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Liquid Chromatographic-Ultraviolet Methods for Furegrelate in Serum and Urine: Preliminary Pharmacokinetic Evaluation in the Dog
Liquid Chromatographic-Ultraviolet Methods for Furegrelate in Serum and Urine: Preliminary Pharmacokinetic Evaluation in the Dog DUANEB. LAKINGS' AND JANICEM. FRIIS Received May 23, 1984, from Drug Metabolism Research, The Upjohn Company, Kalamazoo, MI 49001. 1984. Analytical methods have been developed for the quantitative determination of furegrelate (l),a thromboxane synthetase inhibitor, in dog serum and urine specimens. The methods use ion-pairing reversedphase high-performance liquid chromatography(HPLC)with UV detection at 268 nm and have the necessary specificity, linearity, and sensitivity to quantitate 100 ng/mL of the drug in serum and 20 pg/mL in urine. Preliminary pharmacokinetic and bioavailability evaluations in the dog indicate that 1 was rapidly distributed and had a terminal half-life of 132 min after an intravenous dose. The primary route of elimination was renal with -70% of the parent drug excreted in urine. Fasted dogs dosed orally with the drug in solution or capsule had similar absorption and elimination kinetics and agreed favorably with the intravenous results. The bioavailability of 1 dosed orally was 77-80% based on serum area under curve and >90% based on urinary excretion of the parent compound. The serum kinetics, but not the urinary elimination,of 1 appeared to be altered in fed dogs given drug in capsule. Abstract
Furegrelate, 5-(3'-pyridinylmethyl)benzofuran-2-carboxylic acid ( l ) ,a thromboxane synthetase inhibitor with reported in vivo activity,' inhibits the biosynthesis of thromboxane A', a naturally occurring lipid formed by metabolism of arachidonic acid.' Thromboxane A, stimulates platelet aggregation and has vasoconstrictive effect^.^-^ Thus, furegrelate has potential in the treatment of various cardiovascular disorders. Studies to define the distribution, elimination, and metabolism of drugs in animals and humans require specific, sensitive, and reproducible analytical methods. In an earlier report,6 a high-performance liquid chromatographic (HPLC) procedure was defined for the determination of 1 in serum. This method had reported sensitivity of 0.5 pg/mL for serum, and the calculated pharmacokinetic values from dogs dosed with l varied substantially depending on route of administration and dose. The report demonstrated that thromboxane A' levels were reduced in the dog after dosing with 1 by intravenous bolus or infusion and orally. This report describes an HPLC method for the quantitative determination of 1 in serum and urine. The methods have the necessary specificity and sensitivity to quantitate 100 ng/mL of 1 in serum and 20 pg/mL of 1 in urine. The analytical techniques were used to evaluate the pharmacokinetics and bioavailability of 1 in the dog after intravenous and oral doses.
Experimental Section Apparatus-The HPLC system employed consisted of a ConstaMetric I11 pump, a SpectroMonitor I11 variable-wavelength detector (Laboratory Data Control, Riviera Beach, FL), and a Rheodyne 7126 loop injector mounted on an Upjohn autoinjector. A Supelcosil LC-18, 5-pm particle size, 250 X 4.6mm i.d. (Supelco, Inc., Bellefonte, PA) column and a Whatman Co:Pell ODS, 35-jim particle size, 50 X 2.1-mm i.d. guard column were used. Sample preparation for serum employed CL8 Sep-Paks 0022-3549/85/0400-0455$0 1.OO/O 0 1985, American PharmaceuticalAssociation
Accepted for publication November 20,
(Waters Associates, Milford, MA). Physiological fluid samples were filtered through Gelman Acrodisc-CR 0.45-pm filters prior to injection onto the HPLC system. Reagents-Reference standards of 1 and the serum internal standard 2 were obtained from The Upjohn Co., Pharmaceutical Research and Development. Propiophenone (3;Aldrich Chemical Co., Milwaukee, WI) was used as the urine internal standard. Acetonitrile was distilled-in-glass purity (Burdick and Jackson Laboratories, Muskegon, MI), and the tetrabutylammonium hydroxide (TBA) was 0.4 M titrant grade (Eastman Kodak Co., Rochester, NY). All other reagents and chemicals were of the highest quality available. Chromatographic Conditions-The HPLC conditions defined to give separation of 1 and 2 for serum analyses utilized an eluant of 30:70 (v/v) acetonitri1e:water containing 0.008 M TBA and adjusted to pH 6.0 with concentrated acetic acid. The flow rate was 1.0 mL/min, the UV detector was at 268 nm, and the injection volume was 50 jiL. For urine samples, analyses of 1 and 3 were performed using a n eluant of 32:68 (v/v) acetonitri1e:water containing 0.008 M TBA, p H 6.0.
1
N'
2
Prior to using the TBA, the reagent was filtered three times through a 0.2-pm Nylon-66 filter (Rainin Instrument Co., Woburn, MA) to remove particulate matter. After preparation, the HPLC eluant was deaerated for -45 min with a helium flow of 10-15 mL/min. The prepared, deaerated eluant was stable for 5 d. Stock Solution Preparation-Stock solutions of 1 and 2 were prepared by accurately weighing 10.0 mg each and dissolving with water; the final concentration of 1 was 100 pg/mL and that of 2 was 5 pg/mL. Stock solutions of 3 were prepared by accurately weighing 100 mg and dissolving with HPLC eluant; the final concentration of 3 was 25 pg/mL. Aliquots of these stock solutions were used to prepare reference standard solutions and to fortify serum and urine samples at various levels of 1. Journal of Pharmaceutical Sciences 1 455 Vol. 74, No. 4, April 1985
Sample P r e p a r a t i o n Procedures-The procedure for the determination of 1 in serum was defined to provide analytical sensitivity of 100 ng/mL. The Cla Sep-Pak was first pretreated with 3 mL of acetonitrile and then with 5 mL of 0.008 M TBA in water. A 0.1-1.0-mL aliquot of serum, depending on the expected level of 1, was quantitatively transferred onto the Sep-Pak, and 1.0 mL of the 5.0-pg/mL stock solution of 2 was added. The sample was allowed to adsorb onto the column using slightly reduced pressure to give a flow of 1-2 mL/min. The column was washed with 5 mL of water and then 2 mL of 10:90 (v/v) acetonitri1e:water a t a flow rate of 1-2 mL/min. The column was eluted, by gravity, with 2 mL of 30:70 (v/v) acetonitrile:water. For urine samples, the procedure consisted of quantitatively transferring a 50-pL aliquot of urine into 2.0 mL of HPLC eluant containing 25 pg/mL of 3 (stock solution of 3). The sample was mixed and filtered into an autoinjector vial. The serum and urine procedures were validated for linearity, precision, and accuracy by preparing and analyzing series of fortified samples on four separate days. For serum, 10 concentrations from 50 to 100 ng/mL were employed. For urine, the 10 concentrations ranged from 200 to 20 pg/mL. Samples a n d S a m p l e Handling-Serum and urinary excretion levels of 1 were determined in four beagle dogs, two males and two females. Each dog received the following four doses of 1: (A) 10 mg/kg as a 50-mg/mL sterile saline solution injected intravenously into the cephalic vein; (B) 10 mg/kg as a 50-mg/mL saline solution administered by oral intubation to a fasted animal; (C) 10 mg/kg of 1 in a no. 13 gelatin capsule given orally to a fasted dog; (D) same as treatment C given to a fed animal. The dogs weighed -10 kg each, determined the day before dosing, resulting in an approximate total dose of 100 mg of 1 . The fed dog received food 1 h prior to dosing, and the fasted dogs received no food 16 h prior to dosing. Blood samples were obtained at the following intervals: (treatment A) 0 (predose), 5 , 10, 20, 30, 45 min, 1, 1.5, 2, 3, 4, 6,8, 12, and 24 h; (treatments B, C, and D) 0 (predose), 15, 30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12, and 24 h. Serum was separated from the blood as quickly as possible and was frozen at -30°C until analysis. Urine specimens for each treatment were obtained at 0 (predose), 0-2, 2-4, 4-6, 6-8, 8-12, 12-24, and 24-48 h. The early urine collection intervals were obtained by catheterization, and the later collections from the metabolism cages. The urine specimens were mixed thoroughly, the volume was measured, and an aliquot was frozen and maintained a t -30°C until analysis. Calculations-The validation of the analytical methods and the determination of 1 in serum and urine samples were accomplished using the relative weight response (RWR) to an internal standard calculation method. A concentration series of 1over the level expected in samples (0.2-50 pg/mL for serum and 20-1000 pg/mL for urine) was prepared with each sample set, and the average RWR of the reference solutions (eq. 1)was employed to calculate the level of 1 in the sample aliquot (eq. 2):
RWR=pg l/aliquot =
PH 1 PH IS
X
pg IS/solution pg l/solution
PH 1 sample PH IS
X
pg IS/sample average RWR
(1) (2)
where P H 1 and PH IS are the peak heights of 1 and either 2 or 3 (depending on the sample matrix), and pg 1 and pg IS are the micrograms of 1 and 2 or 3 in the solution or sample aliquot. The pg l/aliquot was converted to pg/mL for serum and mg/collection for urine using the appropriate dilution factors and urine volumes. During validation and sample analysis, a concentration series 456
1 Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
of 1in the physiological fluid (0.1-50 pg/mL for serum and 201000 pg/mL for urine) was prepared and analyzed to determine the accuracy of the analytical procedure. The recovery of 1 in the fortified serum and urine samples was calculated as follows:
%R =
pg/aliquot (fortified) - pg/aliquot (unfortified) pg added
x 100 (3) For both serum and urine, the recovery of 1 was quantitative over the fortified concentration range. Thus, no correction for loss of 1during sample preparation was necessary. The stability of 1 in serum or urine was determined for control samples fortified with a known level of 1 ( 5 and 25 pg for 1.0 mL of serum or 50 pL of urine) and stored at -30°C. P h a r m a c o k i n e t i c Evaluation-The serum and urinary excretion levels of 1 in the dog were used to determine pharmacokinetic parameter estimates by graphic and computer techniques7 The computer program, NONLIN, was for a twocompartment model with either bolus (intravenous) or firstorder (oral doses) input. The initial estimates for the firstorder two-compartment model were obtained from the estimates of the intravenous dose. For the dogs receiving treatment A (intravenous dose), the parameters calculated included the area under the serum curve (AUC) from 0 to infinity, the mean residence time (MRT), the apparent volume of distribution ( Vd, area), the distribution rate constant (a), the elimination rate constant ( p ) , the cumulative amount of 1 excreted in the urine (UT), the percent of dose excreted (%Dose), the elimination rate constant (k,) from urine data, and the total body (CLT) and renal (CLR)clearances. For the dogs receiving oral doses of 1, the parameters determined were AUC, MRT, a, p, the absorption rate constant ( k a ) ,the highest concentration (C,,,), the time (tmaX)required to reach C,,,, UT, %Dose, and k, .
Results and Discussion Chromatographic Conditions Evaluation-The HPLC conditions previously describedfi for the determination of 1 in serum had employed a 10-pm reversed-phase column with an eluant of 20:80 (v/v) acetonitrile:0.005 M tetrabutylammonium phosphate, pH 7. A sharper, better-defined chromatographic peak for 1 was obtained on a 5-pm Supelcosil LC-18 column, and studies to optimize the organic modifier level, pH, and TBA level were conducted. The results are shown in Fig. 1 and indicate that the retention of 1 is sensitive to slight changes in the acetonitrile level but not sensitive to pH changes above pH 5.5 or to TBA concentrations above 0.006 M. Validation of S e r u m and U r i n e S a m p l e P r e p a r a t i o n Procedures-The results for the validated serum sample preparation procedure are summarized in Table I and show linearity over the entire range with a least-squares linear regression equation of y = (1.016 k 0 . 0 0 5 ) ~+ (0.063 0.090) and a correlation coefficient of 0.9995. The precision of the procedure is shown by the RSD of the four independent samples at each fortification level and was excellent, with the highest RSD value being 6.0%. The minimum quantifiable level, i.e., the lowest fortified serum level with a n RSD of
*
4
5
6
7
8
I
I
the detection limit was 2 pg/mL. Representative HPLC-UV (268 nm) chromatograms of a blank dog urine and a 0-2-h urine collection after a 10-mg/kg oral solution dose are shown in Fig. 3. The stability of 1 in serum and urine stored frozen with samples collected from dogs was good with no detectable change in the concentration of 1 after 30 d at -30°C. Pharmacokinetics in the Dog-The serum and urine levels of 1 in the dog were used to determined the distribution and elimination kinetics after intravenous and oral administration of 10 mg/kg of 1. Table I1 summarized the pharmacokinetic parameters calculated for each dog for each dose. The mean and SD for each parameter for the four dogs are also given in the table. The log serum levels versus time from one dog receiving the four doses are shown in Fig. 4. The cumulative urinary excretion levels versus time for this dog are presented in Fig. 5. The pharmacokinetic parameters determined for the intravenously dosed dogs were similar, as shown by the SD value. The data fit the NONLIN model well with all the correlations being >0.99. The distribution phase of 1 after the intravenous dose was rapid (average half-life of 46.2 min) and the Vd, area
9
PH I
I
24
20 I
0
I
I
I
28 32 36 Acetonitrile Level, Yo
I
I
0.002
(
I
J
I
0.004 0.006 TEA Level, M
40
I
'
0.008
I
0.01
1 2 I
Figure 1-Optimization of HPLC eluant composition for furegrelate. Key: (A) pH; (B) acetonifrile; (C) TBA. Table I-Linearity, Fluids
Precision, and Accuracy of I in Physiological
Fortified Serum Samplesa
Fortified Urine Samplesb
Amount Amount Average Amount Amount RSD, Average Added, Found, RSD, Recovery, Added, Found, Recovery, o/o ~~9150~ l 5 0 P/o YO &/mL PrglmL UL rUL -
50.0 25.0 10.0 7.00 4.00 2.00 1.00 0.50 0.20 0.10 0
50.9 25.2 10.7 7.17 4.13 2.07 1.03 0.51 0.22 0.11 0.01
3.0 1.4 6.0 2.7 3.9 2.7 2.2 3.4 4.4 5.2 -
101.8 100.7 107.2 102.2 103.1 103.0 102.2 101.0 103.8 96.7 -
100.0 80.0 60.0 40.0 30.0 20.0 15.0 10.00 8.00 6.00 4.00 2.00 1.00 0
96.1 77.8 59.5 39.8 30.2 20.4 15.4 10.22 8.26 6.13 4.19 2.12 1.14 0
0.5 0.7 0.4 0.5 0.4 0.3 1.2 2.5 2.1 3.9 2.2 3.1 4.4 -
96.1 97.2 99.2 99.4 100.7 102.2 102.7 102.2 103.2 102.2 104.8 106.2 114.5 -
a Linear regression: y = (1.016 f 0.005)~+ (0.063 ? 0.090); r = 0.9995, n = 4. *Linear regression: y = (0.966 f 0.003)~+ (0.580 -t 0.104); r = 0.9998, n = 4.
the urine with HPLC eluant gave an HPLC chromatogram with minimal interference at the elution of 1. However, a urinary component coeluted with 2 , and thus, this compound was not satisfactory as an internal standard for urinary analysis. Since no sample preparation was required other than simple dilution, an internal standard which was not structurally similar to 1 could be used. Propiophenone (3)was selected as the urine analysis internal standard, and the acetonitrile content of the eluant was increased from 30 to 32% to shorten the chromatography time. Validation of the urine assay is presented in Table I. The least-squares linear regression equation was y = (0.966 k 0 . 0 0 3 ) ~ + (0.58 f 0.10) with a correlation coefficient of 0.9998. The precision was excellent, with the highest RSD being 4.4%,and the accuracy was quantitative, as shown by the average %R. The minimum quantifiable level for urine was 20 pg/mL, and
L 0
8
1 6 0
mL
Predcse Serum
8
16
30 - Min Serum
Figure 2-Represenfafive HPLC-UV (268 nm) chromafograms of blank dog serum and a serum sample obtained 30-min after a 10-mg/kg oral solution dose of furegrelate (7); 2 is the internal standard.
I
3
! I 1
l9d.L 0 8 16 mL 0 8 Predose Urine C-2-h
i
16
Urine
Figure 3-Representative HPLC-UV (268 nm) chromatograms of blank dog urine and a 0-2-h urine collection from a dog receiving a 10-mglkg oral solution dose of furegrelate (7); 3 is the internal standard.
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
I
457
Table Il-Pharmacokinetic
Parameter Estimates in Dogs Treatment A, IntravenousInjection
AUC,
09
Dog
Vd, L
min-'
min-'
Corr?
1 2 3 4 x +SD
3.4 2.5 3.6 3.2 3.2 0.5
0.012 0.018 0.016 0.016 0.015 0.003
0.0053 0.0047 0.0050 0.0063 0.0053 0.0007
0.992 0.992 0.998 0.997
ff,
Percent
MRT, min
CLT, mL/min
Dose Of
CIR, mL/min
ke, min-1
rZb
117 174 104 84 120 39
17.8 11.5 18.2 20.1 16.9 3.7
87.5 65.3 64.5 62.4 69.9 11.8
15.6 7.5 11.4 13.0 11.9 3.4
0.0061 0.0028 0.0058 0.0033 0.0045 0.0017
0.98 0.99 0.94 0.84
mL 6150 8546 5197 4495 6097 1768
Treatment B, Oral Solution
1 2 3 4c
0.018 0.017 0.018 0.017 0.018 0.001
0.018 0.016 0.018 0.135 0.017 0.001
x
+SD
0.960 0.952 0.947 1.000
0.0042 0.0025 0.0058 0.0060 0.0042 0.0017
170 196 211 125 192 21
4042 6879 4834 976 5252 1464
65.7 80.5 93.0
-
79.7 13.7
20.6 35.5 21.3 9.0 25.8 8.4
90 90 120 29 100 17
75.5 74.6 57.6 24.1 69.2 10.1
86.3 114.2 89.3 96.6 15.3
0.0031 0.0035 0.0080 0.0069 0.0049 0.0027
0.97 0.95 0.94 0.99
Percent
Bioavail. Urine
k,, min-1
r26
Of
71.6 52.9 61.1 64.7 62.6 7.8
81.8 81.0 94.7 103.7 90.3 10.9
0.0033 0.0031 0.0028 0.0048 0.0035 0.0009
0.94 0.94 0.94 0.98
k,. min-'
r2b
Urine
71.5 92.0 95.8 110.7 92.5 16.2
0.0138 0.0068 0.0021 0.0031 0.0065 0.0053
0.84 0.94 0.86 0.99
Treatment C, Drug in Capsule (Fasted Dog) ka
Dog
MRT,
a,
7
min-'
min-'
min-1
0.041 0.020 0.012 0.018 0.023 0.013
0.013 0.020 0.012 0.018 0.016 0.004
0.0040 0.0032 0.0020 0.0041 0.0033 0.0009
~-
1 2 3 4
x S D
Bioavail.
,C ,,,,
t-,
mL 0.968 0.981 0.968 0.998
Dose
4181 5927 3884 4240 4559 925
191 198 250 161 200 37
68.0 69.4 74.7 94.3 76.6 12.1
22.3 32.1 15.4 23.1 23.2 6.9
120 120 152 90 121 25
Treatment D, Drug in Capsule (Fed Dog) ks.
ff,
Dog
min-1
min-1
1 2 3 4
0.064 0.026 0.038 0.023 0.038 0.019
0.010 0.027 0.016 0.022 0.019 0.007
x +SD
mf;-l
Corr."
0.0003 0.0052 0.0012 0.0002 0.0017 0.0023
0.999 0.999 0.999 0.980
AUC' pg.mn/ mL
MRT, min
Bioavail. Serum
, ,C , pg/mL
2869 3119 3434 5483 3726 1194
1509 150 491 3563 1428 1536
46.6 36.5 66.1 122.0 67.8 38.2
11.9 20.8 18.8 4.7 14.1 7.3
t,,, min 60 63 34 61 55 14
Peyt Bioavail. Dose 62.6 60.1 61.8 69.1 63.4 3.9
Corr.-correlation from NONLIN evaluation of serum data. r2-squared linear regression coefficient for log (U, of administered dose; data not included in calculation of average values. a
2.0
?
1.5 -
5
1.0-
8G
0.5-
-I
E
0-
9 -0.5 -
-0.1
'
I
I
I
I
I
~
- U ) versus time.
Partial loss
of 3.2 L is greater than the plasma volume in the dog (-400 mL/10 kg), but less than the total body water (-4600 mL/10 kg).' The elimination of 1 from serum had an average half-life of 130 min, which is less than the 11.9 h half-life reported earlier for 1.6Evaluation of the figures in that report indicated that the calculated fi and half-life did not correspond to the last three data points, which have an estimated elimination half-life of 2-3 h. The AUC and CLT averages, when converted to similar units, are similar to the values reported in the earlier paper. About 70% of the administered dose was excreted unchanged in the urine. The log ( UT - U ) versus time evaluation of 1 showed a first-order elimination and a one-compartment model [regression coefficient (rZ)> 0.801. The he values from the urine data agreed with the serum /3 for individual dogs; thus, the elimination kinetics of 1 could be determined from both serum and urine results. The pharmacokinetic evaluation of the serum and urine results from dogs dosed with 1 as an oral solution or drug in a capsule to a fasted animal were similar. The average of various rate constants, ( k a , a , /3, and k,) and the other parameters (AUC,MRT, ,,C,, t,,,, %Dose, and bioavailability in serum and urine) were statistically equal (p > 0.2). The bioavailability of 1 for the oral solution and drug in capsule averaged 80 and
I
I
I
I
960
480
0
I
I
I
1
1920
1440
1
1
2400
1
2880
Minutes Figure 5-Cumulative urinary excretion versus time profiles of furegrelate in a dog receiving 10-mglkg doses of 1 given intravenously (A), as an oral solution (B), and in a capsule to a fasted (C) and a fed animal (0). 1.5r
References and Notes 1. Gorman, R. R.; Johnson, R. A.; Spilman, C. H.; Aiken, J. W. Prostaglandins 1983,26, 325-342. 2. Hamberg, M.; Svensson, J.; Samuelsson, B. Proc. Natl. Acad. Sci. USA 1974, 72, 2994-2998. 3. Ellis, E.; Oelz, 0.; Roberts, L. J., 11; Payne, N. A.; Sweetman, B.; Nies, A.; Oates, J. Science 1976,193, 1135-1137. 4. Needlemen, P.; Minkes, M.; Raz, A. Science 1976, 193, 163-165. 5. Needlemen, P.; Kulkarni, P.; Raz, A. Science 1977,195, 409-412. 6. Wynalda, M. A.; Liggett, W. F.; Fitzpatrick, F. A. Prostaglandins 1983,26, 311-324. 7. Metzler, C. M.; Elfring, G. L.; McEwen, A. J. Biometrics 1974,30, 562. 8. Gerlowski, L. E.; Jain, R. K. J . Pharm. Sci. 1983, 72, 1103-1127.
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9- 0.5ci
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0-
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8 -0.5-
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77% for serum data and 97 and 90% for urine results, respectively, when compared to the intravenous values. Fed dogs dosed with drug in capsule did not have uniform serum kinetics, as shown in Fig. 6. Two dogs had a prolonged elimination phase which altered the various pharmacokinetic parameters. The fed dogs, in general, had a faster absorption, , and t,,,, a lower AUC, and an slower elimination, lower,,C increase in MRT when compared with the other oral doses. These serum result observations indicate that the distribution and elimination are altered in the presence of food and may result in drug accumulation with multiple dosing and may prevent a therapeutic concentration in the blood. The urinary excretion results also had some, but not as pronounced as the serum data, variation when compared with the other doses. The absolute bioavailability of 1 in fed dogs averaged 68% for serum and 92% for urine results. Additional studies on the effect of food on the bioavailability and pharmacokinetics of 1 in the dog are planned to assist in the design of human studies.
'
I
I
I
I
I
I
360 480 600 720 Minutes Figure 6-Log serum concentration versus time profiles of furegrelate in dogs receiving 10-mglkg doses in capsules to fed animals. 0
120
240
Acknowledgments The authors thank Chris Asmus and Jay Hansen for their assistance in the collection of samples, Dennis Weber for his helpful suggestions for the pharmacokinetic evaluations, and Marlene Dopheide for typing and editing the manuscript.
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
1 459
Furegrelate, 5-(3'-pyridinylmethyl)benzofuran-2-carboxylic acid ( l ) ,a thromboxane synthetase inhibitor with reported in vivo activity,' inhibits the biosynthesis of thromboxane A', a naturally occurring lipid formed by metabolism of arachidonic acid.' Thromboxane A, stimulates platelet aggregation and has vasoconstrictive effect^.^-^ Thus, furegrelate has potential in the treatment of various cardiovascular disorders. Studies to define the distribution, elimination, and metabolism of drugs in animals and humans require specific, sensitive, and reproducible analytical methods. In an earlier report,6 a high-performance liquid chromatographic (HPLC) procedure was defined for the determination of 1 in serum. This method had reported sensitivity of 0.5 pg/mL for serum, and the calculated pharmacokinetic values from dogs dosed with l varied substantially depending on route of administration and dose. The report demonstrated that thromboxane A' levels were reduced in the dog after dosing with 1 by intravenous bolus or infusion and orally. This report describes an HPLC method for the quantitative determination of 1 in serum and urine. The methods have the necessary specificity and sensitivity to quantitate 100 ng/mL of 1 in serum and 20 pg/mL of 1 in urine. The analytical techniques were used to evaluate the pharmacokinetics and bioavailability of 1 in the dog after intravenous and oral doses.
Experimental Section Apparatus-The HPLC system employed consisted of a ConstaMetric I11 pump, a SpectroMonitor I11 variable-wavelength detector (Laboratory Data Control, Riviera Beach, FL), and a Rheodyne 7126 loop injector mounted on an Upjohn autoinjector. A Supelcosil LC-18, 5-pm particle size, 250 X 4.6mm i.d. (Supelco, Inc., Bellefonte, PA) column and a Whatman Co:Pell ODS, 35-jim particle size, 50 X 2.1-mm i.d. guard column were used. Sample preparation for serum employed CL8 Sep-Paks 0022-3549/85/0400-0455$0 1.OO/O 0 1985, American PharmaceuticalAssociation
Accepted for publication November 20,
(Waters Associates, Milford, MA). Physiological fluid samples were filtered through Gelman Acrodisc-CR 0.45-pm filters prior to injection onto the HPLC system. Reagents-Reference standards of 1 and the serum internal standard 2 were obtained from The Upjohn Co., Pharmaceutical Research and Development. Propiophenone (3;Aldrich Chemical Co., Milwaukee, WI) was used as the urine internal standard. Acetonitrile was distilled-in-glass purity (Burdick and Jackson Laboratories, Muskegon, MI), and the tetrabutylammonium hydroxide (TBA) was 0.4 M titrant grade (Eastman Kodak Co., Rochester, NY). All other reagents and chemicals were of the highest quality available. Chromatographic Conditions-The HPLC conditions defined to give separation of 1 and 2 for serum analyses utilized an eluant of 30:70 (v/v) acetonitri1e:water containing 0.008 M TBA and adjusted to pH 6.0 with concentrated acetic acid. The flow rate was 1.0 mL/min, the UV detector was at 268 nm, and the injection volume was 50 jiL. For urine samples, analyses of 1 and 3 were performed using a n eluant of 32:68 (v/v) acetonitri1e:water containing 0.008 M TBA, p H 6.0.
1
N'
2
Prior to using the TBA, the reagent was filtered three times through a 0.2-pm Nylon-66 filter (Rainin Instrument Co., Woburn, MA) to remove particulate matter. After preparation, the HPLC eluant was deaerated for -45 min with a helium flow of 10-15 mL/min. The prepared, deaerated eluant was stable for 5 d. Stock Solution Preparation-Stock solutions of 1 and 2 were prepared by accurately weighing 10.0 mg each and dissolving with water; the final concentration of 1 was 100 pg/mL and that of 2 was 5 pg/mL. Stock solutions of 3 were prepared by accurately weighing 100 mg and dissolving with HPLC eluant; the final concentration of 3 was 25 pg/mL. Aliquots of these stock solutions were used to prepare reference standard solutions and to fortify serum and urine samples at various levels of 1. Journal of Pharmaceutical Sciences 1 455 Vol. 74, No. 4, April 1985
Sample P r e p a r a t i o n Procedures-The procedure for the determination of 1 in serum was defined to provide analytical sensitivity of 100 ng/mL. The Cla Sep-Pak was first pretreated with 3 mL of acetonitrile and then with 5 mL of 0.008 M TBA in water. A 0.1-1.0-mL aliquot of serum, depending on the expected level of 1, was quantitatively transferred onto the Sep-Pak, and 1.0 mL of the 5.0-pg/mL stock solution of 2 was added. The sample was allowed to adsorb onto the column using slightly reduced pressure to give a flow of 1-2 mL/min. The column was washed with 5 mL of water and then 2 mL of 10:90 (v/v) acetonitri1e:water a t a flow rate of 1-2 mL/min. The column was eluted, by gravity, with 2 mL of 30:70 (v/v) acetonitrile:water. For urine samples, the procedure consisted of quantitatively transferring a 50-pL aliquot of urine into 2.0 mL of HPLC eluant containing 25 pg/mL of 3 (stock solution of 3). The sample was mixed and filtered into an autoinjector vial. The serum and urine procedures were validated for linearity, precision, and accuracy by preparing and analyzing series of fortified samples on four separate days. For serum, 10 concentrations from 50 to 100 ng/mL were employed. For urine, the 10 concentrations ranged from 200 to 20 pg/mL. Samples a n d S a m p l e Handling-Serum and urinary excretion levels of 1 were determined in four beagle dogs, two males and two females. Each dog received the following four doses of 1: (A) 10 mg/kg as a 50-mg/mL sterile saline solution injected intravenously into the cephalic vein; (B) 10 mg/kg as a 50-mg/mL saline solution administered by oral intubation to a fasted animal; (C) 10 mg/kg of 1 in a no. 13 gelatin capsule given orally to a fasted dog; (D) same as treatment C given to a fed animal. The dogs weighed -10 kg each, determined the day before dosing, resulting in an approximate total dose of 100 mg of 1 . The fed dog received food 1 h prior to dosing, and the fasted dogs received no food 16 h prior to dosing. Blood samples were obtained at the following intervals: (treatment A) 0 (predose), 5 , 10, 20, 30, 45 min, 1, 1.5, 2, 3, 4, 6,8, 12, and 24 h; (treatments B, C, and D) 0 (predose), 15, 30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12, and 24 h. Serum was separated from the blood as quickly as possible and was frozen at -30°C until analysis. Urine specimens for each treatment were obtained at 0 (predose), 0-2, 2-4, 4-6, 6-8, 8-12, 12-24, and 24-48 h. The early urine collection intervals were obtained by catheterization, and the later collections from the metabolism cages. The urine specimens were mixed thoroughly, the volume was measured, and an aliquot was frozen and maintained a t -30°C until analysis. Calculations-The validation of the analytical methods and the determination of 1 in serum and urine samples were accomplished using the relative weight response (RWR) to an internal standard calculation method. A concentration series of 1over the level expected in samples (0.2-50 pg/mL for serum and 20-1000 pg/mL for urine) was prepared with each sample set, and the average RWR of the reference solutions (eq. 1)was employed to calculate the level of 1 in the sample aliquot (eq. 2):
RWR=pg l/aliquot =
PH 1 PH IS
X
pg IS/solution pg l/solution
PH 1 sample PH IS
X
pg IS/sample average RWR
(1) (2)
where P H 1 and PH IS are the peak heights of 1 and either 2 or 3 (depending on the sample matrix), and pg 1 and pg IS are the micrograms of 1 and 2 or 3 in the solution or sample aliquot. The pg l/aliquot was converted to pg/mL for serum and mg/collection for urine using the appropriate dilution factors and urine volumes. During validation and sample analysis, a concentration series 456
1 Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
of 1in the physiological fluid (0.1-50 pg/mL for serum and 201000 pg/mL for urine) was prepared and analyzed to determine the accuracy of the analytical procedure. The recovery of 1 in the fortified serum and urine samples was calculated as follows:
%R =
pg/aliquot (fortified) - pg/aliquot (unfortified) pg added
x 100 (3) For both serum and urine, the recovery of 1 was quantitative over the fortified concentration range. Thus, no correction for loss of 1during sample preparation was necessary. The stability of 1 in serum or urine was determined for control samples fortified with a known level of 1 ( 5 and 25 pg for 1.0 mL of serum or 50 pL of urine) and stored at -30°C. P h a r m a c o k i n e t i c Evaluation-The serum and urinary excretion levels of 1 in the dog were used to determine pharmacokinetic parameter estimates by graphic and computer techniques7 The computer program, NONLIN, was for a twocompartment model with either bolus (intravenous) or firstorder (oral doses) input. The initial estimates for the firstorder two-compartment model were obtained from the estimates of the intravenous dose. For the dogs receiving treatment A (intravenous dose), the parameters calculated included the area under the serum curve (AUC) from 0 to infinity, the mean residence time (MRT), the apparent volume of distribution ( Vd, area), the distribution rate constant (a), the elimination rate constant ( p ) , the cumulative amount of 1 excreted in the urine (UT), the percent of dose excreted (%Dose), the elimination rate constant (k,) from urine data, and the total body (CLT) and renal (CLR)clearances. For the dogs receiving oral doses of 1, the parameters determined were AUC, MRT, a, p, the absorption rate constant ( k a ) ,the highest concentration (C,,,), the time (tmaX)required to reach C,,,, UT, %Dose, and k, .
Results and Discussion Chromatographic Conditions Evaluation-The HPLC conditions previously describedfi for the determination of 1 in serum had employed a 10-pm reversed-phase column with an eluant of 20:80 (v/v) acetonitrile:0.005 M tetrabutylammonium phosphate, pH 7. A sharper, better-defined chromatographic peak for 1 was obtained on a 5-pm Supelcosil LC-18 column, and studies to optimize the organic modifier level, pH, and TBA level were conducted. The results are shown in Fig. 1 and indicate that the retention of 1 is sensitive to slight changes in the acetonitrile level but not sensitive to pH changes above pH 5.5 or to TBA concentrations above 0.006 M. Validation of S e r u m and U r i n e S a m p l e P r e p a r a t i o n Procedures-The results for the validated serum sample preparation procedure are summarized in Table I and show linearity over the entire range with a least-squares linear regression equation of y = (1.016 k 0 . 0 0 5 ) ~+ (0.063 0.090) and a correlation coefficient of 0.9995. The precision of the procedure is shown by the RSD of the four independent samples at each fortification level and was excellent, with the highest RSD value being 6.0%. The minimum quantifiable level, i.e., the lowest fortified serum level with a n RSD of
*
4
5
6
7
8
I
I
the detection limit was 2 pg/mL. Representative HPLC-UV (268 nm) chromatograms of a blank dog urine and a 0-2-h urine collection after a 10-mg/kg oral solution dose are shown in Fig. 3. The stability of 1 in serum and urine stored frozen with samples collected from dogs was good with no detectable change in the concentration of 1 after 30 d at -30°C. Pharmacokinetics in the Dog-The serum and urine levels of 1 in the dog were used to determined the distribution and elimination kinetics after intravenous and oral administration of 10 mg/kg of 1. Table I1 summarized the pharmacokinetic parameters calculated for each dog for each dose. The mean and SD for each parameter for the four dogs are also given in the table. The log serum levels versus time from one dog receiving the four doses are shown in Fig. 4. The cumulative urinary excretion levels versus time for this dog are presented in Fig. 5. The pharmacokinetic parameters determined for the intravenously dosed dogs were similar, as shown by the SD value. The data fit the NONLIN model well with all the correlations being >0.99. The distribution phase of 1 after the intravenous dose was rapid (average half-life of 46.2 min) and the Vd, area
9
PH I
I
24
20 I
0
I
I
I
28 32 36 Acetonitrile Level, Yo
I
I
0.002
(
I
J
I
0.004 0.006 TEA Level, M
40
I
'
0.008
I
0.01
1 2 I
Figure 1-Optimization of HPLC eluant composition for furegrelate. Key: (A) pH; (B) acetonifrile; (C) TBA. Table I-Linearity, Fluids
Precision, and Accuracy of I in Physiological
Fortified Serum Samplesa
Fortified Urine Samplesb
Amount Amount Average Amount Amount RSD, Average Added, Found, RSD, Recovery, Added, Found, Recovery, o/o ~~9150~ l 5 0 P/o YO &/mL PrglmL UL rUL -
50.0 25.0 10.0 7.00 4.00 2.00 1.00 0.50 0.20 0.10 0
50.9 25.2 10.7 7.17 4.13 2.07 1.03 0.51 0.22 0.11 0.01
3.0 1.4 6.0 2.7 3.9 2.7 2.2 3.4 4.4 5.2 -
101.8 100.7 107.2 102.2 103.1 103.0 102.2 101.0 103.8 96.7 -
100.0 80.0 60.0 40.0 30.0 20.0 15.0 10.00 8.00 6.00 4.00 2.00 1.00 0
96.1 77.8 59.5 39.8 30.2 20.4 15.4 10.22 8.26 6.13 4.19 2.12 1.14 0
0.5 0.7 0.4 0.5 0.4 0.3 1.2 2.5 2.1 3.9 2.2 3.1 4.4 -
96.1 97.2 99.2 99.4 100.7 102.2 102.7 102.2 103.2 102.2 104.8 106.2 114.5 -
a Linear regression: y = (1.016 f 0.005)~+ (0.063 ? 0.090); r = 0.9995, n = 4. *Linear regression: y = (0.966 f 0.003)~+ (0.580 -t 0.104); r = 0.9998, n = 4.
the urine with HPLC eluant gave an HPLC chromatogram with minimal interference at the elution of 1. However, a urinary component coeluted with 2 , and thus, this compound was not satisfactory as an internal standard for urinary analysis. Since no sample preparation was required other than simple dilution, an internal standard which was not structurally similar to 1 could be used. Propiophenone (3)was selected as the urine analysis internal standard, and the acetonitrile content of the eluant was increased from 30 to 32% to shorten the chromatography time. Validation of the urine assay is presented in Table I. The least-squares linear regression equation was y = (0.966 k 0 . 0 0 3 ) ~ + (0.58 f 0.10) with a correlation coefficient of 0.9998. The precision was excellent, with the highest RSD being 4.4%,and the accuracy was quantitative, as shown by the average %R. The minimum quantifiable level for urine was 20 pg/mL, and
L 0
8
1 6 0
mL
Predcse Serum
8
16
30 - Min Serum
Figure 2-Represenfafive HPLC-UV (268 nm) chromafograms of blank dog serum and a serum sample obtained 30-min after a 10-mg/kg oral solution dose of furegrelate (7); 2 is the internal standard.
I
3
! I 1
l9d.L 0 8 16 mL 0 8 Predose Urine C-2-h
i
16
Urine
Figure 3-Representative HPLC-UV (268 nm) chromatograms of blank dog urine and a 0-2-h urine collection from a dog receiving a 10-mglkg oral solution dose of furegrelate (7); 3 is the internal standard.
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
I
457
Table Il-Pharmacokinetic
Parameter Estimates in Dogs Treatment A, IntravenousInjection
AUC,
09
Dog
Vd, L
min-'
min-'
Corr?
1 2 3 4 x +SD
3.4 2.5 3.6 3.2 3.2 0.5
0.012 0.018 0.016 0.016 0.015 0.003
0.0053 0.0047 0.0050 0.0063 0.0053 0.0007
0.992 0.992 0.998 0.997
ff,
Percent
MRT, min
CLT, mL/min
Dose Of
CIR, mL/min
ke, min-1
rZb
117 174 104 84 120 39
17.8 11.5 18.2 20.1 16.9 3.7
87.5 65.3 64.5 62.4 69.9 11.8
15.6 7.5 11.4 13.0 11.9 3.4
0.0061 0.0028 0.0058 0.0033 0.0045 0.0017
0.98 0.99 0.94 0.84
mL 6150 8546 5197 4495 6097 1768
Treatment B, Oral Solution
1 2 3 4c
0.018 0.017 0.018 0.017 0.018 0.001
0.018 0.016 0.018 0.135 0.017 0.001
x
+SD
0.960 0.952 0.947 1.000
0.0042 0.0025 0.0058 0.0060 0.0042 0.0017
170 196 211 125 192 21
4042 6879 4834 976 5252 1464
65.7 80.5 93.0
-
79.7 13.7
20.6 35.5 21.3 9.0 25.8 8.4
90 90 120 29 100 17
75.5 74.6 57.6 24.1 69.2 10.1
86.3 114.2 89.3 96.6 15.3
0.0031 0.0035 0.0080 0.0069 0.0049 0.0027
0.97 0.95 0.94 0.99
Percent
Bioavail. Urine
k,, min-1
r26
Of
71.6 52.9 61.1 64.7 62.6 7.8
81.8 81.0 94.7 103.7 90.3 10.9
0.0033 0.0031 0.0028 0.0048 0.0035 0.0009
0.94 0.94 0.94 0.98
k,. min-'
r2b
Urine
71.5 92.0 95.8 110.7 92.5 16.2
0.0138 0.0068 0.0021 0.0031 0.0065 0.0053
0.84 0.94 0.86 0.99
Treatment C, Drug in Capsule (Fasted Dog) ka
Dog
MRT,
a,
7
min-'
min-'
min-1
0.041 0.020 0.012 0.018 0.023 0.013
0.013 0.020 0.012 0.018 0.016 0.004
0.0040 0.0032 0.0020 0.0041 0.0033 0.0009
~-
1 2 3 4
x S D
Bioavail.
,C ,,,,
t-,
mL 0.968 0.981 0.968 0.998
Dose
4181 5927 3884 4240 4559 925
191 198 250 161 200 37
68.0 69.4 74.7 94.3 76.6 12.1
22.3 32.1 15.4 23.1 23.2 6.9
120 120 152 90 121 25
Treatment D, Drug in Capsule (Fed Dog) ks.
ff,
Dog
min-1
min-1
1 2 3 4
0.064 0.026 0.038 0.023 0.038 0.019
0.010 0.027 0.016 0.022 0.019 0.007
x +SD
mf;-l
Corr."
0.0003 0.0052 0.0012 0.0002 0.0017 0.0023
0.999 0.999 0.999 0.980
AUC' pg.mn/ mL
MRT, min
Bioavail. Serum
, ,C , pg/mL
2869 3119 3434 5483 3726 1194
1509 150 491 3563 1428 1536
46.6 36.5 66.1 122.0 67.8 38.2
11.9 20.8 18.8 4.7 14.1 7.3
t,,, min 60 63 34 61 55 14
Peyt Bioavail. Dose 62.6 60.1 61.8 69.1 63.4 3.9
Corr.-correlation from NONLIN evaluation of serum data. r2-squared linear regression coefficient for log (U, of administered dose; data not included in calculation of average values. a
2.0
?
1.5 -
5
1.0-
8G
0.5-
-I
E
0-
9 -0.5 -
-0.1
'
I
I
I
I
I
~
- U ) versus time.
Partial loss
of 3.2 L is greater than the plasma volume in the dog (-400 mL/10 kg), but less than the total body water (-4600 mL/10 kg).' The elimination of 1 from serum had an average half-life of 130 min, which is less than the 11.9 h half-life reported earlier for 1.6Evaluation of the figures in that report indicated that the calculated fi and half-life did not correspond to the last three data points, which have an estimated elimination half-life of 2-3 h. The AUC and CLT averages, when converted to similar units, are similar to the values reported in the earlier paper. About 70% of the administered dose was excreted unchanged in the urine. The log ( UT - U ) versus time evaluation of 1 showed a first-order elimination and a one-compartment model [regression coefficient (rZ)> 0.801. The he values from the urine data agreed with the serum /3 for individual dogs; thus, the elimination kinetics of 1 could be determined from both serum and urine results. The pharmacokinetic evaluation of the serum and urine results from dogs dosed with 1 as an oral solution or drug in a capsule to a fasted animal were similar. The average of various rate constants, ( k a , a , /3, and k,) and the other parameters (AUC,MRT, ,,C,, t,,,, %Dose, and bioavailability in serum and urine) were statistically equal (p > 0.2). The bioavailability of 1 for the oral solution and drug in capsule averaged 80 and
I
I
I
I
960
480
0
I
I
I
1
1920
1440
1
1
2400
1
2880
Minutes Figure 5-Cumulative urinary excretion versus time profiles of furegrelate in a dog receiving 10-mglkg doses of 1 given intravenously (A), as an oral solution (B), and in a capsule to a fasted (C) and a fed animal (0). 1.5r
References and Notes 1. Gorman, R. R.; Johnson, R. A.; Spilman, C. H.; Aiken, J. W. Prostaglandins 1983,26, 325-342. 2. Hamberg, M.; Svensson, J.; Samuelsson, B. Proc. Natl. Acad. Sci. USA 1974, 72, 2994-2998. 3. Ellis, E.; Oelz, 0.; Roberts, L. J., 11; Payne, N. A.; Sweetman, B.; Nies, A.; Oates, J. Science 1976,193, 1135-1137. 4. Needlemen, P.; Minkes, M.; Raz, A. Science 1976, 193, 163-165. 5. Needlemen, P.; Kulkarni, P.; Raz, A. Science 1977,195, 409-412. 6. Wynalda, M. A.; Liggett, W. F.; Fitzpatrick, F. A. Prostaglandins 1983,26, 311-324. 7. Metzler, C. M.; Elfring, G. L.; McEwen, A. J. Biometrics 1974,30, 562. 8. Gerlowski, L. E.; Jain, R. K. J . Pharm. Sci. 1983, 72, 1103-1127.
1.0-I
E
9- 0.5ci
8E
0-
$
8 -0.5-
-I
-1.oL
77% for serum data and 97 and 90% for urine results, respectively, when compared to the intravenous values. Fed dogs dosed with drug in capsule did not have uniform serum kinetics, as shown in Fig. 6. Two dogs had a prolonged elimination phase which altered the various pharmacokinetic parameters. The fed dogs, in general, had a faster absorption, , and t,,,, a lower AUC, and an slower elimination, lower,,C increase in MRT when compared with the other oral doses. These serum result observations indicate that the distribution and elimination are altered in the presence of food and may result in drug accumulation with multiple dosing and may prevent a therapeutic concentration in the blood. The urinary excretion results also had some, but not as pronounced as the serum data, variation when compared with the other doses. The absolute bioavailability of 1 in fed dogs averaged 68% for serum and 92% for urine results. Additional studies on the effect of food on the bioavailability and pharmacokinetics of 1 in the dog are planned to assist in the design of human studies.
'
I
I
I
I
I
I
360 480 600 720 Minutes Figure 6-Log serum concentration versus time profiles of furegrelate in dogs receiving 10-mglkg doses in capsules to fed animals. 0
120
240
Acknowledgments The authors thank Chris Asmus and Jay Hansen for their assistance in the collection of samples, Dennis Weber for his helpful suggestions for the pharmacokinetic evaluations, and Marlene Dopheide for typing and editing the manuscript.
Journal of Pharmaceutical Sciences Vol. 74, No. 4, April 1985
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