A c f a pharmacol. e f foxicol. 1981, 49, 116-124.

From the 'Chemical Institute BC, Royal Danish School of Pharmacy, Copenhagen, and the 2Research Laboratories, H. Lundbeck & Co. A/S, Copenhagen, Denmark

Preliminary Studies on the Absorption, Distribution, Metabolism, and Excretion of THIP in Animal and Man using ''C-labelled Compound

BY Birgit Schultz,' Tove Aaes-Jsrgensen,' Klaus Peter Bsgesd and Aksel Jsrgensen2

(Received December 11, 1980; Accepted January 15, 1981)

Absrracf: Distribution of radioactivity in rats, serum levels in human volunteers and rats and elimination of radioactivity in volunteers, rats, and mice following oral administration of ''C-labelled THIP have been investigated. Peak values of radioactivity in the organs and in serum were seen half an hour after administration, indicating a rapid absorption. Highest concentrations of radioactivity were found in the kidneys, but radioactivity was seen in all investigated tissues including the brain. The radioactivity was mainly excreted with urine (84-93%). Thin-layer chromatography of urine from volunteers, rats, and mice showed that most of the excreted radioactivity corresponds to unchanged THIP. Three metabolites were found in urine from rats each in amounts of 2-7 % of the total dose given. Two of these metabolites were also found in urine from the volunteers in amounts of 30-35% and <2%, respectively, and in urine from mice in amounts of 21 % and 6% of the total dose, respectively. No radioactivity corresponding to unchanged THIP was found in faeces indicating complete absorption of THIP following oral administration. One of the metabolites, the main one in man and mouse, seemed to be a glucuronic acid conjugate of THIP, but the chemical structure of the metabolites has not yet been established.

Key-words: THIP - GABA-agonist - biotransformation - rats - mice - man.

Decreased GABA activity seems to be involved in certain neuroleptical and psychiatric disorders (Lloyd et a/. 1975; McGeer & McGeer 1976; Coyle et a/. 1977; Enna e ta / . 1977), for which reason the development of specific GABA agonists with phar- macokinetic and toxicological properties accept- able for use in the human clinic is of great interest.

Muscimol is a potent GABA agonist (Naik et a/. 1976; Scheel-Kriiger et a/. 1977), but too toxic for use in the human clinic. Among a number of muscimol analogues synthetized THIP has been found to be a potent and specific GABA agonist (Scheel-Kriiger et a/. 1977 & 1979; Krogsgaard-

Larsen et a/. 1977). The pharmacological studies have shown that THIP is active after systemic administration in a variety of animal models (Christensen et a/. 1979).

The present publication deals with preliminary studies on serum levels and elimination in man and on the absorption, distribution, metabolism, and excretion of THIP in mouse, and rat.

Materials and Methods

LabeNed compound. Carbon-14 labelled THIP, HBr, ([7a- ''C]-3-Hydroxy-4,5,6,7-tetrahydroisoxazolo [5,4-c] pyri-

*Proofs: Tove Aaes-Jmgensen, H. Lundbeck & Co. A h , Ottiliavej 7-9, DK-2500 Valby, Denmark

BIOTRANSFORMATION O F THIP 117

Fig. 1. Structure ofTHIP, HBr([7a-14C]3-hydroxy-4,5,6,7 -tetrahydroisoxazolo [5,4-c] pyridinium bromide). The asterisk indicates the 14C-labelled atom.

dinium bromide) was synthetized by using bromo [1-14C] acetate as the starting product. The carbon-14 atom was placed in the molecule as shown in fig. 1.

The radiochemical purity as determined by thin-layer chromatography in solvent system I and I 1 was >98%.

The specific activity of the administered compound was 11.6 pCi/mg I4C-THIP, HBr.

Experimental. 1. Rat srudy. elimination. Male rats (Wistar/Af/Han/ Mol (Han 67) SPF) weighing 110-120 g were used. Ten rats were given 10 mg/kg of I4C-THIP, HBr (calculated as the zwitterion) and 190 mg/kg ofunlabelledTHIPand 10 rats were given 5 mg/kg of I4C-THIP, HBr (calculated as the zwitterion). Both doses were given orally in water (10 ml/kg). Urine was collected from both groups in the periods 0-4, 4-8, 8-12, 12-24, and 24-48 hours after administration. From the 5 mg/kg group urine was furthermore collected in the period 48-72 hours. From this group faeces were collected in the same intervals as the urine, while faeces were collected in the periods 0-8, 8-24, and 24-48 hours after administration in the high dose group. Total radioactivity was estimated in all samples and in addition all urine samples, all faeces samples from the high dose group, and faeces from the low dose group collected at 12-24 hours after administra- tion were subjected to thin-layer chromatography (TLC).

2. Mouse study, elimination. Ten male NMRI/BOM, SPF mice weighing 20-25 g were given 5 mg/kg of I4C- THIP, HBr (calculated as the zwitterion) orally in water (10 ml/kg). Urine was collected in the periods 0-4, 4-8, 8-12, 12-24, and 24-48 hours after administration. Faeces were collected 0-8, 8-24, and 24-48 hours after administration. Total radioactivity was estimated in all samples. Furthermore urine samples collected until 24 hours and faeces samples collected in the period 0-8 hours after administration were investigated by TLC.

3. Human study, elimination and serum levels. Two male volunteers participated in the study. Their data were as follows: Subject AJ 40 years 73 kg body weight Subject NL 51 years 60 kg body weight The subjects received a dose of 10 mg I4C-THIP, HBr (calculated as the zwitterion) dissolved in 50 ml tap water followed by 50-100 ml tap water. Urine was collected in

the periods 0-1, 1-2, 2-3, 3-4, 4-6, 6-8, 8-10, 10-12, 12-24, 24-36, and 36-48 hours after administration. Faeces were collected 0-24, 24-48, and 48-72 hours after administration. Blood samples were drawn after y4, %, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours. Serum was obtained by centrifugation. Total radioactivity was measured in all samples. Urine samples collected until 12 hours (AJ) or 24 hours (NL) and serum samples from both subjects collected y4, %, 1, and 2 hours after administration were investigated by TLC.

4. Rat study, distribution and serum levels. Sixteen male Wistar/Af/Han/Mol (Han 67), SPF rats (125-150 g) were given 5 mg 14C-THIP, HBr/kg (calculated as the zwitterion) orally in water (10 ml/kg). Groups of two rats were killed by exsanguination under ether anaesthesia 72, I , 1 %, 2,3,6, 10, and 24 hours after administration. Brain, liver, lungs, kidneys, heart, and spleen were removed. Blood samples were collected at the same times and serum was obtained by centrifugation. Total radioactivity was estimated in all samples. Furthermore, serum samples collected %, 1, and 1% hour after administration and tissue homogenates from animals killed 72 hour (brain, heart, and spleen), y2 and 1 hour (lungs), or %, 1 , l %,and 2 hours (kidneys) after administration were investigated by TLC.

The collected tissue, urine, faeces, and serum samples from all four experiments were stored frozen until analysis.

Preparation of sumples .for liquid scintillation counting. Serum and urine. Aliquots of serum and urine, 50 pl or 100 pl, were counted directly in 10 ml of Pico-Fluor 15 (Packard).

Faeces. The samples were homogenized with 6 (rats), 4 (mice), or 2 (human volunteers) volumes of water in an Ultra Turrax homogenizer (Janke & Kunkel). Two ml of the homogenate were freeze-dried. To 10 mg of the dry matter were added 100 pI water and after 30 min. 400 pI Soluenea 350 solubilizer (Packard). After incubation for one hour at 50", the samples were bleached by adding 200 p1 of isopropanol and 200 pl of 30% H202-soI. The samples were then kept for 10 min. at room temperature, and at 40" for 30 min. After cooling 10 ml Pico-Fluor 15 were added to each sample.

Brain. liver, lungs, kidneys, heart, and spleen. The samples were homogenized with 4 volumes of water (Ultra Turrax). To 200 p1 aliquots of homogenates were added 400 pl of SoIueneB 350. After incubation for one hour at 50", the samples were bleached by adding 200 plof isopropanol and 200 pl of 30% H202-solution. The samples were then kept for 10 min. at room temperature, and at 40" for 30 min. After cooling 10 ml of Pico-Fluor were added.

Preparation of samples f o r thin-layer chromatography (TLC). Five pI of urine were taken for TLC. Faeces homogenates were centrifuged and 5 pl of the supernatant were taken for TLC. Half a ml of serum or tissue homogenate and 2.5 ml methanol were shaked for 5 min. and then centrifuged for 10 min. The supernatant was

118 BIRGIT SCHULTZ ET AL.

evaporated to dryness at 60" under air. After dissolving the residue in 50 pl of water, 5 pl were taken for TLC.

Thin-layer chromatography (TLC). Thin-layer chroma- tography was carried out on glass plates ( 5 x 2 0 cm) precoated with a 250 pm layer of anasil H (Analabe).

The following solvent system were used: I : Butano1:acetic acid:water 4: 1:l

11: 1sobutanol:ethylmethyIketon:acetone: methano1:water:conc. ammonia 40:20:20: I : 14: 15

After development the silica gel was scraped off auto- matically into separate counting vials for each half centimeter beginning at the application spot. The radio- activity was determined in 5 ml of diotol scintillator (Herberg 1960), where 2,5-diphenyloxazole and 1,4-bis (5-phenyl-2-oxazolyl)-benzene were replaced by 14.30 g Permablend I11 (Packard). Unlabelled reference substan- ce of THIP was developed on the same plate. The reference spot was visualized by spraying with a mixture ( I + 1) of 1 % potassium permanganate and 5% sodium carbonate both in water.

It was not possible to perform TLC on all samples either because of too low content of radioactivity or because of too high content of disturbing compounds.

In vitro hydrolysis. Hydrolysis was performed on urine collected 4-8 and 8-12 hours (rats, 5 mg I4C-THIP/kg), 0-4 hours (mice) and 1-2 and 2-3 hours (volunteers) after administration and on faeces collected 8-12 hours (rats, 200 mg l4C-TH1P/kg) after administration of I4C-THIP.

a. Acid hydrolysis. Two ml of urine or faeces homo- genate and 0.6 ml concentrated hydrochloric acid were incubated at 100' for 30 min.

b. Hydrolysis with P-glucuronidase. One ml of urine or faeces homogenate, 0.5 ml 0.1 M phosphate buffer pH 6.8, one drop ofchloroform and 50 mg of P-glucuronidase (bacterial/Sigma Chemical Co., St. Louis, Mo., U.S.A.) was incubated at 37" for 20 hours.

c. Hydrolysis with P-glucuronidase and sulphatase. One ml of urine or faeces homogenate, 1 ml 1 M sodium acetate buffer pH 5.0 and 200 p1 P-glucuronidase with sulphatase activity (Helix pomatia/Sigma Chemical Co.) was incubated at 37" for 20 hours.

After hydrolysis the samples were centrifuged and 5 pI of the supernatant were taken for TLC (system I). Measurement of radioactivity. The radioactive samples were counted in a Beckman liquid scintillation spectro- meter (LS 335). Some of the samples from experiments 1, 2, and 3 were recounted after addition of ''C-toluene as an internal standard in order to determine the counting efficiency, whereas some of the samples from experiment 4 were recounted after addition of 14C-toluene as an internal standard in order to calibrate the external standardization (channel ratio) made by the instrument.

Results

S e r u m levels.

The concentrations of total radioactivity expressed as ng 14C-THIP/ml serum from the two male volunteers given 10 mg I4C-THIP corresponding to about 0.15 mg I4C-THIP/kg are shown in fig. 2 . The peak values, corresponding to 160 and 280 ng THIP/ml, appeared at about half an hour after

H A J

o--O NL

Fig. 2. Concentration of radioactivity in serum expressed as ng ''C-THIP/ml after administration of a single dose at 10 mg j4C-THIP to two male volunteers.

BIOTRANSFORMATION OF THIP 119

r - m - w 0 N * -

0 0 0 0 0 0 c 9 9 T C " l l -

- N w m - h o o o o o c 999-1=

m m - r O P - m N I b Q ' " 0 9 t 2 0 0 0 0 - c

5 m m w - r - a 3 0 0 0 0 6 0 7 l t ? ? " l b .

O \ m m * m - 1?-??1 8 0 0 - - 0

5 c s - 5

.K 3

5 c 2

m a f N

5 c 3

L a

9 c

5 c 9

m

5 .K N

5 c - L a

9

0 c

LI 3

c 3

C

o c L .o m 5 0 .9 E .5 'g E

4

U

3 n E s

120 BIRGIT SCHULTZ ET AL

administration after which time the curves declined very fastly. Thus, 8 hours after administration, the concentration was only about 1/10 of the highest concentration. Twentyfour hours after administra- tion no radioactivity was detectable in serum.

In table 1 are shown serum concentrations expressed as pg/ml of I4C-THIP after administra- tion of 5 mg 14C-THIP/kg to rats. The peak values appeared at about half an hour after administra- tion. As by the volunteers the concentration de- creased very rapidly.

The fractions of radioactivity found as THIP and metabolites in serum from volunteers and rats are shown in table 2. The amount of THIP decreased and the amounts of metabolites increased with time. Two hours after administration of I4C-THIP to the volunteers only about 25% of the radio- activity in serum referred to THIP. This corre- sponded to a concentration of about 30 ng THIP/ ml for the two volunteers.

Distribution. The contents of radioactivity in tissues from rats given 5 mg 14C-THIP/kg expressed as pg I4C- THIP/g and % of dose are given in table 1. As for serum concentrations of total radioactivity peak values were indicated at about % hour after admini- stration in all organs. Highest concentrations were seen in the kidneys. The concentrations found in

0,

1oc

80

6C

LO

20

1 M I C E 5 m g / k g

2 R A T S 2 0 0 m g l k g

01 nose 3 R A T S 5 m g / k g

U R I N E

heart, liver, and spleen were almost identical; while the concentration in lungs was higher and in the brain somewhat lower.

Thin-layer chromatography of the tissue homo- genates from rats showed only small amounts of radioactivity corresponding to THIP (20-30 %) except for the brain, which half an hour after administration contained about 65 YO unchanged THIP and 4-1596 of each of three metabolites. The amounts of the three metabolites found in the other organs varied from 5 to 40%.

Excretion. The cumulative excretion of radioactivity with urine and faeces after administration of 14C-THIP to rats, mice and volunteers is shown in fig. 3. During the collection periods radioactivity corres- ponding to 99% (rats, 5 mg/kg), 93% (rats, 200 mg/kg), and 90% (mice and vo1unteers)ofthe dose given was found in urine and faeces. No radio- activity was found in faeces from the two human volunteers, whereas 6.3%, 9.0% and 1.6% of the total dose was found in faeces from rats ( 5 mg/kg and 200 mg/kg) and mice, respectively. Thus the ratio between the amount of radioactivity excreted with urine and faeces was 9 for rats given 200 mg/kg, 15 for rats given 5 mg/kg, and 56 for mice. The excretion of radioactivity was very fast for volunteers as well as for rats and mice. The

F E C E S

1 3 c

0 A J

0 NL

% o f dose

U R I N E 100

80

60

LO

20

Fig. 3. Cumulative excretion of total radioactivity with urine and faeces after oral administration of I4C-THIP to rats (5 mg/kg and 200 mg/kg), mice (5 mg/kg) and human volunteers (0.15 mg/kg).

BIOTRANSFORMATION OF THIP 121

excretion of radioactivity was faster for the group of rats given 5 mg I4C-THIP/kg than for the group of rats given 200 mg 14C-THIP/kg just as the excretion of radioactivity with faeces was smaller for rats given 5 mg/kg than for rats given 200 mg/kg.

An example of two thin-layer chromatograms of the same urine fraction (rat urine collected 12-24 hours after administration of 200 mg/kg) in two different solvent systems is shown in fig. 4. As can be seen from the lower half of the figure (system 11, hydrophilic alkaline system) the counting of the fractions along the plate gives 4 peaks, a major one corresponding to THIP and three minor ones designated ml, mz, and m3. The upper chromato- gram (system I, hydrophilic acid system) shows three peaks a major one corresponding to the

SYSTEM I1

’” 3 UL.:, + 0 2 L 6 8 10 12 1L CM

Fig. 4. Thin-layer chromatograms of urine collected 12-24 hours after administration of 200 mg “C-THIP/kg to rats. Abscissa: Distance from application spot. Ordi- nate: Radioactivity in each silica gel fraction in per cent of total radioactivity on the plate.

parent compound and two minor peaks designated 1 and 2. Investigation of the chromatograms of the rat urine collected in the different intervals estab- lished that the amount of radioactivity found in one of the small peaks, 1, in system I always correspon- ded to the amount of radioactivity of peak ml in system I1 whereas the amounts of radioactivity of peak 2 in system I corresponded to mz+m3 in system 11. Thus the chromatography of rat urine indicated that at least three metabolites were present.

Thin-layer chromatographic investigations of the urine fractions collected from mice and human volunteers given I4C-THIP (system I and 11) showed 3 spots in both systems corresponding to THIP and two metabolites. For both volunteers and mice the Rf-values for the two metabolites corresponded to the Rf-values for ml and m3 found in rat urine, which seemed to indicate that the same two meta- bolites of THIP (ml and m3) were found in urine from volunteers, rats, and mice, whereas a third metabolite (m2) was found in urine from rats only. However, the studies on in vitro hydrolysis indi- cate that the spot ml in colunteers and mice corresponds to two different compounds.

In urine fractions from both volunteers, rats, and mice only small amounts of radioactivity were found at Rrvalues different from the above men- tioned spots, and it is not possible from these studies to say if this radioactivity corresponded to metabolites.

Fig. 5 shows the cumulative excretion of THIP and metabolites in urine from volunteers, rats, and mice given I4C-THIP. The excretion of unchanged THIP as well as the metabolites was seen to be fast. Most of the radioactivity excreted with urine corresponded to unchanged THIP in all three species. The total amounts of radioactivity excreted as metabolites in urine were 18 % for rats receiving 5 mg I4C-THIP/kg and 13 % for rats receiving 200 mg I4C-THIP/kg. The three metabolites were for both groups of rats excreted in amounts from 2-7% of the total dose, although the relative amounts of the metabolites are different for the two groups. In urine from mice amounts of the metabolites ml and m3 corresponding to 21 % and 6% ofthe total dose, respectively, were found. In volunteers 30-35 % of the total dose was excreted as ml, whereas m3 was excreted in small amounts ( < 2 % of the total dose).

122 BIRGIT SCHULTZ ET AL.

RATS, 5 rng lLC-THIP/kg

t

RATS, 200 rng “C-THIP/kg

80 1

LO

0 1 8 12 16 20 21

M I C E 4 T H I P l g L c - - , rn,

LQK- ---TH’P

.-A rn

rn, m - t *

0 8 16 21 32 LO 48 0 8 16 21

Fig. 5. Excretion of unchanged THIP and metabolites with urine after administration of I4C-THIP to human volunteers (about 0.15 mg/kg), rats (200 mg/kg and 5 mg/kg) and mice (5 mg/kg) as determined by TLC (system 11). THIP(0 ,O) ml(.,O) m(+) m ( A , A ) .

Thin-layer chromatography of faeces homoge- nates from rats and mice showed that almost all the radioactivity (about 80% of the amount of radio- activity excreted in faeces) corresponded to meta- bolites m2 (rats) or m3 (mice). For both species only small amounts of unchanged THIP (<O. 1 % of the total dose) were excreted in faeces.

In vitro hydrolysis. Thin-layer chromatography of urine fractions from volunteers, rats, and mice after hydrolysis with both pure 0-glucuronidase and P-glucuronidase containing sulfatase activity showed disappearance of metabolite ml and corresponding increase in the amount of THIP (volunteers and rats) or THIP and m3 (mice). This suggests that ml from volunteers and rats corresponds to a glucuronic acid conjugate of THIP and that ml from mice in addition to a glucuronic acid conjugate of THIP contains a conjugate of a metabolite. Hydrolysis with hydro- chloric acid showed disappearance of metabolite ml in urine from rats, but only partly disappear- ance of metabolite ml in urine from volunteers and mice.

Thin-layer chromatography of faeces homoge- nate from rats before and after hydrolysis with both enzymes and hydrochloric acid showed no dif- ference.

Biological half-life. A plot of the logarithm of the amount of un- changed drug remaining to be excreted versus time is linear in the elimination phase(Giba1di & Perrier 1975) and has the slope - p/2.303 and can there- fore be used to determine the biological half-life, which is 0.693/p. By this method the biological half-life has been found to be 1.5 (AJ)and 1.9(NL) hours in the two volunteers, 2.8 (rats, 5 mg/kg), 3.0 (rats, 200 mg/kg), and 1.4 (mice) hours in animals.

Discussion

The experiments described in this paper have shown that THIP is completely absorbed by both man, mouse, and rat whenadministered by the oral route as no or very little unchanged THIP is excreted with faeces. It is also found that the drug is absorbed rapidly by man and rat with peak values

BIOTRANSFORMATION OF THIP 123

of total radioactivity in serum appearing as early as half an hour after administration.

The fact that the presence of THIP has been demonstrated in the brain shows that THIP is capable of penetrating the blood-brain barrier. This is in agreement with the results found by whole-body autoradiography of I4C-THIP in mice, which showed presence of radioactivity in the central nervous system (Krogsgaard-Larsen et al. 1981) and to the finding that THIP is pharmaco- logically active after systemic administration in a variety of animal models (Christensen eta/ . 1979; Meldrum & Horton 1980). Concentrations of mus- cimol, a GABA agonist like THIP, in the brain of rats have been measured after intravenous injection of 0.9 mg 3H-muscimol/kg (Baraldi et al. 1978). The highest concentration of muscimol in the brain corresponding to 0.02% of the total dose was observed 30 min. after administration. The highest concentration of THIP in the brain after oral administration of 5 mg I4C-THIP/kg was also found 30 min. after administration, but the relative uptake (0.06% of the total dose) of THIP in the brain was higher than the uptake found for musci- mol.

The radioactivity after administration of I4C- THIP is mainly (rat and mouse) or solely (man) excreted with urine, which could be expected on the basis of the low molecule weight and high polarity (zwitterion structure) of THIP.

Thin-layer chromatography of the collected urine fractions after administration of 14C-THIP seemed to indicate the presence of three metabolites (ml, m2, and m3) of THIP in rat urine and two metabo- lites (probably corresponding to ml, and m3) in urine from man and mouse. The extent of metabo- lism of THIP is low as most of the excreted radioactivity corresponded to unchanged THIP. Rats given 5 mg I4C-THIP and 200 mg I4C- THIP/kg, respectively, have been shown to meta- bolize THIP to almost the same extent, showing that the metabolic capacity is not saturated at 200 mg/kg orally.

The main metabolite of THIP especially by man, ml , seems to be a glucuronic acid conjugate of THIP. The metabolism of another 3-hydroxy- isoxazol, hymexazol(3-hydroxy-5-methyl-isoxazo- le), in rats has been investigated previously (Ando et al. 1974). These authors found the main meta-

bolite to be 3-(~-D-glucopyranuronosyloxy)-5-me- thylisoxazole, excreted in amounts corresponding to 65% of the total dose given. By analogy with these results ml is probably the 3-0-glucuronic acid conjugate of THIP. Attempts to isolate metabolite ml and the two other metabolites m2 and m3 has not been successful and the chemical structure of the metabolites have not yet been established.

Acknowledgement The collaboration of Drs. S. Honor6 Hansen and P. Krogsgaard-Larsen, The Royal Danish School of Pharmacy, Dept. of Chemistry BC, is gratefully acknowledged. Our thanks are also due to Mr. Hans Nyegaard, Mrs. Judith Rasmussen, and Miss Karin Bngvad for skillful technical assistance and to Mrs. Ruth Sparre Anderson for preparation of the manuscript.

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