Metabolism of [14C]-quazodine in the rat, dog, and man

TOXICOLOGY

AND

Metabolism

APPLIED

PHARMACOLOGY

36,307-322

of [14C]-Quazodine

(1976)

in the

Rat,

Dog, and

Man1

M. J. BARTEK AND J. A. LABUDDE Department of Pathology and Toxicology, Mead Johnson Research Center, Evansville, Indiana 47721 Received August 4,1975; accepted December 29,1975

Metabolismof [14C]-Quazodinein the Rat, Dog, and Man. BARTEK, M. J. LABUDDE, J. A. (1976). Toxicol. Appl. Pharmacol. 36, 307-322. The pharmacokineticsof [‘%I-quazodine, a new bronchodilator, were examined in man and dog. Absorption, metabolism,and excretion of quazodine werestudiedin the rat, dog, and man, while distribution of the drug was measuredin rats. After iv dosage,clearanceof unchangeddrug from plasma wasrapid in both dogsand man and followed a biexponentialdecaycurve in accordancewith the equationC, = Ae-“* + Be-O*. A goodfit betweenthe actual data and the computer-generatedcurves was obtained employing a nonlinearregressionanalysiscomputerprogram.After po administration quazodinewasrapidly absorbedin both manand dog, a peak plasmaconcentration beingobservedat 0.5 hr in man andat 1 hr in dogs.The drug did not localize in cerebrospinalfluid of dogs.Radioactivity wasfound in all tissuesof rats at 1 hr after oral dosage,and no evidencefor extremedrug localization or prolongedretention wasfound in any tissueincluding brain. In rats, 71.9% of the dosewasrecoveredin urine and 14.2% in fecesduring the first 3 days after dosing. The 72-hr recoveriesin dog urine and feces were61.4and 25.8%, whereasin humansthesevalueswere84.1and 1.1x, respectively. The major pathway for metabolismof quazodine in man, and to a lesserextent in the dog and rat, was by demethylation at the 7-position of the quinazoline ring-systemfollowed by conjugation with glucuronic acid or sulfate. The glucuronide conjugate accounted for 78.0% of the radioactivity in humanurine, 45.1% in dog, and 27.4% in rat urine. The amount of radioactivity present as the sulfate conjugate was 3.1, 15.3,and 10.5% in human,dog, and rat urine, respectively.

AND

Quazodine, 6,7-dimethoxy-4-ethylquinazoline, has been shown in laboratory animals to be a bronchodilator (Aviado et al., 1967) as well as a cardiac stimulant with vasodilator properties (Carr et al., 1967).As part of the toxicologic evaluation of quazodine, studies on its metabolic fate were undertaken. These studies utilized [14C]-quazodine and included the following: (a) excretion studies and plasma concentration determinations of unchanged drug and metabolites in man and dogs after iv and oral administration, (b) tissue distribution and excretion studies in rats following oral administration, (c) determination of drug distribution between plasma protein, plasma water, and red blood cells in dog and human blood, and (d) separation and identification of metabolites from rat, dog, and human urine. 1Presented in part at the EleventhAnnual Meetingof the Societyof Toxicology, Williamsburg, Virginia, March 5-9, 1972. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

307

308

BARTEK

AND

LABUDDE

METHODS

Synthesis of radioactive quazodine. Quazodine (Fig. 1) was labeled with 14C in the 2-position of the quinazoline ring as described previously (Madding, 1972). The final product, after recrystallization from acetone, had a specific activity of 4.65 ,&i/mg. It was shown to be radiochemically pure by thin-layer chromatography on Eastman 6060 silica gel sheets in a solvent system of Skelly F/acetone (1: 1).

FIG. 1. Structural formula of [2-‘4C]6,7-dimethoxy-4-ethylquinazoline the location of the 14C label.

(quazodine). Asterisk marks

Synthesis of the two desmethyl derivatives of quazodine was briefly as follows. For preparation of the 7-desmethyl derivative, propiovanillone was benzylated to give 4’-benzyloxy-3’-methoxy propiophenone. The latter was cyclized to 4-ethyl-7-benzyloxy-5-methoxy quinazoline which was converted by catalytic hydrogenolysis to 4-ethyl-7-hydroxy-6-methoxyquinazoline (mp 222.5-225°C corr.). The other desmethyl derivative of quazodine, 4-ethyl-6-hydroxy-7-methoxyquinazoline (mp 201-204°C corr.), was prepared in a similar manner from propioisovanillone. Elemental analyses (C,H,N) of the two derivatives were satisfactory; infrared and nuclear magnetic resonance spectra were consistent with the assigned structures. Dog studies.Three mature purebred beagle dogs, weighing lo-14 kg each and fitted with indwelling catheters, were used for iv studies. At 30 min prior to drug administration the animals were given 200 ml of water using a stomach tube. Each dog received an iv dose of 10 mg of [14C]-quazodine/kg; the drug was dissolved in isotonic saline at a concentration of 100 mg/ml and injected into the cephalic vein. Blood samples were collected in Vacutainers at selected times, and urine was collected hourly for 6 hr after which the dogs were maintained in stainless steel metabolism cages for collection of urine and feces. Food and water were permitted ad libitum. Oral dosage studies were conducted in 6 beagle dogs which had been fasted overnight. [14C]-Quazodine was administered at a dose of 10 mg/kg in a soft gelatin capsule, and blood, urine, and feces samples were collected for 72 hr after dosing. Cerebrospinal fluid was collected from two of the dogs at 1, 3, 5, and 24 hr from the cisterna magna by occipital puncture through the median lines of the neck, immediately above the first cervical vertebra. An additional two dogs were given a single po dose of 0.294 mg/kg [14C]-quazodine, the mean po dose used in the human studies. Rat excretion and tissuedistribution. Excretion of [‘4C]-quazodine was measured in three male and three female rats, Charles River CD strain weighing 250-400 g. The animals were fasted overnight and given 5 ml of water 0.5 hr prior to administration of a po 10 mg/kg dose of [14C]-quazodine dissolved in water (5 mg/ml). Animals were maintained in individual metabolism cages for collection of urine and feces. For tissue distribution studies five male and five female rats were each dosed po with 10 mg/kg of [‘4C]-quazodine, and one male and one female were sacrificed at 1, 3, 6, 24, and 72 hr after dosing. Tissues were removed and kept frozen until assayed.

METABOLISM

OF QUAZODINE

309

Human studies. The subjects were healthy male volunteers weighing between 66 and 80 kg. They had not received other drug medication for 2 weeks prior to quazodine administration and, except for intake of clear liquids, fasted overnight before and until 4 hr after drug administration. All six subjects were given a po dose of the drug, and 2 weeks later two of the six received the drug iv. For the po study each subject received a single capsule contaning 21.5 mg of [14C]-quazodine and the mean dose was 0.294 mg/kg. For the iv study each subject was given 2 ml of a solution of isotonic saline containing 10 mg of [‘4C]-quazodine. Blood samples, collected using Na,EDTA as the anticoagulant, were immediately centrifuged, and the plasma frozen until analyzed. Total urines were collected at 4, 8, 24,48, and 72 hr after drug administration, and an aliquot was frozen for later analysis. Feces were collected at 24-hr time intervals for 72 hr. Preparation of samples for liquid scintillation counting. Aliquots of plasma and urine samples were mixed with scintillator solution and counted directly. Fecal samples were homogenized in 9 vol (w/v) of water and a weighed lo@-200-mg aliquot of the homogenate was counted. Tissue samples were prepared for counting according to the quick freeze-pulverization method of Adams et al. (1970). For counting, all samples contained at least 0.6 ml of water plus 15 ml of a scintillator solution containing 5.0 g/liter of 2,5-diphenyloxazole, 0.5 g/liter of p-bis-(O-methylstyryl)-benzene, and 120 g of naphthalene per liter of p-dioxane. The samples were counted in a Packard Tri-Carb Model 3375 liquid scintillation spectrometer with automatic external standardization. Determination qf unchanged drug and chloroform-extractable and chloroformnonextractable metabolites in biological samples. Two milliliters of each plasma and

urine sample were extracted for 15 min with two successive lo-ml portions of heptane. Following centrifugation, the combined heptane extracts were placed in a fresh tube, washed with 2 ml of 0.1 N NaOH, and a portion of each extract was counted. Radioactivity in the heptane extracts was entirely in the form of unchanged drug as verified by TLC. Following heptane extraction the aqueous residues were shaken for 15 min with 10 ml of chloroform, centrifuged, and a portion of the chloroform extract was counted. Corrections were made for the 80 % recovery in the heptane extracts of known amounts of quazodine added to control plasma and urine, and for the amount of unchanged drug (4.3 ‘A) recovered in the chloroform extract. Values for “chloroformnonextractable metabolites” were obtained by subtracting the sum of “unchanged drug” plus “chloroform-extractable metabolites” from the total radioactivity of the sample. Distribution

of quazodine between plasma protein, plasma water,

and erythrocytes.

The distribution in vitro of quazodine among plasma protein, plasma water, and erythrocytes was determined in dog and human blood. Protein binding was determined by vacuum ultrafiltration for 4 hr at 24°C. To determine the equilibrium distribution of quazodine between plasma and erythrocytes, radioactivity was measured in the plasma following incubation of labeled drug with control whole blood. Five milliliters of whole blood were incubated with 50 ,ug of [14C]-quazodine for 30 min at 37°C and after centrifugation the plasma layer was sampled and counted. Similarly, for determining the equilibrium between plasma water and erythrocytes, radioactivity was measured in the buffer phase following incubation of 50 pug of [14C]-quazodine with 2.5 ml of

310

BARTEK

AND

LABUDDE

washed erythrocytes plus 7.5 ml of buffered salt solution containing EDTA (Schmid er al., 1962). Results from these studies were analyzed on the basis of the following 3-compartment model : plasma protein + plasma water % erythrocytes. Separation and ident$cation of urinary metabolites. Samples of the 4- or 8hr urine specimens from rats, dogs, and humans that received [14C]-quazodine were chromatographed on No. 6060 Eastman Chromagram silica-gel TLC sheets. The sheets were developed consecutively in two solvent systems, butanol/acetic acid/water (3 : 1: 1) and chloroform/methanol (4: 1) and scanned on a Varian Berthold series 6000 radioscanner. Urine samples were examined for the presence of glucuronide and sulfate conjugates. Duplicate l-ml urine specimens from each species were extracted with heptane and chloroform as outlined above. The aqueous residues were then adjusted to pH 5 with 0.5 N acetate buffer and one set was incubated with 5000 units of /3-glucuronidase (Ketodase)2 for 18 hr at 37°C. A second set was treated with /?-glucuronidase and aryl sulfatase (Helix pomatia, B Grade)3 at 37°C for 18 hr. Following enzyme treatment, the samples were re-extracted with chloroform to determine the quantity of metabolites released from the conjugated form as a result of enzyme treatment. For large scale isolation of metabolites, 200 ml of urine from dogs given an oral dose of [14C]-quazodine was placed on 5 x 30-cm column of Amberlite XAD-2 resin. Following a water wash (100 ml), the radioactivity was eluted with 1 liter of methanol which was evaporated to dryness, and the residue was redissolved in 10 ml of water. This material was adjusted to pH 5 with acetate buffer and incubated overnight at 37°C with 25,000 units of /I’-glucuronidase. The hydrolysate was extracted twice with chloroform, the extract was taken to dryness, and the residue was redissolved in methanol. The methanol solution was streaked in Analtech preparative TLC plates (silica gel, 1 mm) and developed in chloroform/methanol (9: 1). After radioscanning, the area under the major radioactive peak was scraped from the plate, eluted with methanol, and again subjected to preparative TLC using a solvent system of butanol/acetic acid water (3: 1: 1). The purified metabolite was eluted from the plate, subjected to mass spectrometry, and compared with authentic 6-desmethyl and 7-desmethyl quazodine derivatives on the basis of gas-liquid and thin-layer chromatographic properties. Urine samples from rats and humans were treated in similar fashion. The trimethylsilyl derivatives of the urinary metabolite of quazodine and the two synthetic desmethyl derivatives were made by adding 0.5 ml of hexamethyldisilazane plus two drops of pyridine to the samples dissolved in acetonitrile. The silylated derivatives were separated on a 6-ft x 0.25-in. column of 3.8 % SE-30 on lOOjl20 mesh Gas Chrom Q at 175°C using a Hewlett-Packard Model 402 gas chromatograph. RESULTS

Following iv administration of [14C]-quazodine, clearance of the unchanged drug from plasma was relatively rapid in both dogs and man (Table 1). When plotted semilogarithmically, as illustrated in Fig. 2 for subject LHK and Dog l-PH41, the data appeared to follow a biexponential decay curve in accordance with the equation C, = 2Warner-Chilcott,Morris Plains,NewJersey.

3 Endo Laboratories, Inc., Garden City, New York.

311

METABOLISM OF QUAZODINE TABLE PLASMA

CONCENTRATIONS FOLLOWING

1

OF UNCHANGED DRUG IN MAN AND DOG INTRAVENOUS ADMINISTRATION OF [14C]-Q~~~~~~~~ Plasma concentration @g/ml>

Dose 10 mg Time after dosing

Subject LHK

Subject SBM

5 min 10 min 20 min 30 min 40 min 45 min 1.0 hr 1.5 hr 2 hr 3 hr 4 hr 5 hr 6 hr 8 hr

0.258 0.154 0.097

0.134 0.120 0.091

0.059

0.055

0.044

0.038

0.016

0.013

0.005

0.004

0.002

0.001

Dose 10 mg/kg

Dog 22

Dog # l-PH41

# 2-B141

Dog

7.22 5.30 3.72 2.65

7.46 4.50 3.92 2.66

8.07 5.10 3.28 3.13

2.00 1.40 0.86 0.55 0.27 0.17 0.14 0.11 0.10

2.04 1.50 1.07 0.66 0.32 0.16 0.06 0.08 0.01

2.41 1.48 0.69 0.48 0.23 0.19 0.04 0.02 0.02

Ae-‘l’ + Bemmflf. A nonlinear regression analysis computer program (B. Blumenstein, personal communication) was used to fit the data to the equation. Examples of the fit between the computer-generated curves and the actual data are shown in Fig. 2. On the assumption that clearance of quazodine could be described in terms of a two compartmental open model, apparent rate constants for drug disposition were calculated and are presented in Table 2 using the nomenclature of Riegelman et al. (1968). High concentrations of metabolites were found in plasma shortly after iv dosage as shown in Fig. 3 for man and Fig. 4 for dogs. The plasma metabolites were mostly in TABLE PHARMACOKINETIC

2

PARAMETERS FOR QUAZODINE IN MAN INTRAVENOUS ADMINISTRATXON

Subject

AND

DOGS FOLLOWING

Subject

Constant

LHK

SBM

Dog f2

Dog # l-PH41

Dog f 2-B141

Body weight (kg)

79.4 3.559 3.803 6.429

65.8 1.249 0.800 0.380

11.0 2.129 3.458 2.777

11.5 3.561 5.072 10.002

12.0 3.888 4.784 9.750

18.6

64.5

10.5

6.15

5.79

k cl

k 21 k 12

VP= F

(liters)

312

BARTEK AND LABUDDE

FIG. 2. Computer-generated semilogarithmic plots of the disappearance of unchanged drug from plasma of man and dog administered [“‘Cl-quazodine iv.

1

0

FIG. 3. Mean plasma concentrations [WI-quazodine.

6

of metabolites in man after iv administration

8 of 10 mgrof

313

METABOLISM OF QUAZODINE

1

FIG. 4. Mean plasma concentrations [‘%I-quazodine/kg.

of metabolites in dogs after iv administration

Unchanwd Chlorafarm Chforoform

Extractable Nonextractable

CJr’Jg

of 10 mg of

D-Z-13

t”lemo,,ter

#..

MetabO,,ler

P

. ... ... ... ..+

. ... ... .. ... .

l.:-_i“‘+F,q, .....*....._................ ......... ~ 03

1

2

3

4

5

6

7

8

24

4 48

!iaurr After Drug *dmm,rtrat,on

5. Mean plasma concentrations of unchanged drug, chloroform-extractable, and chloroformnonextractable metabolites in man following oral administration of 0.294 mg of [r4C]-quazodine/kg. FIG.

a polar form, not extractable into chloroform, and presumably consisted of the conjugates of the metabolite, desmethylquazodine, identified as described below in urine. The mean plasma concentration data obtained after oral dosage of 10 mg of [‘“Clquazodine to man are shown in Fig. 5. Maximum concentrations of both unchanged drug and metabolites were obtained a 30 min, the earliest time period studied. Very little radioactivity was present as unchanged drug at any time after dosage. Most of the radioactivity (75-90 %) was in the form of metabolites not extractable into chloroform. Plasma.concentration curves for unchanged drug and metabolites in dogs after oral administration of 10 mg of [‘4C]-quazodine/kg are shown in Fig. 6. When expressed

314

BARTEK AND LABUDDE

FIG. 6. Mean plasma concentrations of unchanged drug, chloroform-extractable, and chloroformnonextractable metabolites in dogs following oral administration of 10 mg of [‘VI-quazodine/kg.

in terms of percentage of total radioactivity, considerably more unchanged drug was found in dog plasma than in human plasma. Again in terms of percentage of total radioactivity, concentrations of chloroform-extractable metabolites in dogs and man were similar, but concentrations of nonextractable metabolites were higher in the human subjects. In dogs, plasma concentrations of unchanged drug and metabolites were proportional to dose. Although not reported, the relative amounts of these components obtained after a 0.294mg/kg dose were the same as those obtained after a lo-mg/kg dose. For example, at peak values, unchanged drug accounted for 26.7 and 28 % of the total after 0.294 and 10 mg/kg respectively, and nonextractable metabolites for 58.8 and 56 % of that total. Cerebrospinal fluid values for radioactivity in dogs were about one-fifth as high as the plasma concentrations at 1, 3, 5, and 24 hr after dosage (Table 3). TABLE 3 PLASMA AND CEREBROSPINAL FLUID CONCENTRATIONS OF RADIOACTIVITY IN Does AFTER A SINGLE ORAL DOSE OF 10 mg [14C]-@JAzoDINE/kg” Hours after drug administration

Plasma

Dog #

0.5

1

2

2

4

5

7

24

48

4A

0.14 1.35

7.06 6.36

5.87 5.84

3.25 3.35

1.84 2.41

1.56 1.95 0.45

1.21 1.80

0.51 0.71 0.14 0.14

0.17 0.17

142

CSF

4A 142

Plasma/CSF

4A 142

1.66

0.89

1.39 4.26 4.58

0.84 3.66

3.99

0.40

3.49

3.45 4.85

a Data are expressed in micrograms of quazodine equivalents per milliliter

5.08 of sample.

METABOLISM

315

OF QUAZODINE

Despite the high lipid solubility of quazodine, when it is equilibrated with whole blood, higher concentrations are found in the plasma than in the erythrocytes (Table 4). To investigate this further, distribution of the drug was first examined in a system TABLE 4 DISTRIBUTION

OF DRUG

Species

BETWEEN PLASMA PROTEIN, AND RED BLOOD CELLS

Percentage in plasma Equilibration

A. Whole Blood Dog Man

PLASMA

WATER

Percentage in RBC

Studies

52.6 64.8

B. Washed RBC’s + Buffered Salt Solution Dog 27.8 Man 28.7 Ultrafiltration Studies

47.4 35.2 72.2 71.3

Species

Percentage in ultrafiltrate

Percentage protein bound

Dois Man

35.0 20.4

65.0 79.6

Three-Compartment model

[Xl

WI VI Calculated percentage of drug in each compartment 47.7 Dog 34.0 18.4 Man 53.4 13.4 33.3 consisting of buffer and washed erythrocytes from either dogs or man. With cells from both species about 2.4 times more drug was present in the erythrocytes than in the buffer. Secondly, the binding of quazodine to plasma proteins was measured. In human plasma a higher percentage of drug was protein-bound than in dog plasma. From these two studies the theoretical distribution of quazodine in whole blood was calculated in accordance with the three compartment system given in Table 4. Good agreement with the actual distribution in whole blood was obtained. It can be seen that at a given whole blood concentration, although the concentration of quazodine is higher in human plasma than in dog plasma, the amount of unbound drug in plasma is considerably lower in man. Table 5 contains results of the rat distribution study. With the exception of small intestine, tissues from rats sacrificed at 1 hr contained substantially more radioactivity

316

BARTEK

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LABUDDE

TABLE 5 DISTRIBUTION

OF RADIOACTIVITY IN THE RAT FOLLOWING OF 10 mg [14C]-QuAzoDrNE/kg”

ORAL

ADMINISTRATION

Time of sacrifice after dosing (hr) Sample

1

3

6

8.04 5.83 10.2 8.69 1.19

Small intestineb

17.8 23.9 22.5 22.9 4.46 6.40 3.50 7.64 4.72 6.95 3.00 4.69 5.52 8.32 (15.1)

Large intestineb

(0.35)

Stomachb

(4.87)

Fat

8.04 9.07 7.71 9.00 0.64 15.9 3.73 5.17 0.65 0.25 4.46 7.01 (8.68)

3.82 2.30 3.34 6.98 0.33 0.45 0.27 0.85 0.33 0.82 0.16 0.24 0.55 1.25 (16.5) (6.03) (0.60) (2.52) (0.10) (0.31) 0.13 0.32 0.59 0.91 1.29 4.31 0.19 0.52

Liver Kidneys Spleen Gonads Heart Brain Lungs

Adrenals Plasma Skeletal muscle Carcass Skin

Urineb n Data

are expressed

in micrograms

1.09 1.43 1.79

1.61 1.66 0.67 0.71 2.14 1.46 (61.3) (20.0) (0.15) (0.14) (0.37) (1.52) 2.45 1.52 1.89 2.39 4.89 4.06 1.74 1.38 0.05 0.14 2.23 1.63 (24.0) (28.9) of quazodine

24 0.52 0.43 0.58 0.50 0.20 0.10

0.15 0.15 0.16 0.14 0.09 0.06 0.09 0.19 (1.W (1.38) (0.43) (0.37) (0.01) (
0.05 0.12 0.13 0.24 0.31 0.07 0.04 0.24 0.11 0.13 0.12 (78.2) (89.6)

0.01 1.03

0.41 0.88 (40.1) (32.5) equivalents

per

0.18 0.14 0.17 0.15 0.04 0.04 0.08 0.0 0.09 0.09 0.09 0.06 0.07 0.10

(0.02) (0.07) (
0.09 0.06 0.14 0.08 0.15 0.17 0.08 0.07 0.08 0.04 0.06 0.15 (97.8) (69.1)

gram.

* Percentage of dose per organ or total urine.

than did tissues from animals sacrificed at 3 hr or longer. No evidence for drug localization or retention was observed. At 24 hr after dosing, the whole body contained only 3 % of the administered radioactivity, and at 72 hr this value had dropped to 0.5 %.

26.1 f 5.1

29.6 f 3.4

Oral”

9.2

35.2 41.0

71.8 104.2

67.2b

6hr

IV

Oral”

IV

Oral”

4 hr

8 hr

84.1 109.4

83.1 f 8.7

24 hr

66.9 + 6.9

59.0 65.7

53.7 f 2.0

92.9 119.2

Urine

75.6 k 8.6

a Each figure represents the mean of six values C SE. b No sample.

Rat

2 94

J’w

LHK SBM

Man

Species test subject Route

68.9 + 6.8

71 .o 77.5

60.8 _+ 3.0

93.5 120.2

84.0 f 8.8

48 hr

71.8 + 6.3

72.0 78.1

61.4 + 3.1

93.8 120.4

84.1 f 8.8

72 hr

11.3 + 2.6

0.14 NS*

13.7 i- 2.2

16.2 13.1

0.02 0.90 25.0 f 3.4

0.7 + 0.25

0.1 f 0.03 NSb 0.04 14.8 f 4.5

48 hr

24 hr

Feces

14.2 + 2.0

19.1 13.4

0.05 2.66 25.8 _+ 3.5

72 hr ____ l.OkO.21

TABLE 6 CUMULATIVE PERCENT EXCRETION OF RADIOACTIVITY IN URINE AND FECES OF MAN, DOGS, AND RATS FOLLOWING ORAL OR INTRAVENOUS ADMINISTRATION OF [‘%I-QUAZODINE

s 2

3: r: $ st; SC 9 lo s

318

BARTEK

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LABUDDE

Concentrations of radioactivity in the brain were markedly lower than the corresponding values in plasma, a result which is consistent with the relatively low amounts of radioactivity found in cerebrospinal fluid in dogs. Table 6 summarizes results on the excretion studies conducted in man, dogs, and rats in terms of cumulative percentage excretion of radioactivity. Quazodine and its metabolites were eliminated very rapidly in urine of human subjects with 67 “,, of the dose excreted during the first 4 hr and 83 % within the first day. Very little radioactivity was recovered in feces of human subjects. Radioactivity was excreted less rapidly by rats and dogs; the 6-hr recoveries in dog and rat urine were 29.6 and 26.1x, respectively. Elimination of unchanged quazodine and its metabolites was similar in man and dogs. Less than 2% of total radioactivity in urine was excreted as unchanged drug regardless of route of administration or species, Chloroform-extractable metabolites accounted for 3-12x of the total and nonextractable metabolites, for 88-97x. As mentioned above, in man, feces contained negligible amounts of radioacitvity and fecal radioactivity in dogs was not characterized. Radioscans of thin-layer chromatograms of human, dog, and rat urine, shown in Fig. 7, suggest the presence of a small amount of unchanged drug plus a single major

FIG. 7. Radiochromatogram scans of urine from rats, dogs, and humans administered [WIquazodine orally.

METABOLISM

OF QUAZODINE

319

metabolite in human urine; two major, and perhaps one or two minor, metabolites in dog and rat urine. As shown in Fig. 8, chromatography of urine specimens in a second solvent system of chloroform/methanol (4 : l), indicated that a large portion of the most rapidly migrating peak on the chromatograms shown in Fig. 7 was actually a relatively nonpolar metabolite rather than exclusively unchanged drug. Table 7 contains results

FIG. 8. Radiochromatogram scansof urine from rats, dogs,and humans administered [%]-quazodine orally.

on the quantification of quazodine and urinary metabolites as measured by solvent extraction prior to and after enzyme treatment. Very little of the total radioactivity was present in urine as apparent unchanged drug, regardless of species. Four to 10 % of the radioactivity, depending on the species, was present as chloroform-extractable metabolites before enzymatic hydrolysis. Quantitatively, the major difference among urine of man, dog, and rat was in the amount of drug present as a glucuronide conjugate. The amount of radioactivity apparently present as the sulfate conjugate was only 3% in human urine, but in dog and rat urine it accounted for 15.3 and 10.5 % of the total, respectively.

320

BARTEK AND LABUDDE

TABLE 7 PERCENTAGEOFTOTAL

RADIOACTIVITYINEACHURINEEXTRACT

Treatment Heptaneextraction of unchangeddrug Chloroform extraction prior to enzymetreatment Chloroform extraction following /3-glucuronidase incubation Chloroform extraction following /?-glucuronidase + aryl sulfataseincubation

Rat urine

Dog urine

Human urine

0.1 27.4

0.2 4.6 45.1

0.1 4.1 78.0

37.9

70.4

81.0

10.1

The radioactivity in chloroform extracts of urine, either before or after incubation with /?-glucuronidase or arylsulfatase, had an R, value equal to that of peak # 1 of Fig. 7, and this peak presumably consisted mainly of a phenolic metabolite of quazodine along with a small amount of unchanged drug. Chromatograms of aqueous residues remaining after /I-glucuronidase treatment and chloroform extraction did not show the presence of peak # 3, indicating that the latter consisted of a glucuronide conjugate of a phenolic quazodine metabolite. Similarly, chromatograms of residues of samples treated with aryl sulfatase were lacking in peak # 2, suggesting its identity as a sulfate conjugate of a metabolite. The minor peaks remain unidentified. The major metabolic intermediate, isolated from human, dog, or rat urine by preparative thin-layer chromatography, was subjected to mass spectral analysis. From the fragmentation pattern, two possible formulas were assigned : 4-ethyl-6-hydroxy-7methoxy quinazoline or 4-ethyl-7-hydroxy-6-methoxy-quinazoline. The trimethylsilyl (TMS) derivatives of the latter two authentic compounds and the urinary metabolite were prepared and subjected to gas-liquid chromatography. The TMS derivatives of 7-OH quazodine and the urinary metabolite had identical retention times of 9.7 min, whereas the 6-OH derivative was eluted at 9.2 min. Thin-layer chromatography in three different solvent systems furnished additional proof that the urinary metabolite was 7-hydroxy quazodine as shown in Table 8. TABLE 8 THIN-LAYER CHROMATOGRAPHYOF URINARY METABOLITE, 6-OH QUAZODINE, AND 7-OH QUAZODINE

Rf values Solvent system Methanol/acetone/triethanolamine (100:100:3) Chloroform/ether (85:15) Chloroform/acetone/aceticacid (100:2:1)

Urinary metabolite

&OH Quazodine

7-OH Quazodine

0.64

0.82

0.66

0.19 0.33

0.37 0.50

0.19 0.33

METABOLISM

OF QUAZODINE

321

DISCUSSION Clearance of quazodine from human or dog plasma was rapid and appeared to follow a biexponential decay curve. The initial decrease in plasma concentrations can be considered as a composite of two rapid processes : uptake of drug into tissues and metabolism of the drug. The relatively large rate constants for both elimination (k,,) and tissue uptake (k12) suggest that both these processes are of importance in the initial rapid clearance of unchanged quazodine from plasma. The pharmacokinetic analysis suggests that the lower initial plasma concentrations of unchanged drug in subject SBM as compared to subject LHK result from a greater apparent volume in the central tissue compartment of subject SBM rather than a more rapid elimination of drug. In dogs the mean volume of distribution (V,,,) as calculated from VP k12 and k,, was about 1.6 times body weight, which suggests a preferential localization of quazodine in tissues as compared to plasma. The calculations were based on total plasma concentration rather than unbound drug concentrations. If the latter were used, a much larger apparent volume of distribution would be obtained. After oral administration, the absorption of drug was rapid, peak plasma concentrations having been obtained at 30 min in man and at 1 hr in dogs. The finding that a negligible amount of unchanged drug was present in human plasma, suggests that quazodine is metabolized more rapidly by man than by dogs. The substantial (34-fold) difference in dose given to the two species might also be responsible for the pharmacokinetic differences. However, plasma concentrations of drug and metabolites in dogs receiving 10 mg/kg or 0.294 mg/kg of [14C]-quazodine indicate that the drug was handled similarly regardless of the amount given. The excretion data also support this postulate; urinary excretion of radioactivity of metabolites was more rapid in man than in the dog or the rat. The concentration of unchanged drug in human plasma after oral administration was less than would be expected on the basis of the data obtained after intravenous dosage, suggesting that extensive first-pass metabolism of the drug occurred. The finding that cerebrospinal fluid contained only one-fifth as much radioactivity as plasma indicates that either quazodine or its metabolites are partially excluded from the central nervous system. The identity of the radioactivity in the cerebrospinal fluid was not investigated. Although quantitative differences among species were observed in the amounts of drug excreted as glucuronide and sulfate conjugates, the presence of 7-OH quazodine in extracts of glucuronidase-treated urine from all three species suggests a common metabolic pathway, O-demethylation of the methoxy group at the 7-position followed by conjugation of the resulting phenolic hydroxyl group with glucuronic acid or sulfate. Little of the free metabolite, 7-hydroxy quazodine, was present in urine because it is rapidly conjugated prior to elimination. Enzymatic O-demethylation followed by conjugation has been shown to be prominent in the metabolism of other dimethoxy heterocyclic compounds such as papaverine (Axelrod et al., 1958), versidyne (Schwartz et al., 1964), benzquinamide (Wiseman et al., 1964), and tetrabenazine (Schwartz et al., 1966). ACKNOWLEDGMENTS Synthesis of [14C]-quazodine wasperformed by Dr. G. Madding, Department of Chemical Development, Mead Johnson Research Center, and synthesis of authentic 6-OH and 7-OH

322

BARTEK

AND

LABUDDE

quazodine was performed by Mr. C. Hanning, Department of Chemical Research, Mead Johnson Research Center. The human metabolism studies were conducted by Dr. J. W. Goldzieher, Southwest Foundation for Research and Education, San Antonio, Texas, under the direction of Dr. M. 0. Greaney, Clinical Research, Mead Johnson Research Center. REFERENCES L. D., CURRY, L. L. AND BARTEK, J. J. (1970). Rapid method for homogenizing tissue samples for liquid scintillation counting. Clni. Chem. 16,60. AVIADO, D. M., FOLLE, L. E. AND PISANTY, J. (1967).The cardiopulmonaryeffectsof a quinazoline (MJ-1988): Cardiac stimulant, pulmonary vasodilator and bronchodilator. .I.

ADAMS,

Pharmacol.

Exp. Ther. 155,76-83.

J., SHOFER, R., INSCOE, J. K., KING, W. M. AND SJOERDSMA, A. (1958).The fate of papaverinein man and other animals.J. Pharmacol. Exp. Ther. 124,9-15. CARR,P. W., COOPER, T., DAGCIETT, W. M., LISH, P. M., NUGENT, G. G. AND POWERS, P. C. (1967).Effects of compound MJ-1988 on myocardial contractility, oxygen consumption, coronary blood flow and vascularresistance.&it. J. Pharmacol. Chemother. 31,56-65. MADDING, G. D. (1972).A study of the cyclization of 2’-formamido-4’,5-dimethoxypropiophenonewith ammonia.J. Org. Chem. 37,1853-1854. RIEGELMAN, S., Loo, J. AND ROWLAND, M. (1968).Concept of a volume of distribution and possibleerrors in evaluation of this parameter.J. Pharm. Sci. 57, 128-133. SCHMID, H. J., JACKSON, D. P. AND CONLEY, C. L. (1962).Mechanismof action of thrombin on platelets.J. Clin. Invest. 41, 543-553. SCHWARTZ, D. E., BRUDERER, H., RIEDER, J. AND BROSSI, A. (1964). Metabolic Studies of versidyne,a newanalgesic,in the rabbit and in man.Biochem. Pharmacol. 13,777-790. SCHWARTZ, D. E., BRLJDERED, H., RIEDER, J. AND BROSSI, A. (1966). Metabolic studiesof tetrabenazine,a psychotropic drug in animalsand man. Biochem. Pharmacol. 15,645-655. WISEMAN, E. H., SCHREIBER, E. C. AND PINSON, R. J. (1964).The distribution excretion and metabolismof benzquinamide.Biochem. Pharmacol. 13,1421-1435. AXELROD,