Xenobiotica the fate of foreign compounds in biological systems

ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20

Metabolism of a Benzothiazine compound (SQ 11,579) by the Intact Rat, Isolated Perfused Rat Liver, and Rat-Liver Microsomes S. J. Lan, A. V. Dean, B. D. Walker & E. C. Schreiber To cite this article: S. J. Lan, A. V. Dean, B. D. Walker & E. C. Schreiber (1976) Metabolism of a Benzothiazine compound (SQ 11,579) by the Intact Rat, Isolated Perfused Rat Liver, and RatLiver Microsomes, Xenobiotica, 6:3, 171-183 To link to this article: http://dx.doi.org/10.3109/00498257609151627

Published online: 30 Sep 2009.

Submit your article to this journal

Article views: 1

View related articles

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [University of Sheffield]

Date: 14 September 2015, At: 02:12

XENOBIOTICA,

1976, VOL. 6,

NO.

3, 171-183

Metabolism of a Benzothiazine compound (SQ 11,579) by the Intact Rat, Isolated Perfused Rat Liver, and Rat-Liver Microsomes S. J. LAN, A. V. DEAN, B. D. WALKER and E. C. SCHREIBER Drug Metabolism Department, The Squihb Institute for Medical Research, New Brunswick, N. J. 08903, U.S.A.

Downloaded by [University of Sheffield] at 02:12 14 September 2015

(Received 30 April 1975)

1. The metabolic dispositions of a benzothiazine compound (SQ 11,579) by the intact rat, isolated perfused rat liver, and rat-liver microsomes have been investigated, and the results compared. 2. The drug was well absorbed after oral administration to rats and was widely distributed in all tissues, which, with the exception of brain, had higher concentrations of the drug, its metabolites, or both, than did plasma. 3. Metabolism by rat-liver microsomes included N-oxidation, N-demethylation, .S-oxidation and aryl hydroxylation. Metabolites hydroxylated in the aromatic ring were excreted only in bile, both by the isolated perfused rat livers and by anaesthetized bile-duct-cannulated rats. 4. Liver perfusion of the benzothiazine or its monodesmethyl analogue (V) resulted in temporary cessation of the flow of perfusate through the organ. The benzothiazine sulphoxide (IV) had only a slight effect on the flow of liver perfusate, but IV followed by I caused the flow of perfusate to cease.

Introduction Of various systems used to study the metabolism of drugs, the most common are tissue homogenate, microsomal preparations, perfusion of isolated organ, and intact animal. Metabolic studies with liver homogenate or microsomes provide simple procedures useful for observation of single enzymic reactions, but because they involve destruction of cellular integrity and variation in the conditions of incubation, they often fail to reveal the complete metabolism of the drug. Studies of drug metabolism in intact animals, on the other hand, provide little information about intermediate biotransformation of the drug, since only ' terminal ' metabolites are excreted in urine and faeces. The perfusion of an isolated organ (ex vim) can provide a system that permits study of the various steps of drug biotransformation while preserving the integrity of cellular structure. Few studies have been made of the comparative metabolism of a single compound by various systems. We now present a study of the metabolism of a benzothiazine compound in four systems, namely, rat-liver microsomes, isolated perfused rat livers, rats with cannulated bile duct, and intact rats. 4- [3- (Dimethy1amino)prop yl] -3,4-dihydro-2-( 1-hydroxyethyl)-3-phenyl2H-1,4-benzothiazine (I, SQ 11,579), is a non-steroidal anti-inflammatory agent with analgesic activities (Krapcho & Turk, 1973 ; Millonig et al., 1973). Metabolism of the benzothiazine I by rat- or dog-liver microsomes, and the isolation and identification of the urinary metabolites from rat, dog, and monkey have been reported (Lan et al., 1973a). The drug is metabolized extensively,

172

S . J . Lan et al.

Downloaded by [University of Sheffield] at 02:12 14 September 2015

both in vitro and in vivo, in all three species, and N-demethylation, sulphoxidation, N-oxidation, aromatic hydroxylation, and conjugation with glucuronic acid, have been demonstrated.

Experimental Materials 4- [3 - (Dimethylamino)propy1]-3,4-dihydro-2-(I-hydro~y[2-~~C]ethy1)-3phenyl-2H-l,4-benzothiazine (14C-SQ 11,579) was synthesized as previously reported (Krapcho & Turk, 1973), and had radiochemical purity of 99.8% by t.1.c. in three solvent systems and by radioautography (Lan et al., 1973 a). T h e sulphoxide (IV) and the monodesmethyl (V) derivatives of SQ 11,579 were synthesized by Dr. J. Krapcho at the Squibb Institute for Medical Research. Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Wilmington, Mass.), bovine serum albumin (Fraction V) (Sigma Chemical Co., St. Louis, Mo.), ketodase (/3-glucuronidase, Warner-Chilcott, Morris Plains, N. J.), Quanta-gram Q-1 -F pre-coated silica-gel thin-layer plates, (Quantum Industries, Fairfield, N. J.), and all other chemicals, of reagent grade, were purchased. Methods Oral or intraperitoneal administration to intact rats : Male rats (approx. 300 g) each received 15 mg of 14C-SQ 11,579 (50 mg/kg, and 0*257pCi/mg)in 3 ml water by gavage daily for 3 days. Rats were housed in individual metabolism cages and urine and faeces were collected daily and frozen immediately after collection. When the drug was administered intraperitoneally, a single dose of 14C-SQ 11,579 (50 mg/kg) in distilled water was given to each rat, and the urine and faeces were collected daily for 3 days. Intravenous administration to anaesthetized bile-duct-cannulated rats : Two male rats, 300-350 g, fasted for 24 h were anaesthetized by injection of 0.1 ml of pentobarbital (6y0w/v). The bile duct was cannulated with PE-10 Intramedic polyethylene tubing (Clay Adams), and bile was collected in a graduated centrifuge tube. T h e urinary bladder was then cannulated with PE-50 Intramedic polyethylene tubing and urine was collected. At hourly intervals, 1 ml of mannitol buffer (K,HPO,, 450 mg ; KH,P0,,100 mg ; mannitol, 50 g ; in 1 litre water) was injected into the abdominal cavity through a catheter. After urine and bile had been collected for 1 h, a dose of 14C-SQ 11,579 (25 mg/kg) was administered via the inferior vena cava. Urine and bile were collected at I-h intervals for 4 h. Tissue distribution: A group of eight male rats, approximately 300g each, were fasted for 24 h then given a single dose of 14C-SQ 11,579 (50 mg/kg in water by gavage). The animals were housed in individual metabolism cages, expired CO, was trapped in a mixture of 2-methoxyethanol, 630 ml ; phenylethylamine, 450 ml ; toluene, 720 ml ; Z,S-diphenyloxazole, (PPO) 10.8 g ; and 1,4-bis-[2-(5phenyloxazolyl)]benzene (POPOP) 0-36g, and the 14C0, measured. At various times after dosing, the rats were sacrificed by exsanguination via the portal vein. The heparinized blood was centrigufed at 2000 r.p.m. for 10 min to separate plasma from red cells. Other tissues were excised and blotted with absorbent paper before radioassay. Urine and faeces were collected when available.

Downloaded by [University of Sheffield] at 02:12 14 September 2015

Metabolism of a Benzothiazine in Rat

173

Liver perfusion : Male rats, 300-400 g, were maintained on laboratory diet and water ad lib. T h e liver perfusion was performed by the method of Miller et al. (1951) with slight modification (Hagino et al., 1968). The perfusate, consisting of 50 ml of Krebs-Henseleit Ringer bicarbonate solution, p H 7-4, (Krebs & Henseleit, 1932) containing 4.5% bovine serum albumin (Fraction V) was oxygenated with a mixture of 0, and CO, (95 : 5 ) . After 15 to 30 min of perfusion, a flow rate of approximately 1 ml/min per gram of liver was established. The temperature in the perfusion chamber was maintained at 38 & 1 '. Experiments were initiated by addition of the test drug to the perfusate reservoir. The liver was perfused for varying lengths of time. A 0.1 ml aliquot of perfusate was removed periodically for measurement of radioactivity. 14C0, evolved during the perfusion was trapped in 20 ml of the previously described scintillant mixture at 30-min intervals. To measure the flow rate of perfusate, the efflux from the liver was disconnected from the reservoir, collected in a graduated cylinder and returned to the reservoir periodically. At various times, the volume of perfusate collected in a period of 2 min was measured. Bile produced by the perfused liver was collected in a graduated centrifuge tube during the entire perfusion period. Measurement of radioactivity : Measurement of radioactivity in blood, plasma, bile, urine, faeces, and tissues was as described previously (Lan et al., 1973 b). Isolation and identiJcation of radioactive metabolites : At the conclusion of the perfusion, the liver was homogenized with 3 vol. (w/v) of distilled water. The homogenate was then adusted to at least pH10 by addition of ammonia soln. (sp. gr. 0.88) and extracted twice with 3 vol. of ethyl acetate. The aqueous fraction, after ethyl acetate extraction, was then brought to 50% (w/v) with ammonium sulphate and the radioactive compounds not previously extracted were extracted into 3 vol. of ether-ethanol mixture (3 :1, v/v). I n this manner, more than 90% of the radioactivity in the liver homogenate was removed from the aqueous phases. The extracts were combined and the organic solvents removed by distillation in vacuo at 45 '. T h e radioactive metabolites in the perfusate or in the urine samples obtained from 2 bile-duct-cannulated rats were extracted by the same method, and extraction from perfusate or urine was found to be as complete as that from liver homogenate. T h e radioactive metabolites in bile were chromatographed directly without extraction. T h e radioactive metabolites from the perfused liver, perfusate, urine, and bile were separated and identified by methods reported elsewhere (Lan et al., 1973 a).

Results Metabolism of 14C-SQ 11,579 by conscious intact rats or anaesthetized bile-ductcannulated rats Rats that had received a 25- or 50-mg/kg dose of 14C-SQ 11,579 orally or intraperitoneally excreted most of the administered 14C in faeces (Table 1). After intravenous administration of 14C-SQ 11,579 to anaesthetized bile-ductcannulated rats (25 mg/kg), 10 and 37'76 of the administered 14C was excreted in urine and bile, respectively, within 4 h (Figure 1). Based on the ratio of 14C

S. J. Lan et al.

174

Downloaded by [University of Sheffield] at 02:12 14 September 2015

Table 1. Percentage recovery of radioactivity in urine and faeces after oral or intraperitoneal administration of 14C-SQ 11,579 to rats

%

Route of administration

Number of animals

Dose (mg/kg)

Duration (day)

Urine

Faeces

Intraperi toneale Oral? Intraperitoneale Oralt

3 4 3 3

50 50 25 25

3 5 3 3

19-6 17.9 24.4 22.6

59.7 66.2 70.2 71.2

recovered Total

79.3 84.1 94.6 93.8

* Each value is the mean obtained from three rats after a single dose of l4C:SQ 11,579. t Each value is the mean obtained from four rats. Each rat was given a dose of 14C-SQ 11,579 daily for 3 days; urine and faeces were collected daily for 5 days.

I

40 -0

c

-

P 10

P

1 2 3 Time after dosage (h)

4

Fig. 1. Cumulative excretion of radioactivity by anaesthetized bile-duct-canmlated ruts after intravenous administration of 14CC-SQ11,579 (25 mg/kg). A A,Bile; 0 0 , Urine.

excreted in urine and faeces after intraperitoneal and oral administrations, it is estimated that at least 90% of the orally administered 14C-labelled drug was absorbed by the rats (Table 1). Eight rats were used for a sequential tissue distribution study. One animal was used for each time point. After oral administration of 14C-SQ 11,579 (50 mg/kg), the plasma level of the unchanged drug and metabolites ranged from 1-3to 2*8pg/mlduring the first 2 h (Table 2). Between 2 and 24 h after administration of the labelled drug the biological half-life (tOJ of radioactivity in plasma was approx. 8h. Among the different tissues analysed, liver, lung, kidney, adrenal, spleen, and heart contained concentrations of radioactivity at least ten times greater than that of plasma (Table 2). The other tissues had concentration

1269 256 3 230 41 1*8 2.4 3 5 67 28 4 20 8 2 7 21 1 3 1 -

-

-

1465 283 10 152 41 1-3 1.7 4 6 110 39 6 19 15 3 7 28 2 3 0.5

-

-

1548 132 8 68 31 1*3 1-6 3 7 63 25 5 11 9 4 3 22 1 2 1 -

12 2 11 37 2 3 0.9

18

601 357 9 159 43 1*7 2.9 7 8 86 29 6

-

8.8%

-

4.2%

o*lyo

13 16 22 2 9 33 4 4 1

34 229 1128 61 31 2.0 3.2 10 10 122

-

2.8 3.3 8 12 131 39 10 25 15 3 10 42 2 5 1

44

1043 245 21 112

$ Including the contents.

$ Concentrations at each time point were determined in tissues taken from a single rat.

t IZgld.

* Expressed as pg/g of tissue.

Urine (% of administered dose) Faeces(% of administered dose) CO, (yoof administered dose)

Lung Adrenal Skeletal muscle Heart Thymus Adipose Bone Spleen Testis Eye Brain

carcass

Stomach$ Small intestines Colon$ Liver Kidney Plasmat Blood? Skin

Hours

0.02%

-

8.4%

26 131 1718 51 26 2.0 34 5 6 76 1.5 7 10 13 2 7 20 4 3 1

0.04%

14.8% 33.8%

3 134 347 21 6 0.7 3.5 1 3 7 2 1 1 1 0.4 2 3 2 0.4 0

Table 2. Concentration of SQ 11,579 and its metabolites in tissues* at various intervals after oral administration of W-SQ 11,579 (50 mg/kg) to rats

Downloaded by [University of Sheffield] at 02:12 14 September 2015

S.J . Lan et al.

Downloaded by [University of Sheffield] at 02:12 14 September 2015

176

of radioactivity equal to or slightly greater than that of plasma. Only the brain had a lower level of radioactivity than plasma. During the first 2 h after administration of drug, the concentrations of SQ 11,579 and its metabolites in liver were equivalent to 68 to 230 pglg. Higher concentrations were also found in lungs and kidneys, 63 to 131 and 31 to 44pg/g, respectively. These results suggest that not only is 14C-SQ 11,579 well absorbed by rats, but it is also widely distributed among various tissues. Figure 2 depicts the structure of the benzothiazine, SQ 11,579, and its metabolites. T h e distribution of radioactivity among the metabolites excreted in urine by rats after oral administration of W - S Q 11,579, as well as in urine and bile excreted by the anaesthetized rats after intravenous injection of the labelled drug, is shown in Table 3. Intact rats excreted less than 5% of the total administered 14C in urine as unchanged drug and approximately 25% as conjugates. Compounds derived from N-demethylation and sulphoxidation (V and VI) accounted for almost all of the known unconjugated metabolites. No N-oxide or aromatic hydroxylated derivatives (I1 and 111) were detected. Similar results were obtained from the urine samples excreted by the anaesthetized bile-ductcannulated rats within 4 h after the intravenous injection of 14C-SQ 11,579. Excretion of a larger percentage of unchanged drug by the cannulated rats than by the intact rats may be due to the elimination of enterohepatic circulation in the former. Again, metabolites derived from N-demethylation and sulphoxidation accounted for all the known unconjugated metabolites. T h e radioactive compounds excreted in bile were all conjugated. Hydrolysis of the conjugates with p-glucuronidase yielded I, 111, V, VI, and VIII. VIII, which yielded a blue spot after being sprayed with phenolic reagent (Folin & Ciocalteau, 1927), is probably the hydroxy derivative of V, but its identity has not yet been firmly

Substituent Compound R1

I I1 I11

IV

V VI VII VIII

R3

R4

R5

CH, CH3 CH3 CH3 CH3 CH3 H CH3

Fig. 2. Structure of T - S Q 11,579 and its metabolites.

177

Metabolism of a Benzothiazine in Rat

Downloaded by [University of Sheffield] at 02:12 14 September 2015

Table 3. Percentage distributionof radioactivity among the metabolites excreted in urine or bile by rats after oral or intravenous administration of 14C-SQ 11,579 Compound

Intact rats* Urine

Unchanged drug I1 I11 IV V VI VIII Conjugates Unknown

4.3 0 0 0 30-2 28.3 0 24.5 12-7

Anaesthetized bile-duct-cannulated ratst Urine Bile

35.2 0 0 9.7 18.4 17.5 0 19.2 0

6-4 0

15.0 0 15.1 10.4 13.3 25.9 13.8

The figures given are percentages of total radioactivity. Pooled 5 day urine samples from a group of four male rats; each received a 50 mg/kg dose of 14C-SQ11,579 by gavage daily for 3 days (containing 18% of the administered dose). These data are published elsewhere (Lan et al., 1973 a). Pooled 4 h urine and bile samples collected from two rats after intravenous administration of a single dose of '*C-SQ 11,579 (25 mg/kg). All radioactive materials excreted in bile were conjugated ; numbers shown here were obtained after incubation with P-glucuronidase.

established. No N-oxide (11) was detected in urine or bile. The aromatic hydroxylated metabolites (I11 and V I I I ) were found to be preferentially excreted in bile. Neither I11 nor V I I I was found in the urine.

Effect of SQ 11,579 or its metabolites on theflow rate of perfusate During our studies of the metabolism of 14C-SQ 11,579 with the isolated perfused rat liver, we observed that the benzothiazine greatly reduced the flow of perfusate through the liver. SQ 11,579 (50pmol in 2 ml of water) was introduced into the perfusate reservoir. Approximately 3min later, the flow of perfusate through the liver began to fall drastically ; in this interval, 50 ml of perfusate completed one circulation through the organ. Five minutes after the introduction of SQ 11,579 into the perfusate, the flow of perfusate was reduced by 60%; after 10 min, the flow of perfusate had decreased by 95 ; it almost ceased for the next 10 to 15min, then gradually returned to normal during the next hour. Addition of a second challenge of the benzothiazine was without effect (Figure 3). The introduction of the monodesmethyl analogue (V), a major metabolite formed by rat-liver microsomes, into the perfusate had an effect similar to, but less extensive than that of SQ 11,579. At 1 mM, V was only approximately 70% as effective as the parent drug. When V was followed by SQ 11,579, no decrease of the flow of perfusate was observed (Figure 3). T h e sulphoxide analogue (IV), another metabolite of SQ 11,579 formed by rat-liver microsomes, had only a slight effect on the flow of perfusate through the liver. At 1 mM, I V was only 23 yoas effective as the parent drug. However,

5'. J . Lan et al.

178 iE 16

14

-g

- 12 E

Y

0)

c

g 10

Downloaded by [University of Sheffield] at 02:12 14 September 2015

3

'c

&

n 0)

c

e

k 6

ii

4

2 I

I

60

I

120 Time (min)

p 0-

L

.-.,

Efect of SQ 11,579, metabolite IV, or metabolite V on thejYow rate of perfusate through the isolated rat liver. ), first challenge with SQ 11,579, metabolite IV, or metabolite V ; ( J. ), second challenge with SQ 11,579.

Fig. 3. (

I

180

0,

SQ 11,579;

A-

A,Metabolite IV;

Metabolite V.

recovery from the decrease in flow of perfusate induced by IV occurred at a much slower rate than that after induction by SQ 11,579. At 1.5 h after the addition of IV to the perfusate, the flow of perfusate had recovered by only 30%, whereas 1.5 h after the addition of SQ 11,579 the flow of perfusate had returned to the predose rate. Unlike SQ 11,579 and V, when IV was followed by the parent drug, the flow was again drastically reduced (Figure 3). These phenomena were observed in at least two separate experiments.

Metabolism of 14C-SQ 11,579 by the isolated perfused rat liver When 50pmol(3-6pCi)of 14C-SQ 11,579 were added to the perfusate (50 ml), the disappearance of radioactivity from the perfusate appeared to be multiphasic. Immediately after the addition of the labelled drug to the perfusate, there was a quick uptake phase. Five minutes later approximately 25% of the added 14C was found to be taken up by the liver. At 5 to 30min after the addition of the labelled drug, there was a lag period for the disappearance of 14C from the

179

Downloaded by [University of Sheffield] at 02:12 14 September 2015

Metabolism of a Benzothiazine in Rat

perfusate, which was due to the cessation of the flow of perfusate as described earlier. After 30 min, the flow of perfusate gradually returned to normal and the uptake of 14Cby the perfused liver was resumed. The radioactivity in the perfusate was found to have a of about 1 h at 30 to 90 min after the addition of lacSQ 11,579. After 2 h of perfusion, approximately 25% of the 14Cwas found in the perfusate and this value did not change in two more hours of perfusion. The of 14C-SQ 11,579 in the perfusate was 36min. Corresponding to the disappearance of 14C-SQ 11,579 from the perfusate, the amount of metabolites in the perfusate gradually increased (Figure 4). The formation of 14C0, from 14C-SQ 11,579 by the isolated perfused rat liver demonstrated the degradation of the 2-( 1-hydroxyethyl) side chain. A linear relationship between the amount of 14C0, formed and the perfusion time was observed after the flow of perfusate had returned to the pre-dose rate. A lag period of 14C0, formation was observed from 0 to 90 min, due to the cessation of

Time (min)

.,

Fig. 4. Disappearance of W - S Q 11,579 and formation of its metabolites in perfusate of isolated perfwed rat liver. @ --- @,

--*,

total radioactivity; A-

A,SQ 11,579; Metabolite VI.

a-

Metabolite V;

~~

26.30

72.08

1.62

-

-

24.38

0.67 73.60

31.25 0.74

-

-

41.61

Liver

2.01

-

0.68

16.74

-

1.45

-

0.06

-

1.60 7.77 2.52

-

-

3.40

0-37

0.69 0.21

Bile$. Perfusate

72.30

0.87

-

-

2.38

-

-

47.24

18.01 3.80

Liver

3.40 0.29

1.43

-

-

1.23

1-57 3.05

Bile$.

10.97

Perfusion 3 (4 h)

t

Expressed as percentage of total radioactivity administered. 14C-SQ 11,579 (15 mg) was used in all three perfusions. $. All radioactive compounds excreted in bile were conjugated; numbers shown here were obtained after incubation with B-glucuronidase.

#

Total

-

0.24

1.94

-

10.48 1.36

-

0.41

20.66 0-94

8.43 2.03

-

11.87

0.89 0.08

47.62 0.92

-

15.84

Unchanged drug I11 IV V VI VII VIII Unhydrolysed Conjugates Unknown

Rile$. Perfusate

Liver

Perfusate

Perfusion 2 (2 h)

Compound

Perfusion 1 (2 h)

Distribution of radioactivity"

Table 4. Percentage distribution of radioactivity among the metabolites isolated from liver, perfusate and bile

Downloaded by [University of Sheffield] at 02:12 14 September 2015

Downloaded by [University of Sheffield] at 02:12 14 September 2015

Metabolism of a Benxothiaxine in Rat

181

the flow of perfusate. After 4 h perfusion, 0.1 yoof the total added 14Cwas metabolized to 14C02by the isolated perfused rat liver. Expiration”of 14C02by conscious intact rats given 14C-SQ 11,579 orally was also detected (Table 2). T h e radioactive materials in the perfusate, liver, and bile samples obtained after perfusion were isolated and identified according to the procedures described under ‘ Methods ’ Several metabolites were obtained. The percentage distribution of radioactivity among the metabolites formed by the’isolated perfused rat liver is shown in Table 4. After 2 h perfusion, approximately 25% of the added 14Cremained in the perfusate, 73 ”/o was found in the liver, and 2% was excreted in bile. After 2 h perfusion, the perfusate contained 16 and 12% of unchanged 14C-SQ 11,579 in two different experiments. Two metabolites (V and VI), derived from N-demethylation and sulphoxidation, were found in the perfusate. After 4h perfusion, the amount of unchanged drug in the perfusate decreased corresponding to an increased formation of metabolites. The metabolites isolated from the liver were the same as those found in the perfusate, mainly V and VI. A hydroxylated metabolite (111) was also found in the liver. No N-oxide was detected. All metabolites excreted in bile were conjugated ; hydrolysis of these conjugates with p-glucuronidase yielded I, 111, V and VIII. T h e hydroxylated metabolites (I11 and VIII) were, again, excreted only in bile.

Discussion 14C-SQ 11,579 was well absorbed after oral administration to rats. The low concentration of the drug in plasma may result from the wide distribution of the drug in all tissues (Table 2) and the quick elimination of the drug and its metabolites (Figure l). The distribution pattern of SQ 11,579 in rats is similar to that of the phenothiazines, e.g. chlorpromazine (Curry, Derr & Maling, 1970; Forrest, Bolt & Serra, 1968 ; Mahju & Maickel, 1969). T h e metabolic patterns of SQ 11,579 and the phenothiazines were also similar (Lan et al., 1973 a ;Emerson, & Miya, 1963 ; Beckett, Beaven & Robinson, 1963 ; Fishman, Heaton & Goldberg, 1963; Fishman & Goldberg, 1965). However, the concentrations of SQ 11,579 and the phenothiazines in brain were different. Phenothiazines have been demonstrated to have a greater concentration in brain than in plasma of rat and dog (Curry et al., 1970 ; Mahju & Maickel, 1969), whereas the brain is the only organ that has a lower concentration of SQ 11,579 and its metabolites than does the plasma of rats. The metabolic picture of SQ 11,579 is complex. Six different metabolic reactions have been demonstrated. Furthermore, the primary metabolites identified, namely, 11, 111, IV, and V, were further metabolized to the secondary metabolites, VI and VII. T h e metabolites formed under three experimental conditions were qualitatively the same, but varied quantitatively. N-Demethylation, sulphoxidation, and conjugation with glucuronic acid are the three major metabolic reactions of SQ 11,579. Using rat-liver microsomes, we were able to demonstrate all the metabolic reactions except conjugation with glucuronic acid (Table 5 ) . Conjugation did not take place because UDP-glucuronic acid was absent from the incubation mixture in these studies. The metabolites formed by rat-liver microsomes were essentially primary metabolites. On the other hand, the metabolites excreted in urine by the intact rats contained a large percentage of

S.J.Lan et a1.

182

Table 5. Percentage distribution of radioactivity among the metabolites found after incubation of rat-liver microsomes with I4C-SQ 11,579 Compound

Downloaded by [University of Sheffield] at 02:12 14 September 2015

Unchanged drug I1 I11 IV V VI Unknown

Total radioactivity Metabolites formed

(%>

(%>

41.3 3-9 5.3 7.4 35.9 2.4 3.8

6.7 9.3 12.6 61.2 4.1 6.5

Incubation system Each millilitre of incubation mixture contained the following amounts of cofactors: MgCl,, 10 pmol ; NADPt, 1 pmol ; glucose-6-phosphate, 10 pmol; glucose-6-phosphate dehydrogenase, 0.5 units; "C-SQ 11,579, 1.3 pmol; and microsomes from 0.1 g of fresh liver. Reaction was carried out at 37" for 3 h under aerobic conditions. These data are published elsewhere (Lan et al., 1973 a).

the secondary metabolite VI. The metabolites of SQ 11,579 formed by the isolated perfused rat liver contained both primary and secondary metabolites. With the isolated perfused rat liver and the anaesthetized bile-duct-cannulated rats, we also demonstrated the preferential excretion of the glucuronide conjugates of I11 and VIII in bile. However, it is not known why the aryl hydroxylated metabolites are excreted exclusively in bile. Perfusion of the isolated liver is not only a useful tool for the study of drug metabolism, but may also provide a means to study the effects of drugs on liver function. SQ 11,579 and its monodesmethyl analogue (V) markedly reduced the flow of perfusate through the liver, then the flow gradually returned to normal. A second challenge with SQ 11,579 was without effect, for reasons that are not apparent. The gradual return of perfusate flow to normal after a temporary cessation could not be attributed to the removal of SQ 11,579 via its conversion to inactive metabolites, for a substantial percentage of the unchanged drug was still present in liver and perfusate after 2 or 4 h of perfusion. Furthermore, the major metabolite V had the same effect as the parent drug on the flow of perfusate. Several phenothiazine derivatives and an anabolic steroid (Norbolethone) have been reported to decrease the flow of perfusate and bile secretion in the isolated perfused rat liver (Plaa et al., 1960 ; Bassan et al., 1971 ;Kendler et al., 1971). A similar effect for mice dosed with chlorpromazine has also been reported (Eckhardt & Plaa, 1962). Plaa et al., (1960) reported that two phenothiazine derivatives, thioridazine and chlorpromazine, increase hepatic vascular resistance in isolated perfused rat liver. These substances produce a decrease in perfusate flow through the organ, an increase in portal pressure, and a decrease in organ weight. Because of similarities between SQ 11,579 and the phenothiazines in metabolic pattern, tissue distribution, and the effect on perfusate flow and bile secretion in the isolated perfused rat liver, it is suggested that the benzothiazine, SQ 11,579 and its metabolites affect the function of liver by the same, as yet

Metabolism of a Benxothiaxine in Rat

183

unknown, mechanism as do the phenothiazines. The decrease in perfusate flow induced by SQ 11,579 and its metabolites may contribute to the hepatotoxicity of the drug and its metabolite, V, that has been observed in rats after the chronic administration of these substances (unpublished).

Reference BASSAN, H., KENDLER, J., HARINASUTA, U. &ZIMMERMAN, H. J. (1971). Biochem. Pharmac.,

Downloaded by [University of Sheffield] at 02:12 14 September 2015

20, 1429. B E ~A. H., , BEAVEN, M. A. & ROBINSON, H. Z. (1963). Biochem. Pharmac., 12, 779. CURRY, S. H., DERR,J. E. & MALING, H. M. (1970). Proc. SOC. exp. Biol. Med., 134, 314. ECKHARDT, E. T.& PLAA,G. L. (1962). J. Pharmac. exp. Ther., 138, 387. EMERSON, J. L. & MIYA,T. (1963). J. Pharm. Sci., 52, 411. FISHMAN, V.,HEATON, A. & GOLDBERG, H. G. (1963). Proc. SOC.exp. BioZ. Med., 112, 501. FISHMAN, V. & GOLDBERG, H. G. (1965). J. Pharmac. exp. Ther., 150,122. FOLIN,0 . & CIOCALTEAU, V. (1927). J. biol. Chem., 73, 627. FORREST, I. S.,BOLT,A. G. & SERRA, M. T. (1968). Biochem. Pharmac., 17, 2061. HAGINO, Y.,LAN,S. J., NG, C. Y. & HENDERSON, L.M. (1968). J. biol. Chem., 243,4980. KENDLER, J., BOWRY, S., SEEFF,L. B. & ZIMMERMAN, H. J. (1971). Biochern. Pharmac.,

20, 2439. KRAPCHO, J. & TURK, C. F. (1973). J. med. Chem., 16, 776. KREBS,H.A. & HENSELJIIT, K. (1932). Z . Phys. Chem., 210, 33. LAN,S. J., CHANDO, T. J., COHEN,A. I., WELIKY,I. & SCHREIBER, E. C. (1973a). Drug Metab. Disp., 1, 619. LAN, S. J., CHANDO, T. J., WELIKY, I. & SCHREIBER, E. C. (1973 b). J. Phurmuc. exp. Ther., 186, 323. MAHJU,M. A. & MAICKEL, R. P. (1969). Biochem. Pharmac., 18, 2701. MILLER, L. L., BLY,C. G., WATSON, M. L.& BALE,W. P. (1951). J. exp. Med., 94,431. MILLONIG, R. C., GOLDLUST, M. B., MAGUIRE, W. E., RUBIN,B., SCHULZE, E., WOJNAR, R. J., TIJRKHEIMER, A. R., SCHREIBER, W. F., BRITTAIN, R. J. (1973). J. med. Chem., 16, 780.

PLM, G. L.,MCGOUGH, E. C., BLACKER, G. J. & FUJIMOTO, J. M. (1960). Am. J. PhySioZ.,

199,793.

Metabolism of a benzothiazine compound (SQ 11,579) by the intact rat, isolated perfused rat liver, and rat-liver microsomes.

1. The metabolic dispositions of a benzothiazine compound (SQ 11,579) by the intact rat, isolated perfused rat liver, and rat-liver microsomes have be...
852KB Sizes 0 Downloads 0 Views