DIAZEPAM AND N-DESMETHYLDIAZEPAM

Transcription

1 Br. J. clin. Pharmac. (1980), 9, DIAZPAM AND N-DSMTHYLDIAZPAM CONCNTRATIONS IN SALIVA, PLASMA AND CSF C. HALLSTROM & M.H. LADR Institute of Psychiatry, Denmark Hill, London S5 8AF S.H. CURRY Medical College, The London Hospital, Turner Street, London l 2AD 1 Salivary and plasma diazepam and nordiazepam concentrations were measured in 51 paired samples from four experimental situations. In seven of the patients CSF samples were estimated. 2 A correlation of 0.89 (P<0.001) was observed between ry and plasma diazepam and 0.81 (P<0.001) between ry and plasma nordiazepam. 3 Mean ry diazepam was 1.6% (+0.3%) of the plasma diazepam. It was found to vary markedly in an acute dosage study. Mean ry nordiazepam was 2.9% (± 1%) of the plasma measure and was dependent on ry flow rate. 4 CSF diazepam was in equilibrium with diazepam and ry diazepam. 5 Mean protein binding of diazepam in vitro was 99.3% with no variations as a function of concentration. 6 The results suggest ry diazepam and nordiazepam measures to be of value in epidemiological studies. However, they do not predict accurately the plasma total or unbound drug concentration from a ry sample in an individual. Introduction Many drugs can with advantage be measured in. Concentrations in sometimes correlate well with concentrations in plasma. xamples are phenytoin, phenobarbitone, primidone, lithium and antipyrine (Schmid & Kupferberg, 1975; Groth, Prellwitz & Jahnchen, 1974; Mucklow, Bending, Kahn & Dollery 1978; Welch, De Angelis, Wingfield & Farmer, 1975). Saliva is easy to obtain without the trauma of venepuncture, and its collection requires less technical expertise. One of the most widely prescribed drugs, diazepam, has been measured in (Giles, Zilm, Frecker, MacLeod & Sellers, 1977; DiGregorio, Piraino & Ruch, 1978), but only after single oral doses. In contrast with other examples, the concentration/time curves for diazepam in and plasma were not superimposable; ry and plasma diazepam concentrations had an inconstant ratio (Giles et al., 1977). It was presumed that time was needed for the drug to diffuse into the and reach equilibrium. Twenty-four hours after the drug was taken, the ry diazepam concentration was 2% of the plasma concentration, suggesting that ry measurements might reflect diazepam protein binding in vivo. However, DiGregorio and his /80/ $01.11 associates (1978) reported a constant ratio between plasma and diazepam concentration over an 8 h period, with a plasma concentration range of 19.6 to 74.8 ng/ml. The /plasma ratio of 2.9% was of the same order as the 2% to 3.5% range of protein binding, but protein binding was found to increase over the 8 hours. We have conducted a series of experiments to elucidate further the relationship between ry and plasma diazepam, both total and unbound, as well as the relationship of N-desmethyldiazepam (nordiazepam), the major metabolite of diazepam in and plasma. We have also studied CSF drug concentrations to examine the possibility that ry diazepam measures could provide an indirect measure of the CSF diazepam concentration. Methods Acute dosage study On the morning after an overnight fast, three male and three female healthy volunteers (mean age 31 years, s.d. 8) swallowed a 10 mg tablet of diazepam 03D Macmillan Journals Ltd 1980

2 334 C. HALLSTROM, M.H. LADR & S.H. CURRY (Valium-Roche), washed down with approximately 50 ml water. Normal meals were allowed 3 h after medication. Blood and samples were obtained before and at 2, 4, 8, 24, 48 and 96 h after taking the drug. Saliva was collected by the subject rinsing out his mouth with water and then chewing on a small rubber band. The produced in the first 5 min was swallowed. A timed collection was then begun, with the subject spitting all the produced into a pre-weighed wide-mouth plastic screw-top container. Collection was continued until approximately 5 ml had been collected or for 5 min, whichever was the longer. The volume and thus flow rate was determined by weighing the container with the assumption that the specific gravity of the approximated to 1 (normal range of to 1.009, Mason & Chisholm, 1975). The ph was determined with a ph meter. Blood (5 ml) was drawn by venepuncture, with a syringe, and put into a lithium heparin plastic tube (15-25 units heparin/ml of blood) (Searle), immediately before or after the ry sample was collected. Both specimens were centrifuged and the plasma and clear analysed immediately or deep frozen at -200C for subsequent analysis. Two unsupervised ry samples were collected by the subject at home approximately 12 and 36 h after medication, and were brought to the laboratory the next morning for centrifugation and storage. Chronic diazepam users ight patients (five men and three women, mean age 40 years s.d. 12 years) who had been taking diazepam for between 2 and 26 weeks at a mean dose of 17 mg per day, s.d. 9 mg, gave ry and blood samples in the manner already described, at least 4 h after the last dose of drugs. Nordiazepam study After fasting for 6 h, five paid female volunteers (mean age 25 years s.d. 3 years) took 15 mg clorazepate (a precursor of nordiazepam) formulated in a gelatin capsule (Tranxene-Boehringer Ingelheim). Salivary and blood samples were obtained 1 and 24 h later. CSF study Seven inpatients (five men and two women-mean age 48 years s.d. 13 years) scheduled for diagnostic lumbar puncture for investigation of low back pain or early presenile dementia were given 10 mg diazepam as premedication the night before. Two were given a further dose 4 h before the investigation. One patient had been taking 6 mg diazepam per day for several years. CSF (4 ml) was obtained shortly after the and blood collections. Informed consent was obtained from all patients. In all studies, age, sex, smoking status, nature and duration of concurrent medication was noted. In vitro studies (a) Protein binding was studied in six specimens of drug-free plasma which were pooled and then subdivided. Serial dilutions of diazepam were added ranging from a concentration of 1000 to 0.25 ng/ml. Protein binding of diazepam was determined using radio-labelled diazepam by a technique of isotope dilution and equilibrium dialysis (at C) as described for chlorpromazine by Curry (1970). Protein binding of nordiazepam was determined using gas chromatographic analyses, after equilibrium dialysis. In all experiments, measurements were made in both dialysate and residual plasma, in order that allowance might be made for any losses by absorption or adsorption onto or into tubes or membranes. The dialysate was checked for protein content by biochemical analysis and when the test gave positive results the sample was rejected. The specific activity of the [3H]-diazepam was 47 Ci mmol-1. The material was purified by thin layer chromatography immediately before use (silica gel; chloroform/acetone-9/1; location with a Birchover Spark Chamber). Approximately 50 pg of the purified material was added to each dialysis tube. (b) ight specimens of plasma containing diazepam, from the set in the previous paragraph, were frozen and thawed twice. The degree of protein binding was determined before and after each thawing. (c) The ph was determined serially in three samples of left at room temperature for 24 h. Analysis of samples Specimens were analysed in batches. and nordiazepam were extracted into ether, which was then evaporated to dryness. After redissolving the drug, the quantities were determined by gas chromatography with electron capture detection (Dasberg, Klein & Guelen, 1974; Hillestad, Hanson & Melson, 1974; Kaplan, Jack & Alexander, 1973). This method has a level of sensitivity of 0.25 ng/ml. Statistics Statistical analysis comprised parametric correlation coefficients. The /plasma ratios were derived from linear regressions calculated by the method of least squares.

3 DIAZPAM AND NORDIAZPAM IN SALIVA, PLASMA AND CSF 335 a N.a_ U, Cu 300 r 250 F 200 F 150 F 100 F 50 F 30 c 25 aa)n X 20 V Xen 15 n a C c Time (h) 96 0 X U) Figure 1 Mean drug concentrations of six subjects over time after acute administration of diazepam: (0) total plasma, (A) and (e) concentrations. Results The mean plasma, plasma unbound and diazepam concentrations over time of samples in the first study are depicted in Figure 1. The time courses of the drug in the plasma and plasma water are similar. The ry drug concentration is of the same order of magnitude as the unbound fraction, but differs from the concentration. The unbound fraction fell from above 1.6% of the total plasma concentration to less than 1% after 8 h. The ry diazepam curve is not superimposable on the other two. At all times the ry concentration was less than the unbound concentration, so that the ry/plasma ratio was always less than the percentage unbound. The unsupervised collection of produced samples whose diazepam concentrations fitted well on the individual subjects' ry curves in eleven out of twelve cases. Half-lives for diazepam were 38.4 h in plasma, 35.7 h in plasma water and 12.7 h in, measured using data points from 2 to 96 h. concentrations were higher in those subjects taking the drug chronically (study 2), with a mean of 395 ng/ml (s.d. 161) in the chronic group as compared to a mean maximum of 152 ng/ml (s.d. 50) *.- _ - _ co a) N.u C,) o.-i r- %&-. RL45 M Plasma diazepam (ng/ml) -- Figure 2 Scattergram, regression line and 0.05 confidence limits for ry diazepam concentration and total plasma diazepam concentration for all subjects. * chronic diazepam users. * acute diazepam studies.

4 336 C. HALLSTROM, M.H. LADR & S.H. CURRY g C N > 15 - u) 5 0 m Plasma nordiazepam (ng/ml) Figure 3 Scattergram, regression line and 0.05 confidence limits for nordiazepam concentration and total plasma nordiazepam concentration for all subjects. * chronic diazepam users. 0 acute clorazepate study. 2 h after the 10 mg dose of diazepam in the single dose group. The percent unbound was consistently between 1 and 2% in these people. Figure 2 shows the relationship between ry and plasma diazepam concentrations for all pairs of samples for which data were available. The correlation coefficient was 0.89 (n = 34; P<0.001), and the ry drug concentration was 1.6% (s.d. 0.3%) of the plasma concentration overall.' After clorazepate (study 3), nordiazepam concentrations averaged 208 ng/ml (s.d. 60) 1 h and 197 ng/ml (s.d. 39) 24 h after ingestion. However, in the chronic diazepam users, nordiazepam concentrations were much higher (692 s.d. 440). The relationship between ry and plasma nordiazepam is shown in the scattergram (Figure 3). The correlation between the two measures was 0.81 (n = 19; P<0.001) and the ry measure corresponded to 2.9% (s.d. 1%) as calculated from the slope of the curve. After acute dosage of diazepam, the /plasma ratio for diazepam itself was less than the percentage of unbound diazepam in the plasma. After diazepam or clorazepate, the /plasma ratio for nordiazepam was considerably higher than its percentage unbound (Table 1). The /plasma ratio for nordiazepam was also higher than the diazepam -plasma ratio in patients after chronic Table I Ratios of concentrations in and in to concentrations of diazepam and nordiazepam (percentage with range). Drug administered (single dose) Clorazepate (single dose) (multiple dose) Nordiazepam Nordiazepam Measurement Ratios 1.17 ( ) 0.9 ( ) 1.40 ( ) 5.78 ( ) 1.48 ( ) 1.47 ( ) 2.2 ( )

5 DIAZPAM AND NORDIAZPAM IN SALIVA, PLASMA AND CSF 337 0) c co a) N co.a_ 11, U- U) lor ! 2! / Saliva diazepam (ng/ml) Figure 4 Scattergram, regression line and 0.05 confidence limits for CSF diazepam concentration and ry diazepam concentration. administration. Because no tritiated nordiazepam was available, protein binding of this drug was determined only with the acute dosage clorazepate specimens, and by gas chromatography. The correlation between the CSF diazepam concentration and the ry measure was 0.72 (n = 7; P <0.05) (Figure 4). The ratio of the CSF to the concentration was 1.28: 1 (s.d. 0.04). A higher correlation of 0.99 (n = 7; P<0.001), with a ratio of 1.1: 1 (s.d ), was found between the CSF diazepam concentration and the unbound fraction of the plasma concentration. Table 2 shows the ratio and correlations between the compartments of diazepam. Correlation coefficients were calculated between the ry/plasma drug ratio and age, sex, smoking status, duration of medication (where appropriate), and whether other medication was taken, and time elapsed since taking the medication (where appropriate), and whether other medication was taken, and time elapsed since taking medication, as well as ry and plasma concentrations of the drugs and ry ph. No correlation reached significance at the 0.05 level. Salivary flow rate correlated 0.82 (n = 19; P<0.05) with the /plasma ratio for nordiazepam, but (n = 22; NS) with the diazepam ry/plasma ratios. Protein binding in vitro was found not to vary significantly in the range of diazepam plasma concentrations from 1000 to 0.25 ng/ml. Protein binding did vary with freezing. The average percent unbound diazepam for eight samples was 0.67 (range ) before freezing, (corresponding to data for the concentration experiment), 0.99 (range ) after one freezing and thawing, and 1.14 (range ) after two freezings and thawings. Some samples were studied for protein binding on the day of collection, i.e., they were never frozen. Some were frozen and thawed once, to minimize any error. It should be noted that the change in binding in vivo, with time (less than 1% to more than 2% and back to less than 1% in the chronic diazepam users) was outside the range of changes associated with freezing and thawing. This was confirmed with samples which had never been frozen. The ph of standing at room temperature did not alter significantly over 24 h. Discussion The experiments were designed to assess the value of ry measures of diazepam and its major Table 2 Ratios and correlation coefficients between benzodiazepine concentrations in, CSF and plasma (a) (b) x y Concentration in X as % of Y (s.d.) Saliva Unbound plasma Saliva Nordiazepam Saliva Unbound plasma CSF CSF Unbound plasma CSF 1.6% (0.3%) 68% (20%) 1.6% (0.8%) 115% (15%) 1.3% (0.2) 129% (144%) Saliva 2.9% (1%) 0.81 n

6 338 C. HALLSTROM, M.H. LADR & S.H. CURRY metabolite nordiazepam as indicators of plasma diazepam and nordiazepam concentrations, and in particular as indirect measures of the unbound drug fraction. The acute and chronic diazepam studies enabled us to span a range of diazepam plasma concentrations, and the clorazepate study allowed us to study a low plasma concentration group of nordiazepam subjects, the higher values coming from the chronic diazepam study. Clorazepate is converted to nordiazepam in the acid medium of the stomach or on absorption (Shader & Greenblatt, 1977). Since the pka for diazepam is 3.3 (Clifford & Smyth, 1974) and for nordiazepam 3.5, both far removed from the ph of plasma and, it was not surprising that variations in ph of did not influence the ry/plasma drug ratio for the range in our subjects (ph ). There were differences in the ages, sex distribution and body weight between the study groups. The differences were not large enough to cause significant biological change, especially since the purpose of the studies was to assess the value of ry measures of diazepam and nordiazepam as an indicator of plasma concentrations of the drugs, in, unbound plasma and CSF. Any differences that the variables could give rise to, would serve to increase the range over which the phenomena were studied. In the analysis of the individual studies, no variation was noted in the /plasma ratio of the drugs or in protein binding attributable to the nature of the group of subjects studied. Table I demonstrates the differences with single and multiple doses in the ratio of the drugs in the ry and unbound fraction. There is a significant and positive correlation between ry flow rate and excretion of nordiazepam but not for diazepam. These factors taken together would suggest differences in the handling of these two compounds. Our observed unbound drug fraction for diazepam was in the range of 0.61%-0.75% (mean 0.67%). This was lower than the 2%-3.5% quoted by DiGregorio et al. (1978), or the 2% figure of Kanto et al. (1975) who used ultracentrifugation. Methods for determining protein binding are contentious, and our figure of a higher degree of protein binding than has been found by other authors may be important. It should be noted, however, that we used equilibrium dialysis with [3H]-diazepam where possible. The labelled diazepam was first purified. We checked for protein in the dialysate and for decomposition during the experiment. We carried out dialysis at room temperature, which may reduce the percent free (although not for reasons explicable in thermodynamic terms) but prevents the bacterial growth, drug decomposition and protein denaturation inevitable at 37 C. Failure to allow for any of these experimental points could cause interlaboratory differences, probably as increases in percent unbound, but should affect within-laboratory comparisons consistently. It has been suggested that heparin can affect benzodiazepine binding (Desmond, Roberts, Wilkinson, Wood, Dunn & Scheikes, 1979), although this is denied by some workers (Sellers, personal communication, 1979). In our experiments, heparin was not used during venepuncture, but all samples were placed in heparin tubes containing the same amount of lithium heparin, so that within-laboratory changes related to this should not have occurred. Differences between laboratories in this may relate to whether or not heparin from multi-dose vials with rubber stoppers is used. Such rubber stoppers have been shown on occasions to release into aqueous solutions materials which affect protein binding of basic drugs (Borga, Piafsky & Nilsen, 1977). Salivary diazepam was calculated as 1.6% (s.d. 0.3%) of the plasma concentration, with nordiazepam at 2.9% (s.d. 1%). Table 2 suggests that allowing for experimental error, drugs in the, CSF, and unbound fraction were more or less the same. The findings of other authors have not differed markedly from our finding of a CSF diazepam concentration of 1.6% of the. The average ratios of diazepam in to, although different in our three studies, ranging from 0.7% to 2.5% (average 1.6%), did not seem to be dependent on whether the drug was given acutely or chronically and repeatedly, but was seen to vary in the time course study. It may be seen that ry measures of diazepam correlate highly overall with both bound and unbound measures despite the time-related changes in the acute studies. This is also the case for nordiazepam in the and plasma. It was also of interest to note that the unsupervised samples gave ry diazepam concentrations close to the predicted values. It is not possible however, in the individual to predict accurately the plasma total or unbound drug concentration from a ry sample. It was shown that the concentration of diazepam and to an even greater degree, the concentration, did reflect the CSF diazepam concentration. The correlation of 0.81 between nordiazepam and plasma diazepam would however, make a knowledge of the ry concentrations of these drugs of sufficient validity to be of value in epidemiological studies (Fraser, Bulpitt, Kahn, Mould, Mucklow & Dollery, 1976). Our thanks go to Dr Hoare and the other doctors of the Maudsley Hospital who allowed us to study the patients in their charge.. Cooke and J. Dalton helped in the statistical analysis.

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