 193-469 (3298. a Mean, grey matter is followed by a longer accumulation phase of diazepam and its metabolites in the white matter. This accumulation is more marked following repeated dosages of the drug; the lipid-rich white mat- ter is proposed as a deep compartment, Measurement of diazepam concentrations in various parts of the brain strongly suggests that there is early preferential distribution in those areas, mainly grey matter, with the highest blood flow. 1.3.3 Adipose Tissue AAs it is highly lipophilic, diazepam might be ex- ‘pected to be readily distributed to, and possibly stored in, adipose tissue, being released during lipolysis or under other conditions of physiological change (Mar- cucci et al., 1968b). However, direct measurement in humans is insufficient to give reliable quantitative evidence of this. Following a single intravenous injec- tion of 10mg of diazepam, concentrations of 300 and 345ng/g adipose tissue were measured in 2 patients at 30min and 200 and 239ng/g at 60min. No desmethyldiazepam was detected at 1 80min (Marcuc- cietal., 19680). 1.4 Plasma Level Profile and Elimination Kinetics A two compartment open model, consisting of a rapid distribution (a), followed by a longer elimina- tion phase (B) is usually employed to describe the dis- appearance profile of diazepam. Recently published studies on single dose intravenous administration (able III) are in substantial agreement with earlier results obtained with an oral preparation for both the a and B phases (De Silva et al., 1966). A third, deep compartment described as a y phase has been pro- posed by Kaplan et al. (1973), but its existence does not seem to be justified and it has not been quan- titatively documented. Overall consideration of available data on the ter~ minal elimination half-life (t)/2g), indicates that values of 1 to 2 days (24 to 48h) are a good reference for clinical application. However, a linear increase of half-life has been reported with increasing age from 20 to 80 years (Klotz et al., 1975). This is not due to Table 1V. Apparent volume of distribution (Vg) and plasma clearance (Ci) of diazepam after single dose administration Author No. Va a pts (U/kg) (ml/min) Klotz et al. (1975) 5 113 286 Andreasen et al. (1976) 4 11635 Hvidberg and Dam (1876) — 1-2 _ Klotz et al. (1976a) H 09 (28Clinical Pharmacokinetics of Diazepam 78 . Table V. Diazepam apparent half-ife efter repeated administration ‘Author Patients Daily dose Route Duration of ty/25 (mg) treatment (h) number age (y) (days) Van der Klein et al. (1971) 5 _ 30 oral 15 20-42 Berlin et al. (1972) 7 24-60 18 oral 10 26-53 Hillestad et al. (19746) a 21-35 18 oral 6 54 ‘Arnold (1978) 16 21-87 10 oral 28 60 Kaplan et al. (1973) 4 25-43 10 v % 21-37 Kiotz et al. (1976a) 5 29-35 _ IV+oral —sub- 53.0217.4 chronic changing plasma clearance values, which were re- ported in the same study to be fairly stable (ranging from 20 to 32ml/min), but rather a larger initial dis- tribution space (V,) reflecting a larger volume of dis- tribution at steady state (V ss) seems the more likely explanation of these findings (Klotz et al., 1975). Values for volume of distribution and plasma clearance after a single dose are summarised in table Iv. ‘Studies performed during multiple dose and long- term treatment suggest a longer elimination half-life and less interindividual variability (table V). The variations, although statistically significant, are not substantial, and have been related to accumulation of the active metabolite desmethyldiazepam having an inhibitory influence on the rate of diazepam metabol- ism (Klotz et al., 1976b). Animal data seem to sup- Port this interpretation (Klotz et al., 1976b). Con- versely, a diminution of both diazepam and desme- thyldiazepam steady state plasma levels has been shown during continuous (1 to 6 weeks) treatment, ‘Suggesting an autoinduction phenomenon (Kanto et al., 1974c). The induction could apply first to the demethylating step; this is suggested by the fact that a higher percentage of desmethyldiazepam is found in previously treated patients given an intravenous dose of diazepam than in patients never exposed to the drug. Accelerated degradation of the desmethylated metabolite could then follow (Kanto et al., 1974c). Another possible explanation for the lower plasma concentrations is the accumulation of diazepam and desmethyldiazepam in erythrocytes after 11 or more ‘weeks of therapy (Zingales, 1973). ‘The steady state plasma level of diazepam depends on the daily dose (table VID. The most striking feature is the report that the values measured over a period of 2 to 5 years were 1/5 to 1/10th those found after 15 days’ treatment (Kanto et al., 1974c). Causes such as lack of patient compliance, cannot be ruled out with certainty. However, the role of metabolic autoinduc- tion discussed above seems a more likely explanation. Changes from fixed to flexible dosage regimens or vice versa are not an important factor in the plasma level profile. This is not surprising in a drug with a long half-life, In its clinical implications, the plasma level profile of diazepam at steady state cannot be considered sepa~ rately from that of its active metabolite desmethyldiazepam. Desmethyldiazepam was shown very early to be the main metabolic degradation pro- duct of diazepam (De Silva et al., 1966; Schwartz et al., 1965; Kvetina et al., 1968), but only recently,Ciinical Pharmacokinetics of Diazepam Table VI. Steady state plasma concentrations of diazepam 79 Author Daily dose Duration of Plasma (mg) ‘treatment concen- tration (ng/mi Van der Kleijn et al. (1971) 30 14 days 700-1600 Borin et al. (1972) 5 10 days 285-395 Garattini et al. (19730) 18 48 days 104-2438 Dasberg et al. (1974) 20 S days 264-647 Kanto et al. (1974c) 18 15 days 200-400 15 2-5 years 42-207 20-40 2.5 years 85-422 Bond et al. (1977) 10 2-4 weeks 70-392 2 Blood concentration. with the development of more sensitive analytical techniques, has it been possible to describe the kinetics of its appearance and accumulation pattern following both single and repeated dose administra- tion of diazepam by all routes (Hillestad et al., 1974a,b; Kanto et al., 1974c; Arnold, 1975). As can be seen from tables VII and VIII, desmethyldiazepam has a longer elimination half-life (51 to'120n) than diazepam (24 to 48h), possibly because of the lower rate of hydroxylation to ox- azepam. Both the protein binding and the volume of distribution of desmethyldiazepam are similar to those for diazepam. Because of this longer half-life, the accumulation of desmethyldiazepam to steady state can continue over a treatment-period of more than 3 weeks. Moreover, dose-dependent elimination kinetics of desmethyldiazepam has been suggested (Tognoni et al., 1975). In 3 out of 6 anxious depressed patients who received long-term desme- thyldiazepam treatment, a biexponential decay was observed with a very slow first component (Kejlh~'] range 0.0019 to 0.0072; t)/2 range 96 to 349 hours) followed by a faster one when plasma levels ap- proached 550ng/ml (Kejlh~'] range 0.0085 to 0.0205; t1/2 range 26 to 33 hours). While the data are too scanty to allow clear conclusions to be Table Vil. Some pharmacokinetic properties of desmethyl- diazepam Author Protein tyas Vg" TCP binding (h) (U/kg) (nl (3%) /kg) Hillestad - 92 = - et al. (1974) Klotz 760 — _ _ et al. (19760) Tognoni = Blt? 11k 16.73 et al. (1975) 0 219 Mahon - 2m _ ct al. (1976) Apparent volume of distribution. b Total body clearance.Cinical Pharmacokinetics of Diazepam 80 Table Vil. Desmethykdiazepam (NDZ) plasma concentration after diazepam (DZ) or its own administration Author Drug given Daily dose Duration of NDZ plasma Ratio (mg) ‘treatment levels ing/mi) between (days) Dz/Noz Garattini et al. (1973p) oz 8 48 120-314 (219235) 20 1 180-850 0.62-0.86 (398 = 125) Dasberg et al. (1974) oz 20 5 172778 - (350+219.2) Bond et al (1977) oz 10 14-28 83 1070 021-17 (3) Tansella ot al. (1975) NOZ 10 7 270-1328 20 Zi 327-2850 Tognoni et al. (1975) Noz 20-30 5 410-1800, (849+ 196) 10 630-1840 (1103+ 139) derived, dose-dependent elimination kinetics can be suggested From the above referred values of elimination half-life, a very long duration of pharmacological effect can therefore be expected after discontinuation of therapy. These facts and the interindividual varia- tion in the ratios of diazepam to desmethyldiazepam plasma levels, ranging from 0.21 to 1.7 (Bond et al., 1977), should be borne in mind when considering the clinical implications of an accumulative effect of the 2 drugs. Methyloxazepam and oxazepam, the other 2 active metabolites of diazepam, are only found in small amounts when B-glucuronidase is added to samples of adult plasma or serum before extraction procedures. (For different findings see sections 1.6 and 2.4), Quantitative measurement of these compounds using a specific and sensitive analysis has only relatively re- cently become possible (Belvedere et al., 1972). The formation of these nietabolites after administration in ‘man does not seem to have any clinical relevance, although in certain animal species they explain the Jong duration of action of diazepam (Marcucci et al., 1968b; Marcucci et al., 1970). The overall considera- tion of data presented up to this point seems to allow ‘a practical suggestion, the validity and efficacy of which could be checked in clinical practice. An in- dividually tailored treatment schedule based on a single daily administration of diazepam at night should be adequate for a prolonged anxiolytic effect over the following day. No significant accumulation should take place following this regimen, thus avoid- ing the risk of excessive sedation during repeated dose treatment mainly in elderly people, and a too long wash-out period after stopping the treatment. 1.5 Plasma Levels and Clinical Response No simple correlation exists between clinical response and plasma levels of diazepam and desmethyldiazepam, either separately or together. Besides the well known problem of obtaining reliable markers for anxiolytics or sedative drugs in a popula-Clinical Pharmacokinetics of Diazepam tion of widely differing individuals, there are also differences between the levels which are effective after single dose and long-term administration. Whereas 400ng/ml produces a clearly detectable clinical effect, after a single dose, no signs of functional impairment or evidence of therapeutic activity could be observed with much higher steady state plasma levels of both diazepam and desmethyldiazepam (Bond et al., 1977; Tansella et al., 1975; Kanto et al., 1974c; Garattini et al., 19736). In only a few cases has a correlation been found between desmethyldiazepam levels and response; as determined by results of psychiatric rat- ing scales (Curry, 1974) or the appearance of autonomic side-effects (Dasberg, 1975). The lack of correlation between plasma levels and clinical effect thas been confirmed in a carefully controlled multiple dose study in hospitalised patients (Tansella et al., 1978). Dasberg et al. (1974) and Zingales (1973) consider that there are advantages in measuring diazepam plasma levels in order to ensure that steady state levels above 400ng/ml are reached and maintained, but this does not seem to reflect clinical experience, where a therapeutic effect is obtained with a wide range of dosage from 2 to 20mg (Bond et al., 1977). Diazepam does have an anticonvulsant effect at a peak plasma level of 400 to 500ng/ml, which is usually reached following a bolus intravenous injec- tion of 10 to 20mg (Booker and Celesia, 1973; Hvid- berg and Dam, 1976). The extensive metabolism of diazepam, the likeli- hood that different clinical conditions will require different plasma concentrations, and the probability that plasma and brain concentrations of diazepam and desmethyldiazepam will vary depending upon whether treatment is short or long-term (Morselli et al., 1973a), all confirm the lack of indication for rolitine measurement of plasma levels of the drug (Garattini et al., 1973b). 1.6 Biliary Excretion The study of biliary excretion of diazepam has received particular attention in recent years, mainly for kinetic reasons; the existence of a substantial en- terohepatic circulation being hypothesised to explain partially the long elimination half-life of the drug and the secondary peak observed during the elimination phase (Van der Kleijn et al., 1971; Baird and Hailey, 1972). However, diazepam is not excreted in bile in significant amounts after single or repeated doses in ost-cholecystectomy patients with a normal liver function (Klotz et al., 1975, 1976), and evidence for the existence of enterohepatic circulation in patients with biliary disease is controversial. The biliary excretion of diazepam in patients un- dergoing biliary tract surgery has been investigated by 2 groups. Sellman et al. (1975b) compared levels of diazepam and desmethyldiazepam in patients whose bile was collected with a T-tube with levels ina group of cholecystectomised controls; they interpreted their results as providing good evidence of the existence of enterohepatic circulation of diazepam but not of desmethyldiazepam. However, the work of Mahon et al. (1976) does not support this conclusion. Using '4C-5-diazepam given as an intravenous bolus to 5 patients with T-tube biliary drainage, these authors found that a mean of only 5.35% (range 1.7 to 7.4%) of the radioactivity was present in the bile over, a period of 5 to 14 days. Even when corrected to a bile flow of 700ml, this value is still too low (15%) to allow for a clinically significant enterohepatic circula- tion. Furthermore, the radioactivity was not due to either diazepam or desmethyldiazepam (which ac- counted respectively for 0.056% and 0.152% of the dose), but rather to the same hydroxylated ‘metabolites which were found in the urine. The exis- tence of an enterohepatic circulation has been con- firmed for free and glucuronated oxazepam by studies in animals (Bertagni et al., 1972, 1978). Indirect evidence of enterohepatic circulation is offered by Linnoila et al. (1975) on the basis of sec- ondary peaks in diazepam plasma levels after a fat- rich meal; the disappearance curve being linear following water ingestion. While excluding any in- fluence of decreased binding to proteins due to higher concentrations of free fatty acids, a more general mechanism of mobilisation from storage sites is pro-Giinical Pharmacokinetics of Diazepam 82 Table 1X. Diazepam pharmacokinetics in different hepato-bilary diseases ‘Author Disease Patients Dose and route ty/25 Va a th) (rg) (oni/min} number age (y) Klow Cirrhosis 8 41-50 O.tmg/kg IV 79.1-1328 1.74 138224 et al. (1975), (105.6 + 15.2) Acute viral 8 18-31 O.tmg/kg IV 478-1292 — - hepatitis (745 +278) Chronic viral 4 23-49 Oimg/kg IV 25.7-76.0 - 1304179 hepatitis (69.7 +230) ‘Andreasen Cirrhosis 9 23-68 10mg IV 38.1-517.2 2.86 m1 et al. (1976) (1640) Mahon Bilary 5 38-77 40-50xCi 30-187 39 314 et al. (1976) diseases (93.2) posed by Korttila and Kangas (1977), who showed the same pattern of transient increase of diazepam levels following a carbohydrate meal. 1.7 Urinary Excretion Data on urinary excretion of diazepam and its metabolites is scanty. 71% of the radioactivity of a 10mg oral dose of *H-diazepam is found in the urine, Only a very small percentage of this is in the free form,, the major part being excreted as either the glucuronide or sulphate. Insufficient quantitative data exist to confirm whether desmethyldiazepam (Ar- nold, 1975) or oxazepam (Schwartz et al., 1965; Kan- to et al., 1974a) is the more important urinary ‘metabolite (see also section 2.4). 2. Influence of Physiological and Disease ‘States on Kinetics 2.1 Liver Diseases ‘As might be expected for a drug like diazepam, which is extensively metabolised by hepatic microsomal enzymes, and exhibits capacity-limited protein binding sensitive hepatic clearance, it is pri- marily in liver disease that significant disease-induced alterations of kinetic properties have been shown (table 1X). Although there is no indication for the routine use of diazepam in patients with liver disease, an understanding of the kinetic changes likely to oc- cur is necessary so that the dose may be adjusted if necessary in a patient in whom the use of diazepam is, thought warranted. Precise guidelines for any dose adjustment cannot be given at this time and clinicians so using diazepam in the presence of liver diseases must titrate dosage on the basis of both careful patient observation and available kinetic findings. Impaired absorption is suggested by the lower peak plasma levels observed in alcoholic patients given oral diazepam (Sellman et al., 197Sa). A larger (50 to 60% increase) volume of distribution in alcoholic cirrhosis (Klotz et al., 1975; Andreasen et al., 1976) and in patients with biliary disease (Mahon et al., 1976), is a possible explanation for their lower steady state plasma levels. Decreased protein binding (from 97.8 to 95.3%) together with diminished (50%) plasma clearance (Klotz et al., 1975; Andreasen et al., 1976) and in-Ciinical Pharmacokinetics of Diazepam 83 creased volume of distribution (Klotz et al., 1975) has also been observed in cirrhotic patients. Values for the elimination half-life are increased about 2-fold in acute viral hepatitis, but return to normal on recovery from the illness (Klotz et al., 1975). A more marked (2 to S-fold) increase in the elimination half-life is seen in patients with alcoholic cirrhosis (Klotz et al., 1975; Andreasen et al., 1976). These authors, however, could not find any correlation between half- life and various hepatic function tests. A delay in the appearance of the active metabolite desmethyldiazepam and of the peak plasma level (from 43 to 85h) has also been reported in cirrhotics (Andreasen et al., 1976), further supporting the decreased rate of metabolism of diazepam in cir- thosis. However, the net consequence of a slower rate of elimination of diazepam and slower production of desmethyldiazepam is probably negligible when diazepam is used intravenously in status epilepticus (Hvidberg and Dam, 1976). Blaschke (1977) has dis- cussed in more detail the clinical consequences of and changes in disposition of diazepam (and other drugs) in liver diseases. 2.2 Hypoalbuminaemic States ‘A larger free drug fraction can be measured in hy- poalbuminaemic states (Thiessen et al., 1976), with a resultant larger volume of distribution and a signifi- cant increase in plasma clearance of diazepam (p< 0.05) [Klotz et al., 1976a]. Thus, the expected in- crease in clinical effects is probably only temporary; the initial increased availability of the free drug being, compensated for by a faster elimination rate, as also Proposed by Thiessen et al. (1976) for tolbutamide in cirthotics. A higher incidence of diazepam side-effects hhas indeed been observed in patients with hy- poalbuminaemia than in patients with normal serum proteins (Greenblatt and Koch-Weser, 1974); the number of patients with liver or renal disease being the same in both groups. An explanation for the in- crease in clinical effects in hypoalbuminaemia may relate to an earlier and greater diffusion in the central nervous system due to the larger free fraction of both diazepam and desmethyldiazepam available for ac- tivity firstly on the grey and then on the white matter of the brain (section 1.3.2). The role of the dose-de- pendent elimination kinetics suggested for desmethyl- diazepam (section 1.4) at higher free drug plasma con- centration could also be considered as a supplemen- lary factor explaining the frequency of side-effects, which could result from an increased accumulation in the cerebral compartment. No linear relationship between albumin concentra- tion and binding capacity of diazepam was observed in patients with acute renal failure (Andreassen, 1974), Elevated serum free fatty acids do not modify the protein binding of diazepam (Tsutsumi et al., 1975). 2.3 Pregnancy and Labour Studies of diazepam metabolism and kinetics in different stages of pregnancy fall under 2 main head- ings, dependent upon the emphasis given to the evaluation of the metabolic activity of the fetal liver, as distinct from the kinetic behaviour of the drug and its metabolites in crossing the placenta. The clinical interest of the data obtained in the first type of study lies mainly in the information provided on matura- tion of the hepatic drug metabolising enzymes of the fetus. On the other hand, knowledge of the extent and pattern of transplacental passage of diazepam follow- ing single and repeated administration, mainly during, the late stage of pregnancy, is important both in monitoring the effects of diazepam and desmethyldiazepam on the fetus and the newborn, and studying their disposition in the first days of life. 2.3.1 Transplacental Passage ‘As may be expected, in view of the changes which take place in the anatomical structure of the placenta and in the uterine circulation as pregnancy advances, transfer of diazepam across the placenta has been shown to be slower in early pregnancy than in the later stages and during labour (Erkkola et al., 1973:Giinical Pharmacokinetics of Diazepam Table X. Diazepam transplacental kinetics after single dose treatment (after Mandell et al, 1975) Dose No. Time course of plasma concentrations cP/MPo pts minutes corresponding levels (ng/ml oz Noz between last dose mother ‘cord and delivery oz oz oz Noz tomgiV 6 12-218 8-19 5-37 0.9~1.94 tomgiv 7 40-160 24816 122-7 272-29 6923 0,77=250 0,343.28 2 Ratio between cord plasma (CP) and mother plasma (MP) concentrations. b DZ = Diazepam. © NDZ = Desmethykiiazepam. Kanto and Erkkola, 1974; Kanto et al., 1974). These authors showed that the ratio of fetal:maternal plasma levels changed from 1.2 in early pregnancy to 1.8 im the later stages, when diazepam was given as a ‘single parenteral dose, and from 0.4 to 0.8 when diazepam was given by repeated oral administration. Measurable amounts of diazepam were found in all samples from the placenta, fetal liver and fetal brain but not in amniotic fluid. This last finding is at variance Witlr the reports of others (Indanpaan-Heik- kila et al., 1971). Concentrations of diazepam and desmethyl- diazepam following repeated treatment were measured in various organ tissues of a fetus aborted at 31 weeks (Mandelli et al., 1975). The ratio of desmethyldiazepam to diazepam was approximately 2:3 in all tissues with 2 notable exceptions: desmethyldiazepam was highly concentrated in both lung and placenta (10:1 cf diazepam), Available knowledge of the transplacental kinetics of diazepam during late pregnancy and labour is sum- marised in tables X and XI, showing data obtained after single dose and long-term administration respec- tively. Distribution from the maternal to fetal com- partment is rapid, after both intravenous and in- tramuscular administration. Long-term treatment leads to accumulation of both diazepam and des- methyldiazepam on the fetal side, 2.3.2 Fetal Drug Metabolising Capacity Human fetal liver microsomes can metabolise diazepam to desmethyldiazepam and methylox- azepam as early as the 1 3th week of gestation (Acker- mann and Richter, 1977). The metabolising capability of the fetus for some major biotransforma- tion pathways of diazepam is further supported by studies conducted at birth, comparing diazepam and desmethyldiazepam in arterial (AC) and venous (VC) cord blood (Mandell et al., 1975). AC/VC ratios of 0.71 + 0.12 and 0.94 + 0.13 were found for diazepam in short and long-term treated cases respec- tively; the corresponding AC/VC ratios found for desmethyldiazepam were 1.75 + 0.38 and 0.87 + 0.05. These figures suggest an important demethylat- ing activity in the fetus, with plasma concentrations reaching an equilibrium in the feto-maternal unit only after repeated dosing 2.4 Neonates, Infants and Children Differences in the kinetics of diazepam in newborn infants and children are shown in table XII. ItClinical Pharmacokinetics of Diazepam Table Xi. Diazepam transplacental kinetics after repeated dose (RO) treatment ‘Author Treatment No. pts Time course of plasma concentrations corresponding levels (ng/mil mother cord oz Noz oz Noz Cree RD < 30mg 18 15 180 181 241 188 et al. (1973) RD > 30mg 14 18 540 321 664 448 Kanto 10-16mg for 5 12.18 - - 10-150 10-400 et al. (19740) 6-21 days Mandell RO 8 = 137-8 198-10 186-12, 172-15 et al. (1975) 2 DZ = Diazepam. b NDZ = Desmethyldiazepam. Table X1l. Some pharmacokinetic properties of diazepam in premetures, newborn infants and chikiren Author Cases Treatment tv Tecl Va (rni/kg/n) (W/k9) Morselt 4 prematures 0.33m9/kg 75237 27.49: 8.53 1.80+0.28 et al. (19736) (28-34 wh) Mandell 11 newborns 10mg IM to the as220 - et al. (1975), (1-2 days) mother before delivery Morsel 5 children 0.33mg/kg 1843 102,109.72 2.60 +053, et al. (19736) (48 y) seems worth recalling that no clear consensus exists with regard to variations in the volume of distribu- tion according to age. Whereas some studies (Mor- selli, 1976) refer to data suggesting a tendency for the volume of distribution to be lower in the newborn (1.40 to 1.82 L/kg) than that calculated in adults (2.20 to 2.60 L/kg), comparison of values in table XII with results reported in table IV indicates no ma- jor differences in the volume of distribution between newborn infants and adults. Some studies have shown diazepam to be less bound to umbilical cord plasma than to the corresponding maternal plasmaClinical Pharmacokinetics of Diazepam (Kanto et al., 1974), but others have found no difference between cord and adult serum in binding properties of diazepam (Krasner and Yaffe, 1975). Conclusive data on the type and extent of diazepam transformation to hydroxylated metabolites, in the newborn are not available, but it is usually con- sidered that hydroxylation of both diazepam and des- methyldiazepam is very limited or lacking in pre- ‘mature and full-term infants, while the hydroxylated compounds are present in subjects over 2 to 3 weeks of age and in children (Morselli et al, 1973b). However, hydroxylating activity in the fetal liver can. apparently be induced by administration of inducing agents to the mother. Thus, administration of phenobarbitone to the mother during pregnancy resulted in an elimination half-life of diazepam in pre- ‘mature infants of around 16h (Morselli et al., 1974); close to the value observed for young children (table XID. A demethylating activity relatively lower than that in children has been observed in premature and full- term infants [rate of demethylation (K) in newborns of 0.097h compared with 0.179h in children] (Mor- selli, 1976). Data on the usually expected low glucuronidation capacity inthe newborn are more clear cut. While desmethyldiazepam, mainly conjugated, represented the main metabolite detected in the urines of newborns (Morselli et al. 1973b, 1974), only small amounts of the conjugated form of both methylox- azepam and oxazepam were found but these were higher when the mother had been treated with Phenobarbitone. The presence of methyloxazepam, both as free and as glucuronide derivative, in cord and in the plasma of newborns, reported only in those cases in which these compounds were also present in maternal plasma, suggests that hydroxylated and con- jugated compounds can cross the placenta (Mandelli et al., 1975). Glucuronised oxazepam constituted about 70% of all diazepam products in the urine of $ newborn in another study, where no glucuronised form of desmethyldiazepam was found (Kanto et al., 1974a,b). In the same study the measurement of free oxazepam in plasma (13 to 220ng/mi with | value of 121ng/mi) is justified by the authors through a low glucuronising capacity in the newborn. 2.5 Breast Feeding Levels of diazepam in human breast milk are of the order of 1/10 of that in plasma, but administra- tion of therapeutic (10mg) doses to the mother leads to fairly high levels in the newborn (491ng/ml and 172ng/ml respectively after 4 days; 601ng/ml and 74ng/ml after 6 days). The low figure in the breast- fed infant after 6 days is possibly due to an increasing rate of metabolism by the infant (Erkkola and Kanto, 1972), As expected, desmethyldiazepam can be found with diazepam in plasma of a newborn breast-fed by a mother on treatment with the drug (Cole and Hailey, 1975), with levels of desmethyldiazepam in milk (12 to 85ng/ml) consistently higher than those of diazepam (17 to 43ng/ml) after 10mg daily for 6 days (Brandt, 1976). There has been a single case re- port of lethargy and weight loss with EEG evidence of sedative medication, in a breast-fed infant of a ‘mother treated with diazepam 10mg 3 times daily. Maternal plasma and milk levels were not measured ‘but oxazepam could be traced in the urine of the in- fant (Patrick et al., 1972). These studies suggest that a daily dose of 10mg diazepam is probably too small to cause untoward effects in the breast-fed infant. However, if higher daily doses of diazepam must be given repeatedly, breast-feeding should probably be discontinued. 2.6 The Elderly ‘As discussed in section 1.4, there is an increase in elimination half-life of diazepam with increasing age such that at age 80 years the half-life is 90h compared with 20h at age 20 years, as a consequence of an in- crease in volume of distribution. Plasma clearance did not change. The therapeutic implications of these kinetic findings are not clear but for other reasonsClinical Pharmacokinetics of Diezepam 87 (eg. risk of postural hypotension, unsteadiness and falls), lower dosage of diazepam should be used, at Jeast initially, in the elderly. 2.7 Renal Diseases Protein binding of diazepam is decreased from 98 to 92% in patients with renal insufficiency (Kangas et al., 1976) but the implications of this finding are not clear. Renal disease does not appear to affect the rate of elimination of diazepam. There are no data on the role of hypoalbuminaemia associated with uraemia on the disposition of diazepam, but Andreassen (1974) has shown that there is no linear relationship between the albumin concentration and binding capacity of diazepam in patients with acute renal failure. 3. Use of Diazepam in Pregnancy and Labour Recent studies (Aarskog, 1975) have raised suspi- cions about the potential dysmorphogenicity of some antianxiety drugs. Pending more definite results ex- pected from prospective studies, the routine use of diazepam should be discouraged during the first tri- mester of pregnancy, as part of the general recom- ‘mendation to avoid the unnecessary use of drugs dur- ing this period. Administration of diazepam should, however, be considered with great caution throughout pregnancy, as both the drug and its metabolites can accumulate in all fetal tissues (section 2.3) and cause problems at birth. The clinical significance of high plasma and tissue levels of these compounds in the newborn should be carefully considered when evaluating vital signs before and during labour and in the presence of behavioural and physiological impairment in the first 10 to 15 days of extrauterine life. Lower Apgar scores, apnoeic spells, hypothermia, reluctance to feed, and impaired metabolic response to cold stress (Cree et al., 1973; Shannon et al., 1972) have been re- ported after large doses, as has respiratory depression (Andre et al., 1973). Alterations of fetal heart rate have been observed (Scher et al., 1972; Sagen and Haram, 1973), but their clinical significance has not been determined (Mandelli et al., 1975). No major complications should be expected following a single dose of diazepam during labour, but certainly the possible effects of diazepam on the newborn must be taken into account when large (40 to 100mg) intravenous doses of diazepam are used in the treatment of eclampsia and severe pre-eclampsia. 4. Pharmacokinetic Drug Interactions The effects of combined ingestion of ethanol and diazepam have been the subject of many publications. The impairment by this combination of various in- dices of performance of driving-related skills has been, well documented (Linnoila and Mattila, 1973), but data on possible underlying kinetic mechanisms of in- teraction are scanty. No modification of absorption, hhas been observed following an alcoholic (dose of alcohol being 0.5¢/kg body weight) bitter ingestion (Linnoila et al., 1974) using diazepam capsules, but approximately 100% higher peak plasma levels were seen when the absorption pattern of an alcoholic solu- tion of diazepam was compared with that of diazepam in distilled water (Hayer et al., 1977). The former ex- periment seems possibly to be closer to the situation in clinical practice and enhancement of absorption should not therefore be considered a major factor in determining the clinical effects of the diazepam- ethanol interaction. The influence of smoking on diazepam metabol- ism has also received some attention. No differences in plasma elimination half-life or steady state levels have been reported between smokers and non- smokers, suggesting that there is no important indue- tion of the metabolic pathway of diazepam elimina- tion (Klotz et al., 1975). These data are, however, at variance with those from the Boston Collaborative Drug Surveillance Program (1973) showing a markedly lower incidence of side-effects (mainly seda- tion) in elderly smokers, compared with non-Clinical Pharmacokinetics of Diazepam 88 smokers. Despite the fact that no plasma levels were obtained in that retrospective study, clinical evidence suggests that lower concentrations of both diazepam and desmethyldiazepam may be due to increased for- mation of glucuronated and readily excreted hydroxy- ated derivatives of diazepam. The existence and clini- cal significance of increased hydroxylation has been, documented in newborn infants of mothers on treat- ‘ment with phenobarbitone (Morselli et al., 1973b; Sereni et al., 1973; see section 2.4). Despite its high protein binding to plasma pro- teins, diazepam does not seem to be able to displace other highly bound drugs such as warfarin (Orme et al., 1972). A displacing effect on bilirubin has not ‘been confirmed and was attributed to a chemical im- purity present in the pharmaceutical preparation (Adoni et al., 1973; Schiff et al., 1971). References ‘Aarskog, D.: Association between maternal intake of diazepam and oral clefts. Lancet 2: 921 (1975). ‘Ackermann, E, and Richter, K.: Diazepam metabolism in human foetal and adult liver. European Journal of Clinical Phar- macology 11: 43-49 (1977). Adoni, A: Kapitulnik, J.; Kaufmann, N.A; Ron, M. and Bonheim, S.A.: Effect of maternal administration of diazepam ‘on the bilirubin binding of cord blood serum. American Jour- nal of Obstetrics and Gynecology 115: 557-579 (1973). Andre, M. Sibout, M.: Petry, J-M. and Vert P.: Depression respiratoire et neurologique chez le premature nouveau-ne de mere traetee par diazepam. Journal de Gynecologie, Obstetr- ue et Biologie de la Reproduction 2: 357-366 (1973). Andreassen, F.: The effects of dialysis on the protein binding of drugs in patients with acute renal failure. Acta Pharmacologica et Toxicologica 34: 284-294 (1974) Andreasen, P.B: Hendel, J: Greisen, G. and Hyidberg, E.F. Pharmacokinetics of diazepam in disordered liver function. European Journal of Clinical Pharmacology 10: 115-120 (1976), Amold, E.: A simple method for determining diazepam and its major metabolites in biological Muids: Application in bioavailability studies. Acta Pharmacologica et Toxicologica 36: 335-352 (1975). Baird, E'S. and Hailey, D.M.: Delayed recovery from a sedative: Correlation of the plasma levels of diazepam with clinical effects after oral and intravenous administration. British Jour- nal of Anaesthesiology 44: 803-808 (1972). Belvedere, G.; Tognoni, G.; Frigerio, A. and Morse, PL: A specific, rapid and sensitive method for gas-chromatographic determination of methyl-oxazepam in small samples of blood. Analytical Letters 5: 531-541 (1972). Berlin, A.B. Siwers, B. Agurell, S Hiort, A: Sjoqvst,F. and Strom, S. Determination of bioavailability of diazepam in various formulations from steady state plasm concentration data. Clinical Pharmacology and Therapeutics 13: 733-744 ast, Beragn, P; Bianchi, R.; Marcucci, Fs Mussini E. and Garattini, S: the enterohepatc circulation of oxazepam-O-glucuronide in ‘guinea pigs. Journal of Pharmacy and Pharmacology. In press ast. Beriagni, P.; Marcucci, F Mussini, E. and Garatini, $: Biliary ‘excretion of conjugated hydroxyl benzodiazepines after admin- istration of several benzodiazepines to rats, guinea pigs, and ‘mice. Journal of Pharmaceutical Sciences 61: 965-966 (1972). Blaschke, TF. Protein binding and kinetics of drugs in liver dis- cases. Clinical Pharmacokinetics 2: 32-44 (1977) Bond, AJ. Hailey. D.M. and Lader. M.H.: Plasma concentrations of benzodiazepines. British Journal of Clinical Pharmacology 4: 51-56 (1977). Booker, HE. and Celesi, G.G.: Serum concentrations of diazepam in subjects with epilepsy. Archives of Neurology 29, 191-194 (1973) Boston Collaborative Drug Surveillance Program: Clinical depres- sion of the central nervous system due to diazepam and chlor- Aiazepoxide in relation to cigarette smoking and age. New England Journal of Medicine 288: 277-280 (1973). Brandt, R.: Passage of diazepam and desmethyldiazepam into breast milk. Arzneimittel-Forschung 26: 454-457 (1976). Cole, A.P. and Hailey, DM. Diazepam and active metaboite in breast milk and ther transfer to the neonate. Archives in Dis- ease in Childhood 50: 741-742 (1975). Costa, E. and Greengard, P. (eds) Mechanism of Action of Benzodiazepines (Raven Press. New York 1975). (Cree, LE; Meyer, J. and Hailey, D.M.; Diazepam in labour: Its metabolism and effect on the clinical condition and ther- rmogenesis ofthe newborn. British Medical Journal 4: 251-255 (1973), Curry, SH: Concentrationeffet relationships with major and minor tranquilizers. Ciinical Pharmacology and Therapeutics 16: 192-197 (1974). Dasberg, H.: Oral diazepam in hospitalized anxiety patients (With ‘observations on concentration-effect-reationships). Phar- ‘makopsychiatrie Neuro Psychopharmakologie 8: 161-170 (1979) Dasberg, HLH; Van der Kleij, E; Guelen, PLR. and Van Praag, HLM: Plasma concentrations of diazepam and of its metaboliteClinical Pharmacokinetics of Diazepam N-desmethyldiazepam in relation to anxiolytic effect. Clinical Pharmacology and Therapeutics 15: 473-483 (1974). De Silva, J.A.: Koechlin, B.A. and Bader, G.: Blood level distribu- tion patterns of diazepam and its major metabolite in man. Journal of Pharmaceutical Sciences 5S: 692-702 (1966) Erkkola, R. and Kanto, J.: Diazepam and Breast-feeding. Lancet | 1235-1236 (1972), Erkkola, R; Kangas, L. and Pekkarinen, A.: The transfer of diazepam across the placenta during labour. Acta Obstetricia et, ‘Gynecologica Scandinavica $2: 167-170 (1973). Fuccella, LM; Bolcioni, G.; Tamassia, V.; Ferrario, L. and Tos- noni, G. Human pharmacokinetics and bioavailability of ‘temazepam administered in soft gelatin capsules, British Jour- ‘al of Clinical Pharmacology. tn press (1977). Gamble, J.AS.; Dundee, JW. and Asaf, RAE: Plasma diazepam levels after single dose oral and intramuscular ad- ministration. Anaesthesia 30: 164-169 (1975), Gamble, J.A.S.; Mackay, J. and Dundee, J.W : Plasma levels of diazepam, British Journal of Anaesthesiology 45: 1085 (1973). Garattini, Sx Marcucei, F.; Morselli, P-L. and Mussini, E.: The significance of measuring blood levels of benzodiazepines: in Davies and Prichard (Eds) Biological Effects of Drugs in Rela- tion to their Plasma Concentrations, pp.21 1-225 (MacMillan, London 19736). Garattini, S Marcucci, F, and Mussini E Biotransformation of drugs to pharmacologically active metabolites; in Eichler, Farah, Herken and Welch (Eds) Handbook of Experimental Phacmacology, Vol. 28, pp.113-119 (Springer-Verlag, New York. 1975). Garattini, S.; Mussini, E. and Randall, LO. (Edsk The Benzodiazepines (Raven Press, New York, 1973. Ghoneim, M.M. and Korttila, K Pharmacokinetics of in- travenous anaesthetics: Implications for clinical use. Clinical Pharmacokinetics 2: 344-372 (1977). Greenblatt, D.J. and Koch-Weser, J.: Clinical toxicity of chlor- diazepoxide and diazepam in relation to serum albumin con- centration: A report from the Boston Collaborative Drug Sur- veillance Program. European Journal of Clinical Phar- rmacology 7: 259-262 (1974) Greenblatt, DJ. and Shader, R..: Benzodiazepines, pt. 1 and pt I New England Journal of Medicine 291: 1011-1015, 1239-1243 (1974). Hayer, S.L.; Pablo, G.: Radomsky, T. and Palmer, R.F.: Ethanol ‘and Oral diazepam absorption. New England Journal of Medicine 296: 186-189 (1977) Hendel, J. Cumulation in cerebrospinal fuid of the N-desmethyl ‘metabolite after long-term treatment with diazepam in man, ‘Acta Pharmacologica et Toxicologica 37: 17-22 (1975). Hillestad, L; Hansen, T.; Melsom, H. and Drivenes, A. Diazepam metabolism in normal man. 1. Serum concentra tions and clinical effects after intravenous, intramuscular, and ‘oral administration. Clinical Pharmacology and Therapeutics, 16; 479-484 (19740) Hillesiad, Lj Hansen, T. and Melsom, H.: Diazepam metabolism in normal man. Il. Serum concentration and clinical effect after oral administration and cumulation. Clinical Phar- macology and Therapeutics 16: 485-489 (1974b). Hvidberg, E.F. and Dam, M.: Clinical pharmacokinetics of anti- convulsants. Clinical Pharmacokinetics 1: 161-188 (1976). danpaan-Heikkila, J.E.; Jouppila, P-1.; Puolakka, J.0. and Vorne, MS.; Placental transfer and fetal metabolism of diazepam in carly human pregnancy. American Journal of Obstetrics and Gynecology 109: 1011-1016 (1971). Kangas, L.; Kanto, J; Forsstrom, J. and lisal, E.: Protein binding of diazepam and N-demethyldiazepam in patients with poor renal function. Clinical Nephrology 5: 114-118 (1976). Kanto, J. and Erkkola, R: The feto-materal distribution of diazepam in early human pregnancy. Annales Chirurgiae et Gynaecologiae Fenniae 63: 489-491 (1974), Kanto, J; Erkkola, R. and Sellman, R.; Distribution and metabol- ism of diazepam in early and late human pregnancy. Postnatal ‘metabolism of diazepam. Acta Pharmacologica et Toxicologica 35: Suppl. 36 (1974a). Kanto, J.; Erkkola, R, and Sellman, R.: Perinatal metabolism of diazepam. British Medical Journal |: 641-642 (1974b). Kanto, J. lisalo, E.; Lehtinen, V. and Salminen, J.: The conoentra- tions of diazepam and its metabolites in the plasma after an ‘acute and chronic administration. Psychopharmacologia 36: 123-131 (1974), Kanto, J.; Kangas. L. and Siirtola, T.: Cerebrospinal-fluid con- ‘centrations of diazepam and its metabolites in man. Acta Phar- ‘macologica ¢t Toxicologica 36: 328-334 (1975). Kaplan, S.A.; Jack, M.L: Alexander, K. and Weinfeld, R-E. Pharmacokinetic profile of diazepam in man following single intravenous and oral and chronic oral administrations. Journal of Pharmaceutical Sciences 62: 1789-1796 (1973). Kesson, CM.; Gray, J.M.B. and Lawson, D.H.: Benzodiazepine ‘drugs in general medical patients. British Medical Journal 1: (680-682 (1976. Kio, U; Antonin, KH. and Bieck. P-R.: Pharmacokinetics and plasma binding of diazepam in man, dog, rabbit, guinea pig. and rat. Journal of Pharmacology and Experimental “Therapeutics 199: 67-73 (1976a) Klow, U.; Antonin, K.L. and Bieck, P.R: Comparison of the pharmacokinetics of diazepam after single and subchronic doses. European Journal of Clinical Pharmacology 10: 121-126 (19760). ‘Klotz, U.; Avant, G.R Hoyumpa, A.; Schenker, S. and Wilkin- son, G.R. The effects of age and liver disease on the disposi tion and elimination of diazepam in adult man, Journal of Clinical Investigation 55: 347-359 (1975). Korttila, K. and Kangas, L.: Unchanged protein binding and the increase of serum diazepam levels after food intake. Acta Pharmacologica et Toxicologica 40: 241-246 (1977). Krasner, J. and Yaffe, S.J: Drug-protein binding in the neonate; in Morseli, Garattini and Sereni (Eds) Basic and TherapeuticClinical Pharmacokinetics of Diazepam ‘Aspects of Perinatal Pharmacology, pp.357-366 (Raven Press, New York 1975). ‘Kvetina, J Marcucci, F, and Fanelli, R.: Metabolism of diazepam in isolated perfused liver of rat and mouse. Journal of Phar- ‘macy and Pharmacology 20: 807-808 (1968) Lasagna, L.: The role of benzodiazepines in nonpsychiatric medi- cal practice. American Journal of Psychiatry 134: 656-658 (1977). Limnoila, M.; Korttila, K. and Mattila, M.J.: Effect of food and repeated injections on serum diazepam levels. Acta Phar- ‘macologica et Toxicologica 36: 181-186 (1975). Linnoila, M. and Mattila, M.J.: Drug interaction on psychomotor skills related to driving: Diazepam and alcohol. European Journal of Clinical Pharmacology 5: 186-194 (1973). Linnoila, M.; Saario, I. and Maki, M.: Effect of treatment with diazepam or lithium and alcohol on psychomotor skills related to driving. European Journal of Clinical Pharmacology 7 337-342 (1974. Mahon, W.A.; Inaba, T.; Umeda, T:; Tsutsumi, E. and Stone, R.: Biliary climination of diazepam in man. Clinical Phar- macology and Therapeutics 19: 443-450 (1976). Mandelli, M.; Morseli, P.L.; Nordio, S.: Pardi, G.: Principi, N.; ‘Sereni, F. and Tognoni, G.: Placental transfer of diazepam and its disposition in the newborn. Clinical Pharmacology and ‘Therapeutics 17: 564-572 (1975). Marcucci, F.; Fanelli, R.; Frova, M. and Morselli, P.L.: Levels of diazepam in adipose tissue of rats, mice and man. European Journal of Pharmacology 4: 464-466 (19686), Marcucci, F.; Fanelli, R. and Mussini, E.: A method for gas ‘chromatographic determination of benzodiazepines. Journal of Chromatography 37: 318-320 (1968a). Marcucci, F.; Fanelli, R.; Mussini, E, and Garattini, S.: Further ‘Studies on species difference in diazepam metabolism. Euro- pean Journal of Pharmacology 9: 253-256 (1970). Maroucei, F.; Guaitani, A.; Kvetina, J, Mussini, E. and Garattini, S. Species difference in diazepam metabolism and anticonvul- sant effect. European Journal of Pharmacology 4: 467-470 (19680). Morseli, PL. Clinical pharmacokinetics in neonates. Clinical Pharmacokinetics 1: 81-98 (1976). Morselli, P.L.: Cassano, G.B.; Placidi, G.F; Muscettola, G.B. and Rizzo, M.: Kinetics of the distribution of “C-diazepam and its ‘metabolites in various areas of cat brain; in Garattini, Mussini ‘and Randall (Eds) The Benzodiazepines, pp.129-143 (Raven Press, New York, 1973. ‘Morseli, P.L.; Mandelli, M.; Tognoni, G.; Principi, N.; Pardi, G. and Sereni, F.: Drug interactions in the human fetus and in the ‘newborn infant; in Morselli, Garattini and Cohen (Eds) Drug Interactions, pp.259-270 (Raven Press, New York, 1974). Morseli,P.Ls Principi, N.; Tognoni, G.: Reali, E.; Belvedere, G.; Standen, S.M. and Sereni, F.: Diazepam elimination in pre- mature and full term infants, and children. Journal of Perinatal Medicine 1: 133-141 (19736) Nicholson, A.N. and Stone, BM. Effect of a metabolite of diazepam, 3-hydroxydiazepam (Temozepam), on sleep in man, British Journal of Clinical Pharmacology 3: 543-550 (1976). Orme, M.; Breckenridge, A. and Brooks, R.V.: Interactions of benzodiazepines with warfarin. British Medical Journal 3; 611-614 (1972). Patrick, M.J.; Tilstone, WJ. and Reavey, P.: Diazepam and breast-feeding. Lancet 1; $42-543 (1972) Sagen, N. and Haram, K.: Diazepam (Valium) as an anaesthetic for operative vaginal delivery. Acta Obstetricia et ‘Gynecologica Scandinavica $2: 153-156 (1973). ‘Scher. J Hailey, D.M. and Beard, R.W.: The effects of diazepam ‘on the fetus. Journal of Obstetrics and Gynaecology of the Bri- tish Commonwealth 79: 635-638 (1972). Schiff, D.; Chang, G. and Stern, L.: Fixed drug combinations and the displacement of bilirubin from albumin. Pediatrics 48; 139-141 (97D. ‘Schwartz, DLE Vecchi, M.; Ronco, A. and Kaiser, K.: Blood levels after administration of 7-chloro-1,3-dihydro-1- ‘methyl-S-phenyl-2H-I,4-benzodiazepine-2-one (diazepam) in various forms. Arzneimittel-Forschung 16: 1109-1110 (1966). Schwartz, M.A.; Koechlin, B.A; Postma, E.; Palmer, 8. and Krol, G.: Metabolism of diazepam in rat, dog and man. Journal of Pharmacology and Experimental Therapeutics 149: 423-435 (1969). Sellman, R.; Kanto, J. and Pekkarinen, J.: Biliary excretion of diazepam and its metabolites in man. Acta Pharmacologica ct Toxioologica 37: 242-249 (1975p) ‘Sellman, R.: Pekkarinen, A.; Kangas, L. and Raijola, E,: Reduced concentrations of plasma diazepam in chronic alcoholic pa- tients following an oral administration of diazepam. Acta Pharmacologica et Toxicologica 36; 25-32 (1975). Sereni, F.; Mandelli, M.; Principi, N.; Tognoni, G.; Pardi, G. and. Morselli, P.L.: Induction of drug metabolizing enzyme ac- tivities in the human fetus and in the newborn infant; in Frey, Knox and Greengard (Eds) Enzyme, pp.318-329 (Karger Basel, 1973), Shader, R.l. and Greenblatt, D.J: Clinical implications of benzodiazepine pharmacokinetics. American Journal of Psy- chiatry 134: 652.656 (1977) Shannon, R.W.; Fraser, G.P.; Aitken, RG. and Harper J.R. Diazepam in preeclamptic toxaemia with special reference to its effect on the newborn infant. British Journal of Clinical Practice 26: 271-275 (1972). ‘Tansella, M.; Siciliani, O.; Burti, L: Schiavon, M.; Zimmermann ‘Tanzella, CH.; Gemma, M.; Tognoni, G. and Morsell, P.L.: N- desmethyldiazepam and amylobarbitone sodium as hypnotics ‘in anxious patients. Plasma levels, clinical efficacy and residual effects. Psychopharmacologica 41: 81-85 (1975), ‘Tansella, M.; Zimmermann, C.H.; Tansella, L; Ferrario, L.; Preziati, L; Tognori, G. and Lader, M.: Plasma concentra- tions of diazepam, nordiazepam and amylobarbitone afterClinical Pharmacokinetics of Diazepam shortterm treatment in anxious patients. Pharmakop- sychiatri. In press (1978). ‘Thiessen, IJ; Sellers, EM.; Denbeigh, P. and Dolman, L. Plasma protein binding of diazepam and tolbutamide in chronic alcoholics. Journal of Clinical Pharmacology 16; 345.351 (1976). ‘Tognoni, G.; Gomeni, R.; De Maio, D.; Alberti, G.G.; Franciosi, . and Scieghi, G.: Pharmacokinetics of N-demethyldiazepam in patients suffering from insomnia and treated with nortrip- tyline. British Journal of Clinical Pharmacology 2: 227-232 (1979). ‘Tsutsumi E.; Inaba, T.; Mahon, W.A. and Kalow, W.: The dis- placing effect of a fatty acid on the binding of diazepam to human serum albumin. Biochemical Pharmacology 24 1361-1362 (1975), Tyrer, P.: The benzodiazepine bonanza. Lancet 2: 709-710 (1974. 9 ‘Van Der Kleijn, E.: Van Rossum, J.M.; Muskens, E.T.J.M. and Rijnjes, N.V.M.: Pharmacokinetics of diazepam in dogs, mice ‘and humans. Acta Pharmacologica et Toxivologica 29 (Suppl. 3): 109-127 (1971). Zingales, 1.A.: Diazepam metabolism during chronic medication. ‘Unbound fraction in plasma, erythrocytes and urine. Journal of Chromatography 75: 55-78 (1973) Authors’ address: Drs Marinella Mandell, Gianni Tognon! and Silvio Garatini, Istituto di Ricerche Farmacologiche “Mario Negri’, 20157 Milan, Via Eritrea, 62 (Italy).