Journal of Pharmacokinetics and Biopharmaceutics, Vol. 3, No. 6, 1975
Plasma and Salivary Concentrations of Isoniazid in Man: Preliminary Findings in Two Slow Acetylator Subjects Harold G. Boxenbaum, 1 lhor Bekersky, ~ Vincent Mattaliano, 1 and Stanley A. Kaplan ~ Received M a y 21, 1 9 7 5 - - F i n a l July 17, 1975
lsoniazid was administered orally to two slow acetylator subjects, and plasma and saliva samples were assayed for intact drug. Saliva~plasma concentration ratios tended to be scattered about the value of 1. The results indicate that salivary level measurements may provide a useful approach for acetylation phenotyping. However, because of instability of isoniazid in frozen specimens, analyses of drug levels had to be initiated within 1 hr from the time of collection. Protein binding studies indicated that I N H is not bound to plasma proteins to any appreciable extent. K E Y W O R D S : i s o n i a z i d ; salivary d r u g levels; a c e t y l a t i o n p h e n o t y p i n g ; p r o t e i n b i n d i n g .
INTRODUCTION
Investigations dealing with the relationships between drug concentrations in plasma and saliva have recently received considerable attention, and several of these studies have been cited by Matin et al. (1). One potential use of salivary drug level measurements heretofore not investigated is as a method of phenotyping individuals with respect to pharmacogenetic differences in disposition. In this regard, the antituberculosis drug isoniazid (INH) appeared to be a suitable candidate for investigation. It is well established that individuals may be classified as either rapid or slow acetylators of isoniazid, and that this capacity to acetylate isoniazid is a permanent hereditary characteristic (2,3). Plasma half-lives in rapid phenotypic subjects range from 45 to 80 min, and in slow phenotypic subjects from 140 to 200 t D e p a r t m e n t of B i o c h e m i s t r y a n d D r u g M e t a b o l i s m , H o f f m a n n - L a Roche, Inc., Nuttey, N e w Jersey 07110. 443 9 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
444
Boxenbaum, Bekersky, Mattaliano, and Kaplan
rain. If one takes the average half-lives for the disappearance of plasma isoniazid in slow and rapid acetylators as 3 and 1.25 hr, respectively (4), areas under the plasma concentration-time curves will be 2.4 times greater (5) in slow acetylators than in rapid acetylators receiving the same dose. The influence of acetylator status on the response of tuberculosis patients to treatment with isoniazid-containing regimens is clinically important when the isoniazid dosage is given once weekly. The nature of this relationship, as well as that between plasma levels and the incidence of peripheral neuritis is grossly malnourished patients in the absence of concomitant pyridoxine administration, has been previously discussed (6,7). Another important consequence of acetylation polymorphism is the increased likelihood of isoniazid-induced hepatitis in rapid acetylators (8). These individuals metabolize isoniazid to relatively greater quantities of acetylisoniazid than do their slow acetylator counterparts. The acetylisoniazid is subsequently hydrolyzed to acetylhydrazine, which in turn is converted to an active metabolite capable of producing hepatitis. In view of the differences in response and toxicity elicited by isoniazid administration, it would be useful to have a simple, rapid, and accurate method for determination of drug half-life as a guide to dosage adjustment in order to individualize patient requirements: this approach has been recommended by Vesell (9). There presently exist a number of simple urine tests for phenotyping rapid and slow acetylators, and these have been reviewed by Jessamine et al. (10). In particular, determination of the acetylator phenotype from the ratio of urinary excreted acetylisoniazid to acidlabile isoniazid has proved highly efficient (11). Alternatively, one could make use of the sulfadimidine (sulfamethazine) method (12,13). This latter method is based on the polymorphic acetylation of sulfadimidine and requires measurement of intact drug and acetylsulfadimidine in urine. For routine clinical purposes, these simple urine tests are satisfactory. However, if one had available a simple, rapid, and accurate method for the determination of INH elimination half-life, this would be a more desirable approach, since it would provide a more direct index of INH disposition. Half-life determinations, however, have heretofore required blood sampling. It was the purpose of this investigation to examine the feasibility of employing salivary INH levels for determination of half-life as an alternative to plasma levels. Alternatively, one could possibly phenotype individuals using INH salivary concentrations 6 hr following an oral dose of 40 mg INH/kg ~ in a manner analogous to that used by Evans et al. (14) for plasma concentrations. It will be reported herein that, in the two slow acetylator subjects studied, plasma and salivary INH levels were similar. Unfortunately, however, INH is not stable in saliva, and attempts to stabilize or recover INH from frozen saliva were unsuccessful. Therefore, the utility of INH salivary
Plasma and Salivary Concentrations of Isoniazid in Man
445
measurements as a means of acetylator phenotyping outside the laboratory environment will depend on overcoming the stability problem exhibited by this drug. MATERIALS AND METHODS H u m a n Experiments
Two healthy male human subjects, drug free for at least 1 month, participated in these investigations. Subject A was a 31-year-old male, height 165 cm and weight 73.6 kg; subject B was a 34-year-old male, height 175 cm and weight 79.5 kg. After an overnight fast, subject A ingested 700 mg INH (7 tablets, 10O mg each, Eli Lilly & Co., Control No. 3DZ51A) with 240 ml tapwater. The subject rinsed his mouth several times with tapwater to remove any drug which may have been deposited in the oral cavity from the tablets. Water was withheld for 2.5 hr and food for 4 hr, after which both were permitted ad tibitum. Subject B followed the same protocol, except that 700 mg INH was dissolved in 240 ml tapwater and immediately ingested. Heparinized venous blood samples were obtained at the times indicated in Tables I and II, and plasma was immediately separated. Saliva samples were collected over 6-min intervals, the midpoint of which was used for blood collection. Salivary flow was stimulated by having each subject suck and/or chew on a teflon plug. Isoniazid was analyzed in plasma and saliva using the colorimetric method of Dymond and Russell (15) as modified by Ellard et al. (16). Calibration curves utilizing either plasma or saliva routinely had correlation coefficients exceeding 0.99, and the average difference between replicate determinations was 4.5 %. Plasma and saliva were analyzed for INH within 1 hr from time of collection, since no more than 5 ~ instability of INH was evident in either plasma or saliva during this period. In order to determine the stability characteristics of isoniazid in biological specimens, samples were also stored in the frozen state for later use. Samples of saliva from subject B were treated with zinc sulfate and barium hydroxide, frozen, and subsequently analyzed for isoniazid by the fluorometric method of Scott and Wright (17). In an attempt to recover plasma and salivary isoniazid possibly lost via hydrazone formation during frozen storage, a mild acid hydrolysis procedure was adopted (18). This procedure has been shown to hydrolyze the pyruvic and ~-ketoglutarie acid hydrazones of INH quantitatively to intact INH. One milliliter of plasma or saliva was added to 0.2 ml of 0.2 N HCI ; the tube was capped and heated in a water bath at 52~ for 60 min. Subsequently, 0.2 ml of 0.2 N NaOH was added, and the solutions were immediately assayed for INH using the colorimetric procedure cited herein.
Plasma concentration (#g/ml)
10.8 9.67 8.73 6.91 5.21 4.80 4.00
Time (min)
128 180 240 308 365 432 484
11.9 10.1 7.92 6.41 5.36 4.54 3.82
Saliva concentration (#g/ml) 6.6 5.5 5.2 6.6 4.7 4.7 5.3
Saliva volume (ml) 1.10 1.04 0.907 0.928 1.03 0.946 0.955
Saliva/plasma concentration ratio
Assayed within 1 hr from time of collection
5.88 5.33 4.81 3.89 3.28 2.57 2.37
Plasma concentration (pg/ml) 9.80 6.80 5.30 --3.10 2.75 1.50
Saliva concentration (pg/ml)
Assayed after 17 days of frozen storage
9.10 7.10 6.00 4.20 3. t5 2.30 1.75
Plasma concentration (#g/ml)
~
4.10 3.60 1.45
gl.
o
~ =
o
10.2 8.35 7.25
Saliva concentration (/~g/ml)
Assayed after 107 days of frozen storage and subsequent to mild acid hydrolysis
Table I. Plasma and Salivary Isoniazid Levels Following Administration of a Single Oral 700-mg Dose to Subject A
Plasma and Salivary Concentrations of Isoniazid in Man
447
Table II. Plasma and Salivary Isoniazid Levels Following Administration of a Singte Oral
700-rag Dose to Subject B Assayed within 1 hr from time of collection Plasma Saliva Saliva Saliva/plasma Time concentration concentration volume concentration (min) (gg/ml) (/*g/ml) (ml) ratio 17 32 47 62 92 122 152 212 272 332 392
14.6 14.2 12.6 11.5 8.92 9.58 8.21 5.65 4.78 3.69 3.08
14.8 16.4 t4.3 13.2 10.4 9.50 8.51 5.65 4.30 3.32 3.00
7.4 8.6 8.0 4.6 8.1 5.2 8.0 8.0 7.6 6.2 8.1
1.01 1.15 1.13 1.15 1.17 0.992 1.04 1.00 0.900 0.900 0.974
ZnSO4-Ba(OH)2 filtrate assayedafter 43 days frozenstorage: saliva concentration (~g/ml) 0.35 0.54 0.41 0.71 0.38 0.75 0.22 0.45 0.44 0.33 O.17
Both the colorimetric and fluorometric methods are accurate and specific for isoniazid. For all determinations, standards were prepared in the biological fluid being assayed. In the mild acid hydrolysis experiments, standards were carried through the entire procedure. P r o t e i n Binding S t u d i e s
Protein binding studies were conducted by ultrafiltration using a model 12 Amicon standard stirred cell employing a P M 10 Diaflo membrane (Amicon Corporation, 21 Hartwell Ave., Lexington, Mass. 02173). The apparatus, contained within a constant-temperature oven, consisted of a stirred plastic chamber containing drug solution with a gas inlet tube atop the solution to maintain a nitrogen pressure of 45 psi. This pressure forced solution through the membrane; it was then collected and analyzed for INH. The membrane used has a nominal molecular weight cutoff value of 10,000. Prior to use, membranes were soaked in distilled water for 1 hr, with three changes of water. Freshly drawn heparinized blood was collected from subjects A and B, plasma was immediately separated and brought to 37~ and the latter was spiked with isoniazid at a concentration of 10 pg/ml. Immediately thereafter, 10 ml of the spiked plasma was added to the stirred cell contained within a 37~ oven, and the first 0.5 ml of ultrafiltrate was collected. Once spiked plasma was placed in the cell, this ultrafiltrate could be collected within 10 min. Binding of I N H to the membrane and/or plastic components of the cell was determined by substituting water for plasma. Immediately upon
448
Boxenbaum, Bekersky, Mattaliano, and Kaplan
collection, ultrafiltrates were assayed for INH using the colorimetric procedure previously cited. Standard curves were prepared by spiking plasma ultrafiltrates with isoniazid. Proteins in plasma and ultrafiltrate were measured by the Mazel (19) modification of the colorimetric method of Lowry et al. (20). Subjects A and B had plasma protein concentrations of 7.3 and 7.8 %, respectively, which are within the 95 % range of 6.5-8.0 % (21). Ultrafiltrates had a protein concentration approximately 1.7 % that of plasma. This value is approximately three-fold greater than that reported by Shah et al. (22) using an analogous microultrafiltration technique. RESULTS AND DISCUSSION Plasma and Salivary Levels
Plasma and salivary isoniazid concentration-time data are presented in Tables I and II and graphically illustrated in Fig. 1. Accurate measurements of INH concentration were obtained by assaying for drug within 1 hr of sample collection: this point will subsequently be discussed in greater detail. The first blood and saliva samples were not collected from subject A until 128 rain following dosage. This permitted sufficient time for the absorption process to be completed and for the so-called distribution equilibrium to be reached (23,24). Consequently, plasma levels declined in a monoexponential fashion. Subject B, however, had plasma and saliva samples collected at much earlier times, the first sample being taken at 17 min. Apparently the absorption process was very rapid, as evidenced by the lack of data points on the upswing of the plasma concentration-time curve. Once the initial plasma levels were achieved, the more rapid decline in the plasma concentration-time curve resulted as a consequence of the drug's so-called distribution phase. In both subjects, saliva/plasma INH concentration ratios were close to unity. The average ratio was 1.02, and the fiducial limits were 0.900-1.17. Figure 2 is a plot of saliva vs. plasma isoniazid concentrations. If the saliva/ plasma ratios were randomly scattered about the value of 1 throughout the course of the experiments, one would expect this plot to pass through the origin and have a slope of 1. The best line was determined by the method of least squares, assuming no error in x values and weighting y values by the factor 1/y 2. Application of the appropriate t statistic (25) indicated that the slope and intercept estimates were significantly different from 1 and 0, respectively (P < 0.05). In an attempt to determine the factor responsible, plots of the saliva/plasma ratio vs. saliva flow rate as well as plasma INH concentration were constructed: these are shown in Figs. 3 and 4, respec-
Plasma and Salivary Concentrations of Isoniazid in Man
g
z 0 (J
ZO &lOI
449
SUBJECT
A =e
SUBJECT
B =&
& & &
8
6
:E
o~
g
4
o N
3
Z 0
2
I
ZO & &
Z 0
&
&
l-
IZ ~J Z 0 0
IO 8
~
18min.
S
X
_1
4 3
z 0
2
I 50
I Ioo
I 15o
I ZOO
TIME
I z5o
I 30o
I 350
I -----[__l 400 45o
500
(minutes)
Fig. 1. Plasma and salivary isoniazid concentrations following oral administration of 700-rag doses to two healthy human subjects. Half-lives were determined by linear least-squares analysis of logarithmically transformed, unweighted concentration data.
tively. The m e t h o d of least squares was applied as discussed previously. The saliva/plasma ratio did not a p p e a r to correlate with saliva flow rate, as the slope was not significantly different from 0 (p > 0.05). However, there was a weak correlation (r = 0.656) between the saliva/plasma ratio and I N H plasma c o n c e n t r a t i o n : i.e., as plasma concentration decreased, there was a trend for the ratio of saliva/plasma I N H to also decrease. Some caution is required in the interpretation of these data, however, as it is quite possible
450
Boxenbaum, Bekersky, Mattaliano, and Kaplan 2C SUBJECT
A 9 9
SUBJECT
El 9 &
& E
15 m
&
&
~= z w z o u
10
z
5- /
/
9
I
I
5 PLASMA
Y = I. IIx -0,684
I0 ISONIAZlO
CONCENTRATION
I
15
I
20
(pg/ml)
Fig. 2. Relationship between plasma and salivary concentrations of isoniazid; correlation coefficient is 0.990.
that the ratios were primarily being influenced by time and not plasma concentrations. One possibility is that residue amounts of ingested isoniazid were still retained in the buccal cavity in the early moments. This might offer some explanation of the finding previously discussed and illustrated in Fig. 1. Estimates of half-lives of isoniazid determined from saliva were approximately 10 ~o shorter than those determined from plasma. However, application of the appropriate t test (25) to the data from both subjects indicated that, statistically, half-lives determined from plasma and saliva data were n o t significantly different (p > 0.05). One obvious shortcoming of this present report is the limited number of subjects investigated, and a complete lack of data from rapid acetylators. In this regard, a larger population sampling, including rapid acetylators, is required before any general statements regarding the utility of salivary measurements for INH phenotyping and dosage adjustment can be made. Unfortunately, INH was not found to be stable in frozen saliva when assayed
Plasma and Salivary Concentrations of Isoniazid in Man
451
&
t.18
O ~-
114
~-
LIO
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A
= ~
SUBJECT
B
= &
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r Lu 2
t.O6
Plasma and Salivary Concentrations of Isoniazid in Man: Preliminary Findings in Two Slow Acetylator Subjects Harold G. Boxenbaum, 1 lhor Bekersky, ~ Vincent Mattaliano, 1 and Stanley A. Kaplan ~ Received M a y 21, 1 9 7 5 - - F i n a l July 17, 1975
lsoniazid was administered orally to two slow acetylator subjects, and plasma and saliva samples were assayed for intact drug. Saliva~plasma concentration ratios tended to be scattered about the value of 1. The results indicate that salivary level measurements may provide a useful approach for acetylation phenotyping. However, because of instability of isoniazid in frozen specimens, analyses of drug levels had to be initiated within 1 hr from the time of collection. Protein binding studies indicated that I N H is not bound to plasma proteins to any appreciable extent. K E Y W O R D S : i s o n i a z i d ; salivary d r u g levels; a c e t y l a t i o n p h e n o t y p i n g ; p r o t e i n b i n d i n g .
INTRODUCTION
Investigations dealing with the relationships between drug concentrations in plasma and saliva have recently received considerable attention, and several of these studies have been cited by Matin et al. (1). One potential use of salivary drug level measurements heretofore not investigated is as a method of phenotyping individuals with respect to pharmacogenetic differences in disposition. In this regard, the antituberculosis drug isoniazid (INH) appeared to be a suitable candidate for investigation. It is well established that individuals may be classified as either rapid or slow acetylators of isoniazid, and that this capacity to acetylate isoniazid is a permanent hereditary characteristic (2,3). Plasma half-lives in rapid phenotypic subjects range from 45 to 80 min, and in slow phenotypic subjects from 140 to 200 t D e p a r t m e n t of B i o c h e m i s t r y a n d D r u g M e t a b o l i s m , H o f f m a n n - L a Roche, Inc., Nuttey, N e w Jersey 07110. 443 9 1975 Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission of the publisher.
444
Boxenbaum, Bekersky, Mattaliano, and Kaplan
rain. If one takes the average half-lives for the disappearance of plasma isoniazid in slow and rapid acetylators as 3 and 1.25 hr, respectively (4), areas under the plasma concentration-time curves will be 2.4 times greater (5) in slow acetylators than in rapid acetylators receiving the same dose. The influence of acetylator status on the response of tuberculosis patients to treatment with isoniazid-containing regimens is clinically important when the isoniazid dosage is given once weekly. The nature of this relationship, as well as that between plasma levels and the incidence of peripheral neuritis is grossly malnourished patients in the absence of concomitant pyridoxine administration, has been previously discussed (6,7). Another important consequence of acetylation polymorphism is the increased likelihood of isoniazid-induced hepatitis in rapid acetylators (8). These individuals metabolize isoniazid to relatively greater quantities of acetylisoniazid than do their slow acetylator counterparts. The acetylisoniazid is subsequently hydrolyzed to acetylhydrazine, which in turn is converted to an active metabolite capable of producing hepatitis. In view of the differences in response and toxicity elicited by isoniazid administration, it would be useful to have a simple, rapid, and accurate method for determination of drug half-life as a guide to dosage adjustment in order to individualize patient requirements: this approach has been recommended by Vesell (9). There presently exist a number of simple urine tests for phenotyping rapid and slow acetylators, and these have been reviewed by Jessamine et al. (10). In particular, determination of the acetylator phenotype from the ratio of urinary excreted acetylisoniazid to acidlabile isoniazid has proved highly efficient (11). Alternatively, one could make use of the sulfadimidine (sulfamethazine) method (12,13). This latter method is based on the polymorphic acetylation of sulfadimidine and requires measurement of intact drug and acetylsulfadimidine in urine. For routine clinical purposes, these simple urine tests are satisfactory. However, if one had available a simple, rapid, and accurate method for the determination of INH elimination half-life, this would be a more desirable approach, since it would provide a more direct index of INH disposition. Half-life determinations, however, have heretofore required blood sampling. It was the purpose of this investigation to examine the feasibility of employing salivary INH levels for determination of half-life as an alternative to plasma levels. Alternatively, one could possibly phenotype individuals using INH salivary concentrations 6 hr following an oral dose of 40 mg INH/kg ~ in a manner analogous to that used by Evans et al. (14) for plasma concentrations. It will be reported herein that, in the two slow acetylator subjects studied, plasma and salivary INH levels were similar. Unfortunately, however, INH is not stable in saliva, and attempts to stabilize or recover INH from frozen saliva were unsuccessful. Therefore, the utility of INH salivary
Plasma and Salivary Concentrations of Isoniazid in Man
445
measurements as a means of acetylator phenotyping outside the laboratory environment will depend on overcoming the stability problem exhibited by this drug. MATERIALS AND METHODS H u m a n Experiments
Two healthy male human subjects, drug free for at least 1 month, participated in these investigations. Subject A was a 31-year-old male, height 165 cm and weight 73.6 kg; subject B was a 34-year-old male, height 175 cm and weight 79.5 kg. After an overnight fast, subject A ingested 700 mg INH (7 tablets, 10O mg each, Eli Lilly & Co., Control No. 3DZ51A) with 240 ml tapwater. The subject rinsed his mouth several times with tapwater to remove any drug which may have been deposited in the oral cavity from the tablets. Water was withheld for 2.5 hr and food for 4 hr, after which both were permitted ad tibitum. Subject B followed the same protocol, except that 700 mg INH was dissolved in 240 ml tapwater and immediately ingested. Heparinized venous blood samples were obtained at the times indicated in Tables I and II, and plasma was immediately separated. Saliva samples were collected over 6-min intervals, the midpoint of which was used for blood collection. Salivary flow was stimulated by having each subject suck and/or chew on a teflon plug. Isoniazid was analyzed in plasma and saliva using the colorimetric method of Dymond and Russell (15) as modified by Ellard et al. (16). Calibration curves utilizing either plasma or saliva routinely had correlation coefficients exceeding 0.99, and the average difference between replicate determinations was 4.5 %. Plasma and saliva were analyzed for INH within 1 hr from time of collection, since no more than 5 ~ instability of INH was evident in either plasma or saliva during this period. In order to determine the stability characteristics of isoniazid in biological specimens, samples were also stored in the frozen state for later use. Samples of saliva from subject B were treated with zinc sulfate and barium hydroxide, frozen, and subsequently analyzed for isoniazid by the fluorometric method of Scott and Wright (17). In an attempt to recover plasma and salivary isoniazid possibly lost via hydrazone formation during frozen storage, a mild acid hydrolysis procedure was adopted (18). This procedure has been shown to hydrolyze the pyruvic and ~-ketoglutarie acid hydrazones of INH quantitatively to intact INH. One milliliter of plasma or saliva was added to 0.2 ml of 0.2 N HCI ; the tube was capped and heated in a water bath at 52~ for 60 min. Subsequently, 0.2 ml of 0.2 N NaOH was added, and the solutions were immediately assayed for INH using the colorimetric procedure cited herein.
Plasma concentration (#g/ml)
10.8 9.67 8.73 6.91 5.21 4.80 4.00
Time (min)
128 180 240 308 365 432 484
11.9 10.1 7.92 6.41 5.36 4.54 3.82
Saliva concentration (#g/ml) 6.6 5.5 5.2 6.6 4.7 4.7 5.3
Saliva volume (ml) 1.10 1.04 0.907 0.928 1.03 0.946 0.955
Saliva/plasma concentration ratio
Assayed within 1 hr from time of collection
5.88 5.33 4.81 3.89 3.28 2.57 2.37
Plasma concentration (pg/ml) 9.80 6.80 5.30 --3.10 2.75 1.50
Saliva concentration (pg/ml)
Assayed after 17 days of frozen storage
9.10 7.10 6.00 4.20 3. t5 2.30 1.75
Plasma concentration (#g/ml)
~
4.10 3.60 1.45
gl.
o
~ =
o
10.2 8.35 7.25
Saliva concentration (/~g/ml)
Assayed after 107 days of frozen storage and subsequent to mild acid hydrolysis
Table I. Plasma and Salivary Isoniazid Levels Following Administration of a Single Oral 700-mg Dose to Subject A
Plasma and Salivary Concentrations of Isoniazid in Man
447
Table II. Plasma and Salivary Isoniazid Levels Following Administration of a Singte Oral
700-rag Dose to Subject B Assayed within 1 hr from time of collection Plasma Saliva Saliva Saliva/plasma Time concentration concentration volume concentration (min) (gg/ml) (/*g/ml) (ml) ratio 17 32 47 62 92 122 152 212 272 332 392
14.6 14.2 12.6 11.5 8.92 9.58 8.21 5.65 4.78 3.69 3.08
14.8 16.4 t4.3 13.2 10.4 9.50 8.51 5.65 4.30 3.32 3.00
7.4 8.6 8.0 4.6 8.1 5.2 8.0 8.0 7.6 6.2 8.1
1.01 1.15 1.13 1.15 1.17 0.992 1.04 1.00 0.900 0.900 0.974
ZnSO4-Ba(OH)2 filtrate assayedafter 43 days frozenstorage: saliva concentration (~g/ml) 0.35 0.54 0.41 0.71 0.38 0.75 0.22 0.45 0.44 0.33 O.17
Both the colorimetric and fluorometric methods are accurate and specific for isoniazid. For all determinations, standards were prepared in the biological fluid being assayed. In the mild acid hydrolysis experiments, standards were carried through the entire procedure. P r o t e i n Binding S t u d i e s
Protein binding studies were conducted by ultrafiltration using a model 12 Amicon standard stirred cell employing a P M 10 Diaflo membrane (Amicon Corporation, 21 Hartwell Ave., Lexington, Mass. 02173). The apparatus, contained within a constant-temperature oven, consisted of a stirred plastic chamber containing drug solution with a gas inlet tube atop the solution to maintain a nitrogen pressure of 45 psi. This pressure forced solution through the membrane; it was then collected and analyzed for INH. The membrane used has a nominal molecular weight cutoff value of 10,000. Prior to use, membranes were soaked in distilled water for 1 hr, with three changes of water. Freshly drawn heparinized blood was collected from subjects A and B, plasma was immediately separated and brought to 37~ and the latter was spiked with isoniazid at a concentration of 10 pg/ml. Immediately thereafter, 10 ml of the spiked plasma was added to the stirred cell contained within a 37~ oven, and the first 0.5 ml of ultrafiltrate was collected. Once spiked plasma was placed in the cell, this ultrafiltrate could be collected within 10 min. Binding of I N H to the membrane and/or plastic components of the cell was determined by substituting water for plasma. Immediately upon
448
Boxenbaum, Bekersky, Mattaliano, and Kaplan
collection, ultrafiltrates were assayed for INH using the colorimetric procedure previously cited. Standard curves were prepared by spiking plasma ultrafiltrates with isoniazid. Proteins in plasma and ultrafiltrate were measured by the Mazel (19) modification of the colorimetric method of Lowry et al. (20). Subjects A and B had plasma protein concentrations of 7.3 and 7.8 %, respectively, which are within the 95 % range of 6.5-8.0 % (21). Ultrafiltrates had a protein concentration approximately 1.7 % that of plasma. This value is approximately three-fold greater than that reported by Shah et al. (22) using an analogous microultrafiltration technique. RESULTS AND DISCUSSION Plasma and Salivary Levels
Plasma and salivary isoniazid concentration-time data are presented in Tables I and II and graphically illustrated in Fig. 1. Accurate measurements of INH concentration were obtained by assaying for drug within 1 hr of sample collection: this point will subsequently be discussed in greater detail. The first blood and saliva samples were not collected from subject A until 128 rain following dosage. This permitted sufficient time for the absorption process to be completed and for the so-called distribution equilibrium to be reached (23,24). Consequently, plasma levels declined in a monoexponential fashion. Subject B, however, had plasma and saliva samples collected at much earlier times, the first sample being taken at 17 min. Apparently the absorption process was very rapid, as evidenced by the lack of data points on the upswing of the plasma concentration-time curve. Once the initial plasma levels were achieved, the more rapid decline in the plasma concentration-time curve resulted as a consequence of the drug's so-called distribution phase. In both subjects, saliva/plasma INH concentration ratios were close to unity. The average ratio was 1.02, and the fiducial limits were 0.900-1.17. Figure 2 is a plot of saliva vs. plasma isoniazid concentrations. If the saliva/ plasma ratios were randomly scattered about the value of 1 throughout the course of the experiments, one would expect this plot to pass through the origin and have a slope of 1. The best line was determined by the method of least squares, assuming no error in x values and weighting y values by the factor 1/y 2. Application of the appropriate t statistic (25) indicated that the slope and intercept estimates were significantly different from 1 and 0, respectively (P < 0.05). In an attempt to determine the factor responsible, plots of the saliva/plasma ratio vs. saliva flow rate as well as plasma INH concentration were constructed: these are shown in Figs. 3 and 4, respec-
Plasma and Salivary Concentrations of Isoniazid in Man
g
z 0 (J
ZO &lOI
449
SUBJECT
A =e
SUBJECT
B =&
& & &
8
6
:E
o~
g
4
o N
3
Z 0
2
I
ZO & &
Z 0
&
&
l-
IZ ~J Z 0 0
IO 8
~
18min.
S
X
_1
4 3
z 0
2
I 50
I Ioo
I 15o
I ZOO
TIME
I z5o
I 30o
I 350
I -----[__l 400 45o
500
(minutes)
Fig. 1. Plasma and salivary isoniazid concentrations following oral administration of 700-rag doses to two healthy human subjects. Half-lives were determined by linear least-squares analysis of logarithmically transformed, unweighted concentration data.
tively. The m e t h o d of least squares was applied as discussed previously. The saliva/plasma ratio did not a p p e a r to correlate with saliva flow rate, as the slope was not significantly different from 0 (p > 0.05). However, there was a weak correlation (r = 0.656) between the saliva/plasma ratio and I N H plasma c o n c e n t r a t i o n : i.e., as plasma concentration decreased, there was a trend for the ratio of saliva/plasma I N H to also decrease. Some caution is required in the interpretation of these data, however, as it is quite possible
450
Boxenbaum, Bekersky, Mattaliano, and Kaplan 2C SUBJECT
A 9 9
SUBJECT
El 9 &
& E
15 m
&
&
~= z w z o u
10
z
5- /
/
9
I
I
5 PLASMA
Y = I. IIx -0,684
I0 ISONIAZlO
CONCENTRATION
I
15
I
20
(pg/ml)
Fig. 2. Relationship between plasma and salivary concentrations of isoniazid; correlation coefficient is 0.990.
that the ratios were primarily being influenced by time and not plasma concentrations. One possibility is that residue amounts of ingested isoniazid were still retained in the buccal cavity in the early moments. This might offer some explanation of the finding previously discussed and illustrated in Fig. 1. Estimates of half-lives of isoniazid determined from saliva were approximately 10 ~o shorter than those determined from plasma. However, application of the appropriate t test (25) to the data from both subjects indicated that, statistically, half-lives determined from plasma and saliva data were n o t significantly different (p > 0.05). One obvious shortcoming of this present report is the limited number of subjects investigated, and a complete lack of data from rapid acetylators. In this regard, a larger population sampling, including rapid acetylators, is required before any general statements regarding the utility of salivary measurements for INH phenotyping and dosage adjustment can be made. Unfortunately, INH was not found to be stable in frozen saliva when assayed
Plasma and Salivary Concentrations of Isoniazid in Man
451
&
t.18
O ~-
114
~-
LIO
SUBJECT
A
= ~
SUBJECT
B
= &
A
r Lu 2
t.O6
