Salivary excretion and pharmacokinetics of sulfapyridine after sulfasalazine The concentrations of sulJapyridine (SP) and N4-acetylsulJapyridine (AcSP) in the plasma and saliva of 5 healthy male adults (3 slow and 2 rapid acetylators) were determined as a function of time after a single 2.0-gm oral dose of sulJasalazine (salicylazosulJapyridine). SP absorption commenced 3.5 to 6 hr after sulJasalazine administration and occurred slowly (apparent absorption tlhs rangedfrom 1.6 to 5 hr) irrespective of acetylator phenotype. Appreciable differences existed between slow and rapid acetylators with respect to the biologic tlh and total body clearance of SP. SP concentrations in the saliva correlated well with those in the plasma. The saliva: plasma concentration ratio for SP was 0.559 ± 0.027 (mean of 5 subjects ± SE) and was independent of plasma concentration and saliva pH. The mean saliva: plasma concentration ratio for AcSP was lower (0.246 ± 0.056), consistent with the pH-partition hypothesis, and showed considerably more intrasubject and intersubject variation than the ratio for SP. These findings suggest that measurement of SP concentrations in the saliva may be a convenient, noninvasive method for monitoring indirectly the steady-state plasma (serum) concentrations of SP in patients with ulcerative colitis or Crohn's disease who are receiving sulJasalazine.
Theodore R. Bates, Ph.D., H. Peter Blumenthal, M.D., and Henry J. Pieniaszek, Jr., B.A. Amherst, N.Y. Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo
Sulfasalazine (SSZ; salicylazosulfapyridine), one of the drugs of choice in the treatment of ulcerative colitis ,8. 10. 15. 29 is poorly absorbed from the gastrointestinal tract of man 25 and is subject to extensive biotransformation by bacteSupported in part by Grant GM 20852 from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Md., and by a Merck Grant for Faculty Development to Dr. Bates from the Merck Foundation. Received for publication June 8, 1977. Accepted for publication Aug. 17, 1977. Reprint requests to: Dr. Theodore R. Bates, Department of Pharo maceutics, School of Pharmacy, State University of New York at Buffalo, H517 Cooke-Hochstetler Complex, Amherst, N. Y. 14260.
rial azo reductases in the colon and cecum to sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA).6. 22 After colonic absorption, SP is metabolized to N4-acetylsulfapyridine (AcSP), sulfapyridine-O-glucuronide (SP-O-G), and N 4_ acetyl sulfapyri dine-O-gl ucuronide (AcS P -0G).5. 7. 25, 28 Like sulfamethazine, isoniazid, and hydralazine, it exhibits acetylation polymorphism.3. 27 Steady-state serum total SP (SP and its metabolites) concentrations of 20 to 50 /-Lg/ml appear to coincide with clinical improvement in adult patients treated with SSZ, while concen-
917
918
Bates, Blumenthal, and Pieniaszek
trations exceeding 50 p.,g/ml are normally associated with adverse effects. 4. 5 The incidence of adverse effects, which can be also correlated with steady-state serum levels of SP,4 seems to be highest in adults phenotyped as slow acetylators of SP. 4, 26 Steady-state serum concentrations of SSZ or 5-ASA bear no apparent relationship to either the clinical efficacy or toxicity of SSZ. 4, 5 After oral administration of SSZ to healthy subjects or to patients with ulcerative colitis, the mean steady-state serum concentrations of SP in slow acetylators are approximately 2 to 5 times those of rapid acetylators. 5. 8, 25. 26 The pharmacokinetics of SP after SSZ administration has not been adequately studied in man. Schroder and Campbe1l 25 reported on the renal clearance of SP and each of its metabolites in healthy subjects, but their determination of the biologic half-life (t12) of SP was incorrectly based on the time course of total SP (SP and its metabolites) in the serum. Hence, although differences in the steady-state levels of SP do exist between the two phenotypes, the reason for these differences is not clear. Such differences may arise from differences in the fraction of the oral dose absorbed, in the apparent volume of distribution of SP, or in the elimination rate constant or biologic half-life of SP. The genetic polymorphism in the acetylation of SP result in appreciable interindividual variations in the steady-state serum concentrations of unmetabolized and total SP. 5, 8, 25, 26 Even within a given phenotype large variations between individuals are evident,5, 8, 25, 26 possibly related to the erratic and incomplete absorption of SP from the colon. Thus, to ensure the safe and effective use of SSZ, it may be appropriate, at least in some patients, to monitor serum or plasma concentrations of SP during the course of drug therapy and to determine acetylator phenotype. 8 Das and Rubin 8 suggest that determinations of SP and AcSP in the serum are sufficient for these purposes since SP-O-G and AcSP-O-G represent an insignificant part of serum total SP. Our preliminary results! indicated the existence of a direct relationship between the concentrations of SP in the plasma and saliva of a
Clinical Pharmacology and Therapeutics
healthy slow acetylator of SP after a single oral dose of SSZ. AcSP was also excreted in the saliva but to a lesser extent. The purposes of our study were to confirm these findings in healthy slow and rapid acetylators of SP and to determine whether saliva SP and AcSP concentration measurements offer a method for indirectly monitoring the plasma concentrations of these SSZ metabolites. The pharmacokinetics of SP in slow and rapid acetylators were also studied to determine directly the reason why the two phenotypes differ with respect to mean steadystate SP levels. Materials and methods
Subjects. Five healthy male subjects, ranging in age from 21 to 31 yr (mean, 25 yr) and in body weight from 67 to 82 kg (mean, 73 kg) participated in this study. The subjects had no previous history of sulfonamide sensitivity or gastrointestinal disease and the results of physical and laboratory examinations were normal. No drug or alcoholic beverage was ingested for 7 days prior to and for 4 days after SSZ administration. The subjects fasted for at least 8 hr before and 4 hr after administration of the drug. At 8 A.M., after the subjects had emptied their bladders and blank blood, mixed saliva, and urine specimens had been collected, a single 2.0-gm dose of SSZ as four 500-mg commercially available uncoated tablets (Azulfidine, Pharmacia) was administered orally with 240 ml of water. Sample collections and analysis. Heparinized blood (10 ml) and mixed saliva samples were obtained at I, 2, 4, 6, 8, 10, 12, 14, 16, 24, 30, 36,48, and 72 hr after SSZ administration. The mixed saliva samples were collected over a 4- min interval simultaneously with blood collections, and the pH was recorded. Salivation was stimulated by having the subjects chew on a small ball of Parafilm. Quantitative urine collections were made at predetermined intervals for 4 days after drug administration and the pH and volume of each collection were recorded. All plasma, mixed saliva, and urine specimens were stored at - 20° C pending assay.
Volume 22 Number 6
Plasma and saliva concentrations of SP and AcSP were determined by the specific colorimetric method of Hansson and Sandberg17 as modified by Bates and Pieniaszek2 to increase by 4-fold the sensitivity of the original method. Vrine specimens were assayed for SSZ, SP, AcSP, SP-O-G, and AcSP-O-G by specific colorimetric methods 17, 24 with the use of Glusulase (,8-glucuronidase and sulfatase) to hydrolyze the glucuronide conjugates of SP enzymatically. Pharmacokinetic analysis. Plasma and saliva concentrations of SP were plotted semilogarithmically as a function of time and the apparent elimination rate constant K for SP was estimated from the slope of the terminal linear segment of such plots by the method of least squares. The biologic t% of SP was obtained by dividing K into 0.693. The time course of SP concentrations in the plasma was adequately described pharmacokinetic ally by a one-compartment open model with apparent first-order absorption and an apparent lag time (tJ. 14, 31 The apparent absorption rate constant k and to for SP were estimated by the method of residuals or feathering 14 , 31 with the use of least-squares regression analysis. SP and its metabolites are primarily excreted in the urine of man,8, 25, 28 the mean percent of dose excreted (n = 4) being 90% (range, 88% to 95%) after 1.841-gm intravenous dose of SP as the sodium salt. 12 Thus, the total body clearance of SP (product of K and the apparent volume of distribution of SP, V) may be estimated from the ratio of the amount of SP and all of its metabolites ultimately excreted in the urine (assuming this to be equivalent to the amount of SP absorbed after SSZ administration) to the total area under the concentration vs time curve (AVC) for plasma SP as measured by the trapezoidal rule. 14, 31 The renal clearance of SP and AcSP were determined by dividing the plasma AVe value for each species into the cumulative amount of that species excreted in the urine. 14 , 31 The subjects were phenotyped as slow or rapid acetylators of SP based on the magnitude of the formation rate constant for acetylated SP
Salivary excretion of sulfapyridine
919
or on percent acetylation (urine) values calculated from the following relationship:3 % acetylation = AAcSP
+ AAcSP-O-G + AAcSP + AAcSP-O-G
------~~--~~--~-----x Asp
+
A SP- o - G
100
where A represents the cumulative amount ultimately excreted in the urine. The distinguishing boundary between slow and rapid acetylators was taken as 63% for urine. 3 Mean saliva to plasma concentration ratios for SP and AcSP were calculated for each species by averaging the concentration ratios obtained over the entire experimental period. A similar ratio was obtained for each species by dividing the saliva AVe by the plasma AVe. Results and discussion
Plasma and saliva concentration-time profiles for SP and AcSP after oral administration of a 2.0-gm dose of SSZ to a slow acetylator (Subject J. L.) and to a rapid acetylator (Subject M. e.) are depicted in Figs. 1 and 2. Measurable concentrations of SP appeared in the plasma 3.5 to 6 hr after SSZ administration (Table I) which is consistent with normal transit times for fluids to the colon and cecum where SSZ undergoes bacterial azo reduction. The observed times of peak plasma concentrations of SP and AcSP were similar (Table I) and peak levels declined monoexponentially with time (Figs. 1 and 2). The plasma SP concentration-time data for each subject were fitted to the integrated rate expression for a one-compartment open model with apparent first-order absorption and an apparent absorption lag time. 14, 31 The pharmacokinetic parameters derived from such analyses and pertinent urinary excretion data appear in Table I. The mean apparent t% for the appearance of SP in the plasma was 4 hr (range, 1.6 to 5 hr). The apparently slow absorption of SP after SSZ administration is probably due to the existence of a rate-determining dissolution step for SSZ (due to its very low aqueous solubility at gastrointestinal pH values)21 which precedes its metabolic conversion (azo reduction) to SP in the colon. The incomplete absorption of SP (Table II) may be due to the higher degree of ionization of SP (pKa 8.43)20 and the limited
920
Bates, Blumenthal, and Pieniaszek
Clinical Pharmacology and Therapeutics
Table I. Pharmacokinetic parameters for sulfapyridine (SP) and N4- acetylsulfapyridine (AcSP) estimated from plasma concentration and urinary excretion data after a single 2.0-gm oral dose of sulfasalazine Subject Parameter
Peak plasma concentration, observed (p,g/ml) SP AcSP* Time of peak plasma concentration, observed (hr) SP AcSP Total area under plasma concentration vs time curve (p,g-hr/ml) SP AcSP* Acetylation of SP (%)t Apparent onset of SP absorption, to (hr) Apparent absorption rate constant for SP, k (hc l) Absorption half-life (hr) Apparent elimination rate constant for SP, K (hc l ) Biologic half-life (hr) Apparent volume of distribution of SP, V (L1kg) Apparent urinary excretion rate constant for SP, ke (hr-l) Apparent formation rate constant for acetylated SP,§ k f (hr- l ) Acetylator phenotype Clearances (ml/min/kg) Renal (SP) Renal (AcSP) Total body (SP) Extrarenal (SP)
P. B. (3Jyr;82kg)
E.M. (23yr;67kg)
18.6 5.08
14.9 7.56
12.7 2.52
10 10
24 24
16 16
327.5 120.8 33.6 4.1 0.425 1.63 0.0654 10.6 0.439
616.4 385.0 37.6 5.8 0.166 4.17 0.0415 16.7 0.397
474.6 119.2 25.5 3.5 0.161 4.30 0.0457 15.2 0.449
4.99 6.28 24 24
6.19 4.06 24 24
119.5 147.8 62.3 5.3 0.139t
166.5 123.7 52.3 6.2 0.149t
4.98t 0.110
4.66t 0.115
6.30 0.653
6.02 0.651
0.0156
0.0130
0.0127
0.0169
0.0154
0.0199
0.0189
0.0180
0.0726
0.0650
Slow
Slow
Slow
Rapid
Rapid
0.114 0.176 0.478 0.365
0.0863 0.183 0.275 0.189
0.0951 0.299 0.342 0.247
0.184 0.334 1.19 1.01
0.167 0.321 1.25 1.08
*Expressed in tenns of SP. t AcSP to (AcSP + SP) plasma concentration ratio at 36 hr. tSubject to methodological error since the ratio k/K approximates unity.'· § Fraction of total SP excreted in the urine as total AcSP (AcSP + AcSP-O-G) times K.
mucosal surface area in the colon as compared with the small intestines. 13 This explanation is supported by the observation that when SP is administered to man orally, it is rapidly and efficiently absorbed from the small intestines.8, 25 Das and associates 3 , 8 suggest that patients on SSZ therapy can be classified as rapid or slow acetylators of SP based on the percent acetylation of SP in plasma. They 3, 8 recommend that
42.5% acetylation be used as the distinguishing boundary, but this method of phenotyping is applicable only to plasma concentration data from subjects with normal renal function. In patients with impaired renal function (in whom accumulation of the acetylated metabolite[s] of SP in the plasma is possible), phenotyping should be based on the formation rate constant of acetylated SP. The subjects participating in the present study had normal renal function and
Salivary excretion of sulfapyridine
Volume 22 Number 6
921
Sulfapyridine
20.0.
N4 -Acetylsulfapyridine
10..0
ao.
E
.....
6.0.
g:4.0.
t
fi2 a: .0.
as u I-
§ to.
u 0..8
0..6 0..4
0 ' . 2 - L - - - - r - - , - - - . - - - - - r - - r ..J._ _, - _ - , _ - ,_ _, - _ - . 45 75 15 30. 60. 15 30 45 60. 75 TIME, hours
Fig. 1. Plasma (0) and salivary (0) co.ncentratio.ns o.f sulfapyridine and N4- acetylsulfapyridine as a functio.n o.f time after oral administration of 2.0 gm o.f sulfasalazine to. a slo.w acetylato.r (Subject 1. L.). 8.0.
SulfopYridine
S.o. E ....
4.0.
'"::1..2.0.
t
g to. as u
0..8
§o.s u
0..4
0.2
0..1
10.
20.
30.
40.
50. TIME, hours
10.
20.
30
40.
50
Fig. 2. Plasma (0) and salivary (0) concentratio.ns o.f sulfapyridine and N4- acetylsulfapyridine as a function o.f time after o.ral administration of 2.0 gm o.f sulfasalazine to a rapid acetylato.r (Subject M. C.).
the plasma percent acetylation value for each subject appears in Table I. Based solely on the magnitude of this parameter, Subjects P. B., 1. L., and B. G. would be c1 as si fled as slow acetylators of SP and Subjects M. C. and E. M. as rapid acetylators of SP. The validity of this method is questionable, however, since the higher plasma percent acetylation values for Subjects M. C. and E. M. could have resulted,
in part, from the fact that the apparent volume of distribution of SP in these subjects was approximately 50% larger than that in Subjects P. B., 1. L., and B. G. (Table I). Consequently, the apparent formation rate constant for acetylated SP, estimated from the product of the fraction of total SP excreted in the urine as AcSP and AcSP-O-G (Table II) and the apparent elimination rate constant for SP (Table I), was
922
Bates, Blumenthal, and Pieniaszek
Clinical Pharmacology atuf Therapeutics
Table IT. Urinary excretion of sulfasalazine and its sulfapyridine (SP) metabolites after a single 2.0-gm oral dose of sulfasalazine Subject Parameter
P. B.
1. L.
B. G.
M.C.
E.M.
Cumulative amount excreted Sulfasalazine* Total SP* sPt AcSPt SP-O-Gt AcSP-O-Gt Total excretion* Acetylation of SPt Acetylator phenotype
1.56 61.9 23.9 13.6 45.4 16.9 63.5 30.5 Slow
1.47 57.9 31.5 41.7 23.0 3.89 59.4 45.6 Slow
4.26 57.1 27.8 22.0 32.9 17.3 61.4 39.3 Slow
8.39 50.9 15.4 34.6 18.6 31.4 59.3 66.1 Rapid
1.47 67.2 13.4 19.0 29.7 37.9 68.7 56.9 Rapid
*Expressed as percent of dose. tExpressed as percent of total SP (SP and its conjugated metabolites).
Table ITI. Pharmacokinetic parameters for sulfapyridine obtained from plasma and saliva concentration data after a single 2.0-gm oral dose of sulfasalazine Subject Parameter
P. B.
Peak concentration, observed (l-tg/ml) 18.6 Plasma 8.22 Saliva Time of peak concentration, observed (hr) 10 Plasma Saliva 10 Biologic half-life (hr) 10.6 Plasma 9.65 Saliva Total area under the concentration vs time curve (l-tg-hr/ml) 327.5 Plasma 161.3 Saliva used to phenotype 3 subjects as slow acetylators and 2 subjects as rapid acetylators of SP (Table I). The average biologic tYz of SP was approximately twice as long for slow acetylators as for rapid acetylators (Table I). Similar differences have been observed with sulfamethazine. 30 Also, AcSP was cleared by the kidneys about twice as rapidly as SP, irrespective of acetylator phenotype; but the total body and extrarenal (hepatic) plasma clearances of SP were substantially higher in rapid acetylators than in slow acetylators of SP (Table I). Such pharmacokinetic information is required for the design of appropriate multiple dosage regimens for SSZ.
I
1. L.
I B. G. I M.C. I E.M. 4.99 2.82
Paired t test
14.9 7.84
12.7 8.64
24 24
16 16
16.7 15.7
15.2 15.8
6.32 7.08
6.02 7.36
NS (p > 0.7)
616.4 340.2
474.6 301.7
119.5 74.00
166.5 81.91
P < 0.025
24 24
6.19 2.64
P < 0.Q25
24 24
Since the dosage regimen currently used in clinical practice (usually 0.5 to 1.0 gm of SSZ every 6 hr)8 does not take into account the difference in the biologic tVz or the total body clearance of SP between the two phenotypes, it is not surprising that unmetabolized and total SP reach higher and sometimes toxic levels in slow acetylators. 4 • 8. 26 The urinary excretion of SSZ and its SP metabolites is given for each subject in Table II. In accordance with the findings of other investigators,25.28 only 1.5% to 8.4% of the dose of SSZ was ultimately excreted in the urine in an unmetabolized form, while about 60% of the dose was recovered as SP and its three metabo-
Salivary excretion of sulfapyridine
Volume 22 Number 6
Slow Acetylator
~
g:
Rapid Acetylator
8.0
o 24
r'f'O l5
3.2
,2
24
10
O
923
2.4
w
1.6 48
~2.0
0.8
...J
~
36 6
48
4.0
ao
12.0
16.0
1.6
3.2
4.8
PLASMA CONCENTRATION, /Lg/ml
Fig. 3. Relationship between the concentrations of sulfapyridine in the saliva and plasma for a slow acetylator (Subject J. L.; r = 0.995, p < 0.01) and a rapid acetylator (Subject M. C.; r = 0.998, p < 0.0 I). The numbers indicate the time (hr) after sulfasalazine administration. lites. Based on available urinary and fecal excretion data for SSZ and its SP metabolites ,8. 25, 28 the less than quantitative urinary recovery of the dose of SSZ (Table II) appears to be due primarily to incomplete absorption of SP from the colon rather than to incomplete azo reduction and absorption of SSZ. Subjects phenotyped as slow or rapid acetylators based on the formation rate constant for acetylated SP (Table I) were similarly classified by means of percent acetylation values for urine (Table II), the sole exception being Subject E. M. whose acetylation capability as determined in urine (Table II) was slightly below that for a rapid acetylator (56.9% vs 63% or greater3). The time course of SP concentrations in the saliva paralleled that in the plasma (Figs. 1 and 2). The observed times of occurrence of peak SP concentrations in the plasma and saliva were coincident for each subject but showed appreciable intersubject variation, ranging from 10 hr to 24 hr (Table III). Peak salivary SP concentrations and the total area under the salivary SP concentration-time curves for the 5 subjects were significantly lower than those obtained from plasma concentration data (Table III). The biologic tV2S of SP were calculated from the least-squares slope of the terminal linear portion of log plasma or saliva concentration vs time plots similar to those depicted in Figs. 1 and 2 and are listed for each subject in Table III. No statistically significant difference existed between plasma and saliva SP tlhs in the 5 subjects (Table III). Therefore, saliva instead of
plasma concentrations of SP can be used to monitor the elimination rate of SP. As illustrated in Fig. 3 for a typical slow acetylator (Subject J. L.) and a typical rapid acetylator (Subject M. C.) of SP, there was excellent linear relationship between SP concentrations in the saliva and plasma. The saliva: plasma SP concentration ratios for all 5 subjects are summarized in Table IV. Within each subject the mean concentration ratio remained essentially constant (over a wide range of plasma concentrations) during both the absorption and elimination phases of SP (suggestive of rapid equilibration of SP between the plasma and saliva) and was in close agreement with the saliva: plasma SP ratio determined from total area under the curve measurements (Table IV). The saliva: plasma concentration ratio for SP showed little intersubject variation and was apparently independent of acetylator phenotype and of saliva pH over the range of 6.8 to 7.5 (Table IV). On the average, the concentration of SP in the saliva was 56% of that in the plasma. After an initial lag time of 4 to 6 hr, the acetylated metabolite of SP appeared simultaneously in the plasma and the saliva, reached peak concentrations in both fluids at similar times, and was eliminated from both fluids in a monoexponential and parallel fashion (Figs. I and 2 and Table V). AcSP was excreted in the saliva to an appreciably lesser extent than was SP. The average saliva: plasma concentration ratio for AcSP (0.246 ± 0.056) was approxi-
924
Bates, Blumenthal, and Pieniaszek
Clinical Pharmacology and Therapeutics
Table IV. Relationship between the concentration of sUljapyridine in the saliva and in the plasma after a single 2.0-gm oral dose of suljasalazine Subject Parameter Acetylator phenotype Saliva: plasma concentration ratio Mean ratio* No. S.E. C.V (%)t Area ratio:j: No. S.E. C.V. (%) Saliva pH Mean§ No. S.E. C.V. (%)
P. B.
J. L.
Slow
Slow
B. G.
M.C.
E.M.
Slow
Rapid
Rapid
Mean
0.497 9 0.021 12 0.493
0.560 8 0.013 6.7 0.552
0.638 10 0.015 7.7 0.636
0.597 7 0.029 13 0.619
0.502 9 0.020 12 0.492
0.559 5 0.027 11 0.558 5 0.030 12
7.41 13 0.063 3.0
6.84 13 0.060 3.2
7.50 15 0.040 2.1
7.11 13 0.053 2.7
7.18 15 0.067 3.6
7.21 5 0.12 3.6
" Average of all the ratios obtained over the entire experimental period.
t Coefficient of variation. t Obtained by dividing the total area under the salivary concentration vs time curve by the total area under the plasma concentration vs time curve. § Average of all the pH values obtained over the entire experimental period.
Table V. Pharmacokinetic parameters for N 4-acetylsuljapyridine obtained from plasma and saliva concentration data after a single 2.0-gm oral dose of suljasalazine Subject Parameter Acetylator phenotype Peak concentration, observed (JLg/ml)* Plasma Saliva Time of peak concentration, observed (hr) Plasma Saliva Total area under the concentration vs time curve (JLg-hr/ml)* Plasma Saliva Saliva:plasma concentration ratio Mean ratiot No. SE CV (%):j: Area ratio§ No. SE CV (%)
P. B.
J. L.
B. G.
M.C.
E.M.
Slow
Slow
Slow
Rapid
Rapid
5.08 1.40
7.56 3.36
2.52 0.46
6.28 0.79
4.06 0.54
10 16
120.8 40.09 0.326 8 0.Dl5 13 0.332
24 24 385.0 146.2 0.422 8 0.020 14 0.380
16 16 119.2 16.55 0.188 8 0.Dl8 27 0.139
24 24 147.8 22.12 0.175 6 0.Dl8 25 0.150
Mean
24 16 123.7 14.32 0.120 0.246 5 8 0.Dl5 0.056 50 35 0.116 0.223 5 0.055 55
"Expressed in terms of SP.
t Average of all the ratios obtained over the entire experimental period .
.t Coefficient of variation. §Obtained by dividing the total area under the salivary concentration vs time curve by the total area under the plasma concentration vs time curve.
Salivary excretion of sulfapyridine
Volume 22 Number 6
925
Table VI. Partition coefficients (chloroform/aqueous buffer) and dissociation constants for sulfapyridine (SP) and N 4-acetylsulfapyridine (AcSP)
Parameter Partition coefficient at 37° (chloroform/ aqueous buffer*) Observed (Co) Intrinsic (Ca)t Apparent dissociation constant (Ka>t pKa Percent unionized in plasma (pH 7.4)
SP
AcSP
0.879 (pH 7.4) 0.999 (pH 6.4) 1.02 6.5 x 10-9 8.2+
0.202 (pH 7.4) 0.293 (pH 6.5) 0.313 2.2 x 10-8 7.7
86
67
*Isotonic phosphate buffer, United States Pharmacopeia XIX. tFrom observed partition coefficient (Col data at two different pH values and solution of the following two simultaneous equations for the partition coefficient (intrinsic) of the unionized moiety (Cal and the dissociation constant (Ka)at pHI: CO • I = Ca[H+h/Ka tReported pKa value for SP at 25° is
+ [H+]I;
mately 44% of the average ratio for SP (0.559 ± 0.027) and showed considerably more intrasubject and intersubject variation (Table IV and V). A review of the literature suggests that for most drugs (including several sulfonamides), 11, 16, 18, 23 only the non-protein-bound, nonionized fraction of a drug in plasma can readily diffuse across the epithelium of the salivary gland. For a weakly acidic drug possessing excellent lipid solubility characteristics, the saliva to total plasma (protein-bound and unbound) concentration ratio is given by Equation 1:11 R
' =
I I
+ +
at pH,: Co.,
= Ca[H+],/Ka + [H+],.
8.43. 19
10,pHs-pKa) lO'pHp pKa) •
fp
(1)
where pHs and pHp are the pH of the saliva and plasma, respectively, and fp is the fraction of unbound drug in the plasma. The pKa values for SP and AcSP were estimated from chloroform/aqueous buffer partition coefficient data at 37° C and were 8.2 and 7.7, respectively (Table VI). The former value is in good agreement with the literature value of 8.43 determined at 25° C.19 As demonstrated for SP (Table VI) and for other sulfonamides by Despopoulos and Callahan , 9 N4- acetylation apparently increases the acidity of most sulfonamides. Equation 1 does not account for the degree of lipid solubility of the drug, which has been shown for several sulfonamides 16 , 18,23 and barbiturates 23 to strongly affect drug diffusion
across the salivary epithelium. In contrast to AcSP, the diffusibility of SP (product of the percentage of SP nonionized in the plasma and its lipid solubility, as reflected by the intrinsic chloroform/water partition coefficient data presented in Table VI)23 appears to be high. This observation may explain, in part, why the saliva: total plasma concentration ratio for SP (0.559; Table IV) is on the average greater and less variable than that found experimentally for AcSP (0.246; Table V). Differences in the degree of binding of SP and AcSP to plasma protein could also account for the observed differences in the salivary excretion of these two species, a possibility which remains to be explored. The results of our study show that there are appreciable differences between rapid and slow acetylators with respect to the kinetics of SP elimination. This observation suggests the need to individualize SSZ dosage regimens based on the biologic tlh or the total body clearance of SP. There was excellent direct relationship between SP concentrations in the saliva and total SP plasma concentrations (free and proteinbound). The saliva: plasma concentration ratio for SP was apparently independent of total plasma concentration of SP, saliva pH, and acetylator phenotype and exhibited little intersubject variation. It is now necessary to determine whether similar results can be realized in adult and pediatric patients treated with SSZ. If such is the case, then salivary SP concentration
926
Bates, Blumenthal, and Pieniaszek
measurements may offer a convenient, noninvasive method for monitoring the clinical and toxicologic status of these patients. The utility of salivary AcSP concentration measurements for phenotyping purposes remains to be determined. References 1. Bates, T. R., Blumenthal, H. P., and Pieniaszek, H. J., Jr.: Time course of free and N4_ acetylated sulfapyridine concentrations in the plasma and saliva of man after sulfasalazine (salicylazosulfapyridine) administration: Preliminary findings, Res. Commun. Chern. Pathol. Pharmacol. 15:183-188, 1976. 2. Bates, T. R., and Pieniaszek, H. J., Jr.: A modified colorimetric method for the determination of sulfapyridine and its metabolites in plasma and saliva after sulfasalazine administration, Clin. Chern. (In press.) 3. Das, K. M., and Eastwood, M. A.: Acetylation polymorphism of sulfapyridine in patients with ulcerative colitis and Crohn's disease, CUN. PHARMACOL. THER. 18:514-520, 1975. 4. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: Adverse reactions during salicylazosulfapyridine therapy and the relation with drug metabolism and acetylator phenotype, N. Eng!. 1. Med. 298:491-495, 1973. 5. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: The metabolism of salicylazosulphapyridine in ulcerative colitis. I. The relationship between metabolites and the response to treatment in in-patients. II. The relationship between metabolites and the progress of the disease studied in out-patients, Gut 14:631-641, 1973. 6. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: The role of the colon in the metabolism of salicylazosulfapyridine, Scand. 1. Gastroenterol. 9:137-141, 1974. 7. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: Salazopyrin metabolism in ulcerative colitis, Gut 13:840, 1972. 8. Das, K. M., and Rubin, R.: Clinical pharmacokinetics of sulphasalazine, Clin. Pharmacokinet. 1:406-425, 1976. 9. Despopoulos, A., and Callahan, P. X.: Molecular features of sulfonamide transport in renal excretory processes, Am. J. Physiol. 203:19-26, 1962. 10. Dissanayake, A. S., and Truelove, S. C.: A controlled therapeutic trial of long-term maintenance treatment of ulcerative colitis with sulphasalazine (salazopyrin), Gut 14:923-926, 1973. II. Dvorchik, B. H., and Vesell, E. S.: Pharmaco-
Clinical Pharmacology and Therapeutics
12.
13. 14. 15. 16.
17.
18.
19.
20. 21.
22.
23. 24. 25.
kinetic interpretation of data gathered during therapeutic drug monitoring, Clin. Chern. 22:868-878, 1976. Frisk, A. R.: Blood concentration, acetylation and urinary excretion of sulfapyridine and sulfathiazole after various sulfapyridine and sulfathiazole derivatives administered by different routes, Acta Med. Scand. 106:369-403, 1941. Gibaldi, M.: Introduction to biopharmaceutics, Philadelphia, 1971, Lea & Febiger, Publishers, pp. 8-30. Gibaldi, M., and Perrier, D.: Pharmacokinetics, New York, 1975, Marcel Dekker, Inc., pp. 1-43. Goldman, P., and Peppercorn, M. A.: Sulfasalazine, N. Eng I. J. Med. 293:20-23, 1975. Griineisen, A., and Witzgall, H.: Salivary excretion of sulfonamides. Correlation of drug levels in saliva and ultrafiltrate of plasma, Eur. J. Clin. Pharmacol. 7:77-79, 1974. Hansson, K. A., and Sandberg, M.: Determination of sulfapyridine and its metabolites in biological materials after administration of salicylazosulfapyridine, Acta Pharm. Suec. 10:87-92, 1973. Killmann, S. A., and Thaysen, J. H.: The permeability of the human parotoid gland to a series of sulfonamide compounds, paraaminohippurate and inulin, Scand. J. Clin. Lab. Invest. 7:86-91, 1955. Koizumi, T., Arita, T., and Kakemi, K.: Absorption and excretion of drugs. XIX. Some pharmacokinetic aspects of absorption and excretion of sulfonamides (I). Absorption from rat stomach, Chern. Pharm. Bull. 12:413-420, 1964. Notari, R. E.: Biopharmaceutics and pharmacokinetics, New York, 1971, Marcel Dekker, Inc., pp. 55-74. Nygard, B., Olofsson, J., and Sandberg, M.: Some physico-chemical properties of salicylazosulphapyridine, including its solubility, protolytic constants, and general spectrochemical and polarographic behavior, Acta Pharm. Suec. 3:313-342, 1966. Peppercorn, M. A., and Goldman, P.: The role of intestinal bacteria in the metabolism of salicylazosulfapyridine, J. Pharmacol. Exp. Ther. 181:555-562, 1972. Rasmussen, F.: Salivary excretion of sulfonamides and barbiturates by cows and goats, Acta Pharmacol. Toxicol. 21: 11-19, 1964. Sandberg, M., and Hansson, K.-A.: Determination of salicylazosulfapyridine in biological materials, Acta Pharm. Suec. 10: 107-110, 1973. Schroder, H., and Campbell, D. E. S.: Absorption, metabolism, and excretion of salicylazosulfapyridine in man, CUN. PHARMACOL. THER. 13:539-551, 1972.
Volume 22 Number 6
26. Schader, H., and Evans, D. A. P.: Acetylator phenotype and adverse effects of sulphasalazine in healthy subjects, Gut 13:278-284, 1972. 27. Schroder, H., and Evans, D. A. P.: The polymorphic acetylation of sulphapyridine in man, J. Med. Genet. 9:168-171, 1972. 28. Schroder, H., Lewkonia, R. M., and Evans, D. A. P.: Metabolism of salicylazosulfapyridine in healthy subjects and in patients with ulcerative colitis, CUN. PHARMACOL. THER. 14:802-809, 1973.
Salivary excretion of sulfapyridine
927
29. Smith, G. H., and Tong, T. G.: Ulcerative colitis, J. Am. Pharm. Assoc. NSI5:202-212, 1975. 30. Van Oudtshoorn, M. C. B., and Potgieter, F. J.: Determination of pharmacokinetic parameters for rapid and slow acetylators of sulphadimidine, J. Pharm. Pharmacol. 24:357360, 1972. 31. Wagner, J. G.: Fundamentals of clinical pharmacokinetics, Hamilton, Ill., 1975, Drug Intelligence Publications, pp. 57-82.
Theodore R. Bates, Ph.D., H. Peter Blumenthal, M.D., and Henry J. Pieniaszek, Jr., B.A. Amherst, N.Y. Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo
Sulfasalazine (SSZ; salicylazosulfapyridine), one of the drugs of choice in the treatment of ulcerative colitis ,8. 10. 15. 29 is poorly absorbed from the gastrointestinal tract of man 25 and is subject to extensive biotransformation by bacteSupported in part by Grant GM 20852 from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Md., and by a Merck Grant for Faculty Development to Dr. Bates from the Merck Foundation. Received for publication June 8, 1977. Accepted for publication Aug. 17, 1977. Reprint requests to: Dr. Theodore R. Bates, Department of Pharo maceutics, School of Pharmacy, State University of New York at Buffalo, H517 Cooke-Hochstetler Complex, Amherst, N. Y. 14260.
rial azo reductases in the colon and cecum to sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA).6. 22 After colonic absorption, SP is metabolized to N4-acetylsulfapyridine (AcSP), sulfapyridine-O-glucuronide (SP-O-G), and N 4_ acetyl sulfapyri dine-O-gl ucuronide (AcS P -0G).5. 7. 25, 28 Like sulfamethazine, isoniazid, and hydralazine, it exhibits acetylation polymorphism.3. 27 Steady-state serum total SP (SP and its metabolites) concentrations of 20 to 50 /-Lg/ml appear to coincide with clinical improvement in adult patients treated with SSZ, while concen-
917
918
Bates, Blumenthal, and Pieniaszek
trations exceeding 50 p.,g/ml are normally associated with adverse effects. 4. 5 The incidence of adverse effects, which can be also correlated with steady-state serum levels of SP,4 seems to be highest in adults phenotyped as slow acetylators of SP. 4, 26 Steady-state serum concentrations of SSZ or 5-ASA bear no apparent relationship to either the clinical efficacy or toxicity of SSZ. 4, 5 After oral administration of SSZ to healthy subjects or to patients with ulcerative colitis, the mean steady-state serum concentrations of SP in slow acetylators are approximately 2 to 5 times those of rapid acetylators. 5. 8, 25. 26 The pharmacokinetics of SP after SSZ administration has not been adequately studied in man. Schroder and Campbe1l 25 reported on the renal clearance of SP and each of its metabolites in healthy subjects, but their determination of the biologic half-life (t12) of SP was incorrectly based on the time course of total SP (SP and its metabolites) in the serum. Hence, although differences in the steady-state levels of SP do exist between the two phenotypes, the reason for these differences is not clear. Such differences may arise from differences in the fraction of the oral dose absorbed, in the apparent volume of distribution of SP, or in the elimination rate constant or biologic half-life of SP. The genetic polymorphism in the acetylation of SP result in appreciable interindividual variations in the steady-state serum concentrations of unmetabolized and total SP. 5, 8, 25, 26 Even within a given phenotype large variations between individuals are evident,5, 8, 25, 26 possibly related to the erratic and incomplete absorption of SP from the colon. Thus, to ensure the safe and effective use of SSZ, it may be appropriate, at least in some patients, to monitor serum or plasma concentrations of SP during the course of drug therapy and to determine acetylator phenotype. 8 Das and Rubin 8 suggest that determinations of SP and AcSP in the serum are sufficient for these purposes since SP-O-G and AcSP-O-G represent an insignificant part of serum total SP. Our preliminary results! indicated the existence of a direct relationship between the concentrations of SP in the plasma and saliva of a
Clinical Pharmacology and Therapeutics
healthy slow acetylator of SP after a single oral dose of SSZ. AcSP was also excreted in the saliva but to a lesser extent. The purposes of our study were to confirm these findings in healthy slow and rapid acetylators of SP and to determine whether saliva SP and AcSP concentration measurements offer a method for indirectly monitoring the plasma concentrations of these SSZ metabolites. The pharmacokinetics of SP in slow and rapid acetylators were also studied to determine directly the reason why the two phenotypes differ with respect to mean steadystate SP levels. Materials and methods
Subjects. Five healthy male subjects, ranging in age from 21 to 31 yr (mean, 25 yr) and in body weight from 67 to 82 kg (mean, 73 kg) participated in this study. The subjects had no previous history of sulfonamide sensitivity or gastrointestinal disease and the results of physical and laboratory examinations were normal. No drug or alcoholic beverage was ingested for 7 days prior to and for 4 days after SSZ administration. The subjects fasted for at least 8 hr before and 4 hr after administration of the drug. At 8 A.M., after the subjects had emptied their bladders and blank blood, mixed saliva, and urine specimens had been collected, a single 2.0-gm dose of SSZ as four 500-mg commercially available uncoated tablets (Azulfidine, Pharmacia) was administered orally with 240 ml of water. Sample collections and analysis. Heparinized blood (10 ml) and mixed saliva samples were obtained at I, 2, 4, 6, 8, 10, 12, 14, 16, 24, 30, 36,48, and 72 hr after SSZ administration. The mixed saliva samples were collected over a 4- min interval simultaneously with blood collections, and the pH was recorded. Salivation was stimulated by having the subjects chew on a small ball of Parafilm. Quantitative urine collections were made at predetermined intervals for 4 days after drug administration and the pH and volume of each collection were recorded. All plasma, mixed saliva, and urine specimens were stored at - 20° C pending assay.
Volume 22 Number 6
Plasma and saliva concentrations of SP and AcSP were determined by the specific colorimetric method of Hansson and Sandberg17 as modified by Bates and Pieniaszek2 to increase by 4-fold the sensitivity of the original method. Vrine specimens were assayed for SSZ, SP, AcSP, SP-O-G, and AcSP-O-G by specific colorimetric methods 17, 24 with the use of Glusulase (,8-glucuronidase and sulfatase) to hydrolyze the glucuronide conjugates of SP enzymatically. Pharmacokinetic analysis. Plasma and saliva concentrations of SP were plotted semilogarithmically as a function of time and the apparent elimination rate constant K for SP was estimated from the slope of the terminal linear segment of such plots by the method of least squares. The biologic t% of SP was obtained by dividing K into 0.693. The time course of SP concentrations in the plasma was adequately described pharmacokinetic ally by a one-compartment open model with apparent first-order absorption and an apparent lag time (tJ. 14, 31 The apparent absorption rate constant k and to for SP were estimated by the method of residuals or feathering 14 , 31 with the use of least-squares regression analysis. SP and its metabolites are primarily excreted in the urine of man,8, 25, 28 the mean percent of dose excreted (n = 4) being 90% (range, 88% to 95%) after 1.841-gm intravenous dose of SP as the sodium salt. 12 Thus, the total body clearance of SP (product of K and the apparent volume of distribution of SP, V) may be estimated from the ratio of the amount of SP and all of its metabolites ultimately excreted in the urine (assuming this to be equivalent to the amount of SP absorbed after SSZ administration) to the total area under the concentration vs time curve (AVC) for plasma SP as measured by the trapezoidal rule. 14, 31 The renal clearance of SP and AcSP were determined by dividing the plasma AVe value for each species into the cumulative amount of that species excreted in the urine. 14 , 31 The subjects were phenotyped as slow or rapid acetylators of SP based on the magnitude of the formation rate constant for acetylated SP
Salivary excretion of sulfapyridine
919
or on percent acetylation (urine) values calculated from the following relationship:3 % acetylation = AAcSP
+ AAcSP-O-G + AAcSP + AAcSP-O-G
------~~--~~--~-----x Asp
+
A SP- o - G
100
where A represents the cumulative amount ultimately excreted in the urine. The distinguishing boundary between slow and rapid acetylators was taken as 63% for urine. 3 Mean saliva to plasma concentration ratios for SP and AcSP were calculated for each species by averaging the concentration ratios obtained over the entire experimental period. A similar ratio was obtained for each species by dividing the saliva AVe by the plasma AVe. Results and discussion
Plasma and saliva concentration-time profiles for SP and AcSP after oral administration of a 2.0-gm dose of SSZ to a slow acetylator (Subject J. L.) and to a rapid acetylator (Subject M. e.) are depicted in Figs. 1 and 2. Measurable concentrations of SP appeared in the plasma 3.5 to 6 hr after SSZ administration (Table I) which is consistent with normal transit times for fluids to the colon and cecum where SSZ undergoes bacterial azo reduction. The observed times of peak plasma concentrations of SP and AcSP were similar (Table I) and peak levels declined monoexponentially with time (Figs. 1 and 2). The plasma SP concentration-time data for each subject were fitted to the integrated rate expression for a one-compartment open model with apparent first-order absorption and an apparent absorption lag time. 14, 31 The pharmacokinetic parameters derived from such analyses and pertinent urinary excretion data appear in Table I. The mean apparent t% for the appearance of SP in the plasma was 4 hr (range, 1.6 to 5 hr). The apparently slow absorption of SP after SSZ administration is probably due to the existence of a rate-determining dissolution step for SSZ (due to its very low aqueous solubility at gastrointestinal pH values)21 which precedes its metabolic conversion (azo reduction) to SP in the colon. The incomplete absorption of SP (Table II) may be due to the higher degree of ionization of SP (pKa 8.43)20 and the limited
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Clinical Pharmacology and Therapeutics
Table I. Pharmacokinetic parameters for sulfapyridine (SP) and N4- acetylsulfapyridine (AcSP) estimated from plasma concentration and urinary excretion data after a single 2.0-gm oral dose of sulfasalazine Subject Parameter
Peak plasma concentration, observed (p,g/ml) SP AcSP* Time of peak plasma concentration, observed (hr) SP AcSP Total area under plasma concentration vs time curve (p,g-hr/ml) SP AcSP* Acetylation of SP (%)t Apparent onset of SP absorption, to (hr) Apparent absorption rate constant for SP, k (hc l) Absorption half-life (hr) Apparent elimination rate constant for SP, K (hc l ) Biologic half-life (hr) Apparent volume of distribution of SP, V (L1kg) Apparent urinary excretion rate constant for SP, ke (hr-l) Apparent formation rate constant for acetylated SP,§ k f (hr- l ) Acetylator phenotype Clearances (ml/min/kg) Renal (SP) Renal (AcSP) Total body (SP) Extrarenal (SP)
P. B. (3Jyr;82kg)
E.M. (23yr;67kg)
18.6 5.08
14.9 7.56
12.7 2.52
10 10
24 24
16 16
327.5 120.8 33.6 4.1 0.425 1.63 0.0654 10.6 0.439
616.4 385.0 37.6 5.8 0.166 4.17 0.0415 16.7 0.397
474.6 119.2 25.5 3.5 0.161 4.30 0.0457 15.2 0.449
4.99 6.28 24 24
6.19 4.06 24 24
119.5 147.8 62.3 5.3 0.139t
166.5 123.7 52.3 6.2 0.149t
4.98t 0.110
4.66t 0.115
6.30 0.653
6.02 0.651
0.0156
0.0130
0.0127
0.0169
0.0154
0.0199
0.0189
0.0180
0.0726
0.0650
Slow
Slow
Slow
Rapid
Rapid
0.114 0.176 0.478 0.365
0.0863 0.183 0.275 0.189
0.0951 0.299 0.342 0.247
0.184 0.334 1.19 1.01
0.167 0.321 1.25 1.08
*Expressed in tenns of SP. t AcSP to (AcSP + SP) plasma concentration ratio at 36 hr. tSubject to methodological error since the ratio k/K approximates unity.'· § Fraction of total SP excreted in the urine as total AcSP (AcSP + AcSP-O-G) times K.
mucosal surface area in the colon as compared with the small intestines. 13 This explanation is supported by the observation that when SP is administered to man orally, it is rapidly and efficiently absorbed from the small intestines.8, 25 Das and associates 3 , 8 suggest that patients on SSZ therapy can be classified as rapid or slow acetylators of SP based on the percent acetylation of SP in plasma. They 3, 8 recommend that
42.5% acetylation be used as the distinguishing boundary, but this method of phenotyping is applicable only to plasma concentration data from subjects with normal renal function. In patients with impaired renal function (in whom accumulation of the acetylated metabolite[s] of SP in the plasma is possible), phenotyping should be based on the formation rate constant of acetylated SP. The subjects participating in the present study had normal renal function and
Salivary excretion of sulfapyridine
Volume 22 Number 6
921
Sulfapyridine
20.0.
N4 -Acetylsulfapyridine
10..0
ao.
E
.....
6.0.
g:4.0.
t
fi2 a: .0.
as u I-
§ to.
u 0..8
0..6 0..4
0 ' . 2 - L - - - - r - - , - - - . - - - - - r - - r ..J._ _, - _ - , _ - ,_ _, - _ - . 45 75 15 30. 60. 15 30 45 60. 75 TIME, hours
Fig. 1. Plasma (0) and salivary (0) co.ncentratio.ns o.f sulfapyridine and N4- acetylsulfapyridine as a functio.n o.f time after oral administration of 2.0 gm o.f sulfasalazine to. a slo.w acetylato.r (Subject 1. L.). 8.0.
SulfopYridine
S.o. E ....
4.0.
'"::1..2.0.
t
g to. as u
0..8
§o.s u
0..4
0.2
0..1
10.
20.
30.
40.
50. TIME, hours
10.
20.
30
40.
50
Fig. 2. Plasma (0) and salivary (0) concentratio.ns o.f sulfapyridine and N4- acetylsulfapyridine as a function o.f time after o.ral administration of 2.0 gm o.f sulfasalazine to a rapid acetylato.r (Subject M. C.).
the plasma percent acetylation value for each subject appears in Table I. Based solely on the magnitude of this parameter, Subjects P. B., 1. L., and B. G. would be c1 as si fled as slow acetylators of SP and Subjects M. C. and E. M. as rapid acetylators of SP. The validity of this method is questionable, however, since the higher plasma percent acetylation values for Subjects M. C. and E. M. could have resulted,
in part, from the fact that the apparent volume of distribution of SP in these subjects was approximately 50% larger than that in Subjects P. B., 1. L., and B. G. (Table I). Consequently, the apparent formation rate constant for acetylated SP, estimated from the product of the fraction of total SP excreted in the urine as AcSP and AcSP-O-G (Table II) and the apparent elimination rate constant for SP (Table I), was
922
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Clinical Pharmacology atuf Therapeutics
Table IT. Urinary excretion of sulfasalazine and its sulfapyridine (SP) metabolites after a single 2.0-gm oral dose of sulfasalazine Subject Parameter
P. B.
1. L.
B. G.
M.C.
E.M.
Cumulative amount excreted Sulfasalazine* Total SP* sPt AcSPt SP-O-Gt AcSP-O-Gt Total excretion* Acetylation of SPt Acetylator phenotype
1.56 61.9 23.9 13.6 45.4 16.9 63.5 30.5 Slow
1.47 57.9 31.5 41.7 23.0 3.89 59.4 45.6 Slow
4.26 57.1 27.8 22.0 32.9 17.3 61.4 39.3 Slow
8.39 50.9 15.4 34.6 18.6 31.4 59.3 66.1 Rapid
1.47 67.2 13.4 19.0 29.7 37.9 68.7 56.9 Rapid
*Expressed as percent of dose. tExpressed as percent of total SP (SP and its conjugated metabolites).
Table ITI. Pharmacokinetic parameters for sulfapyridine obtained from plasma and saliva concentration data after a single 2.0-gm oral dose of sulfasalazine Subject Parameter
P. B.
Peak concentration, observed (l-tg/ml) 18.6 Plasma 8.22 Saliva Time of peak concentration, observed (hr) 10 Plasma Saliva 10 Biologic half-life (hr) 10.6 Plasma 9.65 Saliva Total area under the concentration vs time curve (l-tg-hr/ml) 327.5 Plasma 161.3 Saliva used to phenotype 3 subjects as slow acetylators and 2 subjects as rapid acetylators of SP (Table I). The average biologic tYz of SP was approximately twice as long for slow acetylators as for rapid acetylators (Table I). Similar differences have been observed with sulfamethazine. 30 Also, AcSP was cleared by the kidneys about twice as rapidly as SP, irrespective of acetylator phenotype; but the total body and extrarenal (hepatic) plasma clearances of SP were substantially higher in rapid acetylators than in slow acetylators of SP (Table I). Such pharmacokinetic information is required for the design of appropriate multiple dosage regimens for SSZ.
I
1. L.
I B. G. I M.C. I E.M. 4.99 2.82
Paired t test
14.9 7.84
12.7 8.64
24 24
16 16
16.7 15.7
15.2 15.8
6.32 7.08
6.02 7.36
NS (p > 0.7)
616.4 340.2
474.6 301.7
119.5 74.00
166.5 81.91
P < 0.025
24 24
6.19 2.64
P < 0.Q25
24 24
Since the dosage regimen currently used in clinical practice (usually 0.5 to 1.0 gm of SSZ every 6 hr)8 does not take into account the difference in the biologic tVz or the total body clearance of SP between the two phenotypes, it is not surprising that unmetabolized and total SP reach higher and sometimes toxic levels in slow acetylators. 4 • 8. 26 The urinary excretion of SSZ and its SP metabolites is given for each subject in Table II. In accordance with the findings of other investigators,25.28 only 1.5% to 8.4% of the dose of SSZ was ultimately excreted in the urine in an unmetabolized form, while about 60% of the dose was recovered as SP and its three metabo-
Salivary excretion of sulfapyridine
Volume 22 Number 6
Slow Acetylator
~
g:
Rapid Acetylator
8.0
o 24
r'f'O l5
3.2
,2
24
10
O
923
2.4
w
1.6 48
~2.0
0.8
...J
~
36 6
48
4.0
ao
12.0
16.0
1.6
3.2
4.8
PLASMA CONCENTRATION, /Lg/ml
Fig. 3. Relationship between the concentrations of sulfapyridine in the saliva and plasma for a slow acetylator (Subject J. L.; r = 0.995, p < 0.01) and a rapid acetylator (Subject M. C.; r = 0.998, p < 0.0 I). The numbers indicate the time (hr) after sulfasalazine administration. lites. Based on available urinary and fecal excretion data for SSZ and its SP metabolites ,8. 25, 28 the less than quantitative urinary recovery of the dose of SSZ (Table II) appears to be due primarily to incomplete absorption of SP from the colon rather than to incomplete azo reduction and absorption of SSZ. Subjects phenotyped as slow or rapid acetylators based on the formation rate constant for acetylated SP (Table I) were similarly classified by means of percent acetylation values for urine (Table II), the sole exception being Subject E. M. whose acetylation capability as determined in urine (Table II) was slightly below that for a rapid acetylator (56.9% vs 63% or greater3). The time course of SP concentrations in the saliva paralleled that in the plasma (Figs. 1 and 2). The observed times of occurrence of peak SP concentrations in the plasma and saliva were coincident for each subject but showed appreciable intersubject variation, ranging from 10 hr to 24 hr (Table III). Peak salivary SP concentrations and the total area under the salivary SP concentration-time curves for the 5 subjects were significantly lower than those obtained from plasma concentration data (Table III). The biologic tV2S of SP were calculated from the least-squares slope of the terminal linear portion of log plasma or saliva concentration vs time plots similar to those depicted in Figs. 1 and 2 and are listed for each subject in Table III. No statistically significant difference existed between plasma and saliva SP tlhs in the 5 subjects (Table III). Therefore, saliva instead of
plasma concentrations of SP can be used to monitor the elimination rate of SP. As illustrated in Fig. 3 for a typical slow acetylator (Subject J. L.) and a typical rapid acetylator (Subject M. C.) of SP, there was excellent linear relationship between SP concentrations in the saliva and plasma. The saliva: plasma SP concentration ratios for all 5 subjects are summarized in Table IV. Within each subject the mean concentration ratio remained essentially constant (over a wide range of plasma concentrations) during both the absorption and elimination phases of SP (suggestive of rapid equilibration of SP between the plasma and saliva) and was in close agreement with the saliva: plasma SP ratio determined from total area under the curve measurements (Table IV). The saliva: plasma concentration ratio for SP showed little intersubject variation and was apparently independent of acetylator phenotype and of saliva pH over the range of 6.8 to 7.5 (Table IV). On the average, the concentration of SP in the saliva was 56% of that in the plasma. After an initial lag time of 4 to 6 hr, the acetylated metabolite of SP appeared simultaneously in the plasma and the saliva, reached peak concentrations in both fluids at similar times, and was eliminated from both fluids in a monoexponential and parallel fashion (Figs. I and 2 and Table V). AcSP was excreted in the saliva to an appreciably lesser extent than was SP. The average saliva: plasma concentration ratio for AcSP (0.246 ± 0.056) was approxi-
924
Bates, Blumenthal, and Pieniaszek
Clinical Pharmacology and Therapeutics
Table IV. Relationship between the concentration of sUljapyridine in the saliva and in the plasma after a single 2.0-gm oral dose of suljasalazine Subject Parameter Acetylator phenotype Saliva: plasma concentration ratio Mean ratio* No. S.E. C.V (%)t Area ratio:j: No. S.E. C.V. (%) Saliva pH Mean§ No. S.E. C.V. (%)
P. B.
J. L.
Slow
Slow
B. G.
M.C.
E.M.
Slow
Rapid
Rapid
Mean
0.497 9 0.021 12 0.493
0.560 8 0.013 6.7 0.552
0.638 10 0.015 7.7 0.636
0.597 7 0.029 13 0.619
0.502 9 0.020 12 0.492
0.559 5 0.027 11 0.558 5 0.030 12
7.41 13 0.063 3.0
6.84 13 0.060 3.2
7.50 15 0.040 2.1
7.11 13 0.053 2.7
7.18 15 0.067 3.6
7.21 5 0.12 3.6
" Average of all the ratios obtained over the entire experimental period.
t Coefficient of variation. t Obtained by dividing the total area under the salivary concentration vs time curve by the total area under the plasma concentration vs time curve. § Average of all the pH values obtained over the entire experimental period.
Table V. Pharmacokinetic parameters for N 4-acetylsuljapyridine obtained from plasma and saliva concentration data after a single 2.0-gm oral dose of suljasalazine Subject Parameter Acetylator phenotype Peak concentration, observed (JLg/ml)* Plasma Saliva Time of peak concentration, observed (hr) Plasma Saliva Total area under the concentration vs time curve (JLg-hr/ml)* Plasma Saliva Saliva:plasma concentration ratio Mean ratiot No. SE CV (%):j: Area ratio§ No. SE CV (%)
P. B.
J. L.
B. G.
M.C.
E.M.
Slow
Slow
Slow
Rapid
Rapid
5.08 1.40
7.56 3.36
2.52 0.46
6.28 0.79
4.06 0.54
10 16
120.8 40.09 0.326 8 0.Dl5 13 0.332
24 24 385.0 146.2 0.422 8 0.020 14 0.380
16 16 119.2 16.55 0.188 8 0.Dl8 27 0.139
24 24 147.8 22.12 0.175 6 0.Dl8 25 0.150
Mean
24 16 123.7 14.32 0.120 0.246 5 8 0.Dl5 0.056 50 35 0.116 0.223 5 0.055 55
"Expressed in terms of SP.
t Average of all the ratios obtained over the entire experimental period .
.t Coefficient of variation. §Obtained by dividing the total area under the salivary concentration vs time curve by the total area under the plasma concentration vs time curve.
Salivary excretion of sulfapyridine
Volume 22 Number 6
925
Table VI. Partition coefficients (chloroform/aqueous buffer) and dissociation constants for sulfapyridine (SP) and N 4-acetylsulfapyridine (AcSP)
Parameter Partition coefficient at 37° (chloroform/ aqueous buffer*) Observed (Co) Intrinsic (Ca)t Apparent dissociation constant (Ka>t pKa Percent unionized in plasma (pH 7.4)
SP
AcSP
0.879 (pH 7.4) 0.999 (pH 6.4) 1.02 6.5 x 10-9 8.2+
0.202 (pH 7.4) 0.293 (pH 6.5) 0.313 2.2 x 10-8 7.7
86
67
*Isotonic phosphate buffer, United States Pharmacopeia XIX. tFrom observed partition coefficient (Col data at two different pH values and solution of the following two simultaneous equations for the partition coefficient (intrinsic) of the unionized moiety (Cal and the dissociation constant (Ka)at pHI: CO • I = Ca[H+h/Ka tReported pKa value for SP at 25° is
+ [H+]I;
mately 44% of the average ratio for SP (0.559 ± 0.027) and showed considerably more intrasubject and intersubject variation (Table IV and V). A review of the literature suggests that for most drugs (including several sulfonamides), 11, 16, 18, 23 only the non-protein-bound, nonionized fraction of a drug in plasma can readily diffuse across the epithelium of the salivary gland. For a weakly acidic drug possessing excellent lipid solubility characteristics, the saliva to total plasma (protein-bound and unbound) concentration ratio is given by Equation 1:11 R
' =
I I
+ +
at pH,: Co.,
= Ca[H+],/Ka + [H+],.
8.43. 19
10,pHs-pKa) lO'pHp pKa) •
fp
(1)
where pHs and pHp are the pH of the saliva and plasma, respectively, and fp is the fraction of unbound drug in the plasma. The pKa values for SP and AcSP were estimated from chloroform/aqueous buffer partition coefficient data at 37° C and were 8.2 and 7.7, respectively (Table VI). The former value is in good agreement with the literature value of 8.43 determined at 25° C.19 As demonstrated for SP (Table VI) and for other sulfonamides by Despopoulos and Callahan , 9 N4- acetylation apparently increases the acidity of most sulfonamides. Equation 1 does not account for the degree of lipid solubility of the drug, which has been shown for several sulfonamides 16 , 18,23 and barbiturates 23 to strongly affect drug diffusion
across the salivary epithelium. In contrast to AcSP, the diffusibility of SP (product of the percentage of SP nonionized in the plasma and its lipid solubility, as reflected by the intrinsic chloroform/water partition coefficient data presented in Table VI)23 appears to be high. This observation may explain, in part, why the saliva: total plasma concentration ratio for SP (0.559; Table IV) is on the average greater and less variable than that found experimentally for AcSP (0.246; Table V). Differences in the degree of binding of SP and AcSP to plasma protein could also account for the observed differences in the salivary excretion of these two species, a possibility which remains to be explored. The results of our study show that there are appreciable differences between rapid and slow acetylators with respect to the kinetics of SP elimination. This observation suggests the need to individualize SSZ dosage regimens based on the biologic tlh or the total body clearance of SP. There was excellent direct relationship between SP concentrations in the saliva and total SP plasma concentrations (free and proteinbound). The saliva: plasma concentration ratio for SP was apparently independent of total plasma concentration of SP, saliva pH, and acetylator phenotype and exhibited little intersubject variation. It is now necessary to determine whether similar results can be realized in adult and pediatric patients treated with SSZ. If such is the case, then salivary SP concentration
926
Bates, Blumenthal, and Pieniaszek
measurements may offer a convenient, noninvasive method for monitoring the clinical and toxicologic status of these patients. The utility of salivary AcSP concentration measurements for phenotyping purposes remains to be determined. References 1. Bates, T. R., Blumenthal, H. P., and Pieniaszek, H. J., Jr.: Time course of free and N4_ acetylated sulfapyridine concentrations in the plasma and saliva of man after sulfasalazine (salicylazosulfapyridine) administration: Preliminary findings, Res. Commun. Chern. Pathol. Pharmacol. 15:183-188, 1976. 2. Bates, T. R., and Pieniaszek, H. J., Jr.: A modified colorimetric method for the determination of sulfapyridine and its metabolites in plasma and saliva after sulfasalazine administration, Clin. Chern. (In press.) 3. Das, K. M., and Eastwood, M. A.: Acetylation polymorphism of sulfapyridine in patients with ulcerative colitis and Crohn's disease, CUN. PHARMACOL. THER. 18:514-520, 1975. 4. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: Adverse reactions during salicylazosulfapyridine therapy and the relation with drug metabolism and acetylator phenotype, N. Eng!. 1. Med. 298:491-495, 1973. 5. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: The metabolism of salicylazosulphapyridine in ulcerative colitis. I. The relationship between metabolites and the response to treatment in in-patients. II. The relationship between metabolites and the progress of the disease studied in out-patients, Gut 14:631-641, 1973. 6. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: The role of the colon in the metabolism of salicylazosulfapyridine, Scand. 1. Gastroenterol. 9:137-141, 1974. 7. Das, K. M., Eastwood, M. A., McManus, J. P. A., and Sircus, W.: Salazopyrin metabolism in ulcerative colitis, Gut 13:840, 1972. 8. Das, K. M., and Rubin, R.: Clinical pharmacokinetics of sulphasalazine, Clin. Pharmacokinet. 1:406-425, 1976. 9. Despopoulos, A., and Callahan, P. X.: Molecular features of sulfonamide transport in renal excretory processes, Am. J. Physiol. 203:19-26, 1962. 10. Dissanayake, A. S., and Truelove, S. C.: A controlled therapeutic trial of long-term maintenance treatment of ulcerative colitis with sulphasalazine (salazopyrin), Gut 14:923-926, 1973. II. Dvorchik, B. H., and Vesell, E. S.: Pharmaco-
Clinical Pharmacology and Therapeutics
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23. 24. 25.
kinetic interpretation of data gathered during therapeutic drug monitoring, Clin. Chern. 22:868-878, 1976. Frisk, A. R.: Blood concentration, acetylation and urinary excretion of sulfapyridine and sulfathiazole after various sulfapyridine and sulfathiazole derivatives administered by different routes, Acta Med. Scand. 106:369-403, 1941. Gibaldi, M.: Introduction to biopharmaceutics, Philadelphia, 1971, Lea & Febiger, Publishers, pp. 8-30. Gibaldi, M., and Perrier, D.: Pharmacokinetics, New York, 1975, Marcel Dekker, Inc., pp. 1-43. Goldman, P., and Peppercorn, M. A.: Sulfasalazine, N. Eng I. J. Med. 293:20-23, 1975. Griineisen, A., and Witzgall, H.: Salivary excretion of sulfonamides. Correlation of drug levels in saliva and ultrafiltrate of plasma, Eur. J. Clin. Pharmacol. 7:77-79, 1974. Hansson, K. A., and Sandberg, M.: Determination of sulfapyridine and its metabolites in biological materials after administration of salicylazosulfapyridine, Acta Pharm. Suec. 10:87-92, 1973. Killmann, S. A., and Thaysen, J. H.: The permeability of the human parotoid gland to a series of sulfonamide compounds, paraaminohippurate and inulin, Scand. J. Clin. Lab. Invest. 7:86-91, 1955. Koizumi, T., Arita, T., and Kakemi, K.: Absorption and excretion of drugs. XIX. Some pharmacokinetic aspects of absorption and excretion of sulfonamides (I). Absorption from rat stomach, Chern. Pharm. Bull. 12:413-420, 1964. Notari, R. E.: Biopharmaceutics and pharmacokinetics, New York, 1971, Marcel Dekker, Inc., pp. 55-74. Nygard, B., Olofsson, J., and Sandberg, M.: Some physico-chemical properties of salicylazosulphapyridine, including its solubility, protolytic constants, and general spectrochemical and polarographic behavior, Acta Pharm. Suec. 3:313-342, 1966. Peppercorn, M. A., and Goldman, P.: The role of intestinal bacteria in the metabolism of salicylazosulfapyridine, J. Pharmacol. Exp. Ther. 181:555-562, 1972. Rasmussen, F.: Salivary excretion of sulfonamides and barbiturates by cows and goats, Acta Pharmacol. Toxicol. 21: 11-19, 1964. Sandberg, M., and Hansson, K.-A.: Determination of salicylazosulfapyridine in biological materials, Acta Pharm. Suec. 10: 107-110, 1973. Schroder, H., and Campbell, D. E. S.: Absorption, metabolism, and excretion of salicylazosulfapyridine in man, CUN. PHARMACOL. THER. 13:539-551, 1972.
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26. Schader, H., and Evans, D. A. P.: Acetylator phenotype and adverse effects of sulphasalazine in healthy subjects, Gut 13:278-284, 1972. 27. Schroder, H., and Evans, D. A. P.: The polymorphic acetylation of sulphapyridine in man, J. Med. Genet. 9:168-171, 1972. 28. Schroder, H., Lewkonia, R. M., and Evans, D. A. P.: Metabolism of salicylazosulfapyridine in healthy subjects and in patients with ulcerative colitis, CUN. PHARMACOL. THER. 14:802-809, 1973.
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29. Smith, G. H., and Tong, T. G.: Ulcerative colitis, J. Am. Pharm. Assoc. NSI5:202-212, 1975. 30. Van Oudtshoorn, M. C. B., and Potgieter, F. J.: Determination of pharmacokinetic parameters for rapid and slow acetylators of sulphadimidine, J. Pharm. Pharmacol. 24:357360, 1972. 31. Wagner, J. G.: Fundamentals of clinical pharmacokinetics, Hamilton, Ill., 1975, Drug Intelligence Publications, pp. 57-82.
