metabolism, distribution and excretion of flupenthixol

Acta pharmacol. et toxicol. 1969, 27, 301-3 13

From the Research Laboratories, H. Lundbeck & Co. A/S, Copenhagen/Valby, Denmark

Metabolism, Distribution and Excretion of Flupenthixol

BY

Aksel Jergensen, Villy Hansen, UUa Dahl Larsen and A. Hauf Khan

(Received October 25, 1968)

Flupenthixol (fluanxol 8) (44 3-(2-trifluormethylthioxanthen-9-ylidene) propy1)-1 -piperazine-ethanol) was synthesized as a member of a series of thiaxanthene derivatives (PETERSEN & M ~ L L E R NIELSEN 1964) and selected for clinical trial on the basis of its pharmacological properties ( M ~ L L E R NIELSEN 1967). The present publication deals with the biotransformation of flupenthixol in dogs and rats and the quantitative aspects of the distribution and excretion of the compound in rats.

Materials and Methods A. Labelled compound.

Flupenthixol was labelled by the Wilzbach method as described by BERGSTROM & LIND- STEDT (1957). About 500 mg of the finely powdered HC1-salt was exposed to 1 Curie tritium-gas for 3-4 weeks. The substance was purified by recrystallization and precipitated as a HCI-salt. The radiochemical purity was found to be >90 by thin-layer chromatography.

The specific activities of the compound used were: Distribution study: 8.9 pci/mg. Excretion study on oral administration: 10.6 pci/mg. Excretion study on intravenous administration : 9.0 pci/mg. Bile collection study: 9.0 pci/mg.

B. Animal studies. Biotransformation studies. Male Wistar rats were given a single dose of 25 mg/kg of

unlabelled flupenthixol by intraperitoneal injection and the urine and faeces collected for the following 24 hours.

Two beagle dogs were given a daily dose of 5 mg/kg of unlabelled flupenthixol by intramuscular injection for four days and the urine was collected during this period. All urine samples were pooled before analysis.

22 Acta pharmacologica. vol. 27. fasc. 4

302 A. J0RGENSEN. V. HANSEN, U. D. LARSEN A N D A. R. KHAN

Distribution study. Male Wistar rats (90-1 10 g) fasted for 16 hours were given a single oral dose of 10 mg/kg of labelled flupenthixol in distilled water (10 ml/kg). Groups of three rats were then sacrificed by exsanguination under ether anesthesia 30 minutes, 1, 2, 4, and 8 hours, and I , 2, 3, 6, and 10 days after administration of the drug. Blood samples were collected in tubes containing EDTA to prevent clotting. The brain, lungs, liver, heart, kidneys, spleen, epididymal fat, gastro-intestinal tract and carcass were examined.

Excrefion studies. 10 male Wistar rats (90-1 10 g) fasted for 16 hours were given I0 mg/kg of labelled flupenthixol orally or intravenously in distilled water (10 ml/kg). The urine and faeces were collected separately. In the study with oral administration, the collection times were 1, 2, 4, 8, and 24 hours and thereafter each day until ten days after administration. In the investigation with intravenous injection the times of collection were 1, 4, 8, 24, 48, 72, 96, and 120 hours.

Bile collection srirdy. Four male Wistar rats (210-250 g) fasted for 16 hours were an- esthetized with urethane (1.5 g/kg i.p.). A polythene drain was inserted into the common bile duct and the bile was collected. The bile collected for the first hour served as control bile. The rats were then given 2 mg/kg of labelled flupenthixol intravenously and the bile was collected for five hours.

C. Isotope technique. Samples were measured in a Beckman Liquid Scintillation Counter with automatic

external standardization. With the exception of the blood samples, the scintillation medium used was 10 ml of the dioxane-methanol-toluene-naphthalene mixture (diotol) as described by HERBERG (1960). modified by replacing POPOP with dimethyl-POPOP. With the blood samples a toluene/triton X-100 (2/1) medium was used containing 6 g/I PPO and 0.3 g/l dimethyl-POPOP.

D. Extraction In the studies with the unlabelled compound, extractions were made on the urine and

faeces of rats and on the urine of dogs at basic and acidic pH. Extraction was performed with three portions of ether or ethylene dichloride (EDC) at each pH. The organic phases were dried over anhydrous sodium sulphate and evaporated to dryness at low temperature in vacuum. The remaining material was redissolved in 1-2 ml EDC or ethanol and used for chromatography. Acid- or P-glucuronidase hydrolysis was performed on urine before or following basic extraction.

In the studies with labelled compound urine as well as faeces- and tissue homogenates were extracted 3-4 times with EDC at pH 9. The combined extracts were concentrated by evaporation in vacuum, an aliquot was taken for counting of radioactivity and another one used for chromalography. Acid- or P-glucuronidase hydrolysis was made following extraction and another extraction made as described above.

E. Chronratogruphy. Thin-layer chromatography (TLC) was performed on glassplates (20 x 20 cm) coated

with a 2 5 0 p layer of Silica Gel G according to STAHL (Merck) and activated at 110" for 30 minutes.

The following solvent systems were used: I . Benzene: diethylamine: dimethylformamide

(unsaturated) 80: 10 10

FATE O F FLUPENTHIXOL 303

FPT - SO T r i f l u o r m e thy1 thiaxanthone

Fig. I . Reference substances. Flupenthixol (FPT), flupenthixol-sulphoxide (FPT-SO), 2-trifluormethyl-9-(3-N-piperazInyl-propyliden)-thiaxanthene (HN-FPT) and 2-trifluor-

methyl-thinxanthone.

Acetone: heptane: diuthylaniine 60:40: 10 (saturated) Ether: diethylamine 90: 10 (saturated) Butanol: acetic acid: water 80: 20: 20 (saturated) Isopropanol: benzene: I ammonia 35 :45: 20 (unsaturated) (upper phase was used). For the detection of spots the following spray reagents were used:

Sulphuric acid, conc. (UV light) (all compounds) Persulphate reagent (sulphoxides) (SMITH 1960) 5 FeCI3-solution (phenoles) (STAHL 1967) Fast Blue Salt B (phenoles) (STAHL 1967) lodoplatinate reagent (N-compounds) (SMITH 1960)

Flupenthixol (FPT), flupenthixol-sulphoxide (FPT-SO), 2-trifluormethyl-Y-(3-N-piperazinyl-propy1iden)-thiaxanthene (HN-FPT) and 2-trifluormethyl-thiaxanthone were used as reference substances (for formulas see fig. 1).

FPT and HN-FPT were exposed to gentle oxidation by heating an 0.5% ethanolic solution adjusted to pH 4 with one tenth the volume of 3bg; Hz07-solution over night i n an oven at 40". The solution was chromatographed directly.

After chromatography of radioactive exlracts the silica gel was scraped off into a separate counting vial for each centimeter beginning at the starting point, and the radioactivity determined.

F. U. V . absorptiori curves.

U.V. absorption curves of reference substances were made in 0.1 N-HCI-solutions. Absorption curves of spots on the chromatogram were made by scraping off the spot containing area, eluting i t with 0.1 N-HCI-solution and filtering through a glass filter G 4. A blank made from the plate was used for correction. The absorption curves were read in a Zeiss U.V. PMQ spectrophotometer against 0.1 N-HCI-solution.

22.

304 A. JORGENSEN, V. HANSEN, U. D . LARSEN A N D A. R. K H A N

Table I . Rf-values of Mo, MI, MII, Mi11 and reference substances.

Mn . . . . . . . . . . . . . . . . . . . . . .

MI . . . . . . . . . . . . . . . . . . . . . . M I I . , . . . . . . . . . . . . . . . . . . . .

M I I I , . . . . . . . . . . . . . . . . . . . .

FPT . . . . . . . . . . . . .

FPT-SO. . . . . . . . . . . . . . . . . . HN-FPT . . . . . . . . . . . . . . . . .

FPT + H 2 0 2 . . . . . . . . . . . . . HN-FPT + H 2 0 2 . . . . . . . . .

Trifluormethyl-thiaxanthone

System 1

0.85

0.77 0.51 0.41 0.41 0.32 0.84

0.74 0.52 0.42 0.74 0.41 0.32 0.98

System 2 ~~ ~

0.57

0.45 0.30 0.25 0.20 0.15 0.58

0.45 0.30 0.23 0.45 0.20 0.15 0.86

System 3 ~

0.27

0.14

0.06

0.29

0.15 0.11

System 4

0.37 0.33 0.17 0.45

0.29

0.37 0.33 0.17 0.45

0.17 0.29

System 5

0.60

0.39

0.07

0.60

0.38 0.22

0.38 0.07

Results Biotransformation of Pupenthixol.

Chromatography of urine extracts indicated the presence of four compounds, named Mo, MI, MII, and MIII. Rf-values are given in table 1. Mo, MI and MII had Rf-values and gave colour-reactions with the spray reagents identical to authentic FPT, FPT-SO and HN-FPT respectively. The U.V. absorption curve of MI was shown to be identical to the U.V. absorption curve of authentic FPT-SO (fig. 2), with a shoulder at about 250 mp and a maximum at 216 mp. Authentic FPT and HN-FPT had maxima at 204 mp and 228 mp and a shoulder at about 265 mp. The U.V. absorption curve of MII~ (fig. 3) showed the same characteristics as the curve of MI and authentic FPT-SO, indicating that MIII might be a sulphoxide. In addition it gave orange fluorescence in U.V. light after spraying with conc. sulphuric acid, and green fluorescence after spraying with persulphate reagent as did authentic FPT-SO, while FPT and HN- FPT gave a yellow fluorescence with the two spray reagents. Oxidation by H 2 0 2 of FPT and HN-FPT gave, in addition to traces of the parent compounds, spots on the chromatograms which had Rf-values identical to authentic FPT-SO and MIII respectively. This formation of MIII from

FATE OF FLUPENTHIXOL

LOO

300

200

LOO

-

.

ZOO I

100 I

305

. ---L.- 270 2€0 250 2 L O 230 220 210

- . 200 o n a

L . RO 310 360 2 9 0 % 0 50 260 2 k l 2 i O G r 2 z -26-%o-nm-

Fig. 2. U . V . absorption curves of authentic FPT-SO (a) and MI (b).

Fig. 3. U.V. absorption curve of MIII.

306 A. JQRGENSEN, V. HANSEN, U. D. LARSEN AND A. R. KHAN

FLUPENTHIXOL SULFOXILE W

FLUPEMTHIXOL W

H N -FPT

-1

Fig. 4. Biotransformation of Rupenthixol.

HN-FPT, in addition to the U.V. absorption characteristics and colour reactions of MIII, makes it reasonable to assume that the metabolite MIII is identical with HN-FPT-SO.

Thus the four excretion products are considered to be FPT, FPT-SO, HN-FPT and HN-FPT-SO, indicating the biotransformation shown in fig. 4. In all chromatograms a fifth spot corresponding to trifluormethyl- thiaxanthone (for formula see fig. 1 ) is seen, but this substance is most likely not a metabolite, as it is always formed in small quantities by chemical manipulations with the thiaxanthenes.

In urine from rats, but not from dogs, more FPT and HN-FPT are extractable after acid- or 0-glucuronidase hydrolysis, indicating that these two compounds are to some extent bound as glucuronides in urine from rats. No phenolic metabolites could be detected.

Urine from rats and dogs contains mainly the two sulphoxides and only small amounts of FPT and HN-FPT in the free form. In the faeces only FPT and small quantities of HN-FPT could be detected.

Distribution study. The distribution of radioactivity following oral administration of 3H

labelled flupenthixol is shown in table 2 and fig. 5. The blood concentrations are shown in table 3.

Apart from the gastro-intestinal tract, which contained unabsorbed as

Tabl

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308 A. JP)RGENSEN, V. HANSEN, U. D. LARSEN A N D A. R. KHAN

1 2 4 8 2L 72 hours

Fig. 5. Distribution of radioactivity following oral administration of 3H-flupenthixol. Ordinate: Radioactivity expressed as pg Rupenthixol/g wet weight.

well as excreted substance, the greatest concentrations were seen in the liver and the lungs. The lowest concentrations were found in the brain and the blood. Ten days after administration radioactivity could be determined only in the liver, kidneys and spleen.

Extraction and chromatography were performed on pooled organs. The organ, time after administration and per cent of total radioactivity extractable at pH 9 are shown in table 4. It is seen that in the brain, the extractable part of radioactivity, decreased with time. Acid hydrolysis of brain homogenates after the first extraction did not release more radioactivity for extraction, indicating that the remaining radioactivity was strongly bound. Chromatography of the extracts showed that the brain and the liver contained almost exclusively the unchanged FPT, with only traces of metabolites, while the lungs as well as the parent compound contained a small quantity of FPT-SO.

Excretion studies. a. Oral administralion. The cumulative excretion of radioactivity in

the urine and faeces following oral administration of 3H-flupenthixol is shown in fig. 6. Total excretion of radioactivity was greatest in the faeces, in which more than 63 % of the administered dose was found. The urinary

FATE OF FLUPENTHIXOL 309

Table 3. Blood concentrations expressed as

pg flupenthixol/ml (n = 3).

I pg/ml I S.D. I

I 30 min.. . . .

I hour. . . . 2 hours.. . . 4 hours . . . . 8 hours.. . . 1 d a y . . . . . 2 days. . . . . 3 days. . . . . 6 days. . . . .

10 days.. . . .

0.37 i 0.05 0.90 i 0.02 0.84 1- 0.13 1.14 i 0.01 1.24 4 0.17 0.71 t 0.06 0.46 _t 0.09 0.38 k 0.00 0.26 t 0.04 0.10 1- 0.02

excretion accounted for 17.4% of the dose. 2.2% remained in the body at the end of the ten days period. Excretion in the urine was greatest between 4 and 8 hours (about 1% of the dose/hour). Excretion in the faeces was greatest in the period 8 to 24 hours being about 1.6%/hr.

About fifty per cent of the radioactivity present in urine could be extracted without hydrolysis. Hydrolysis using P-glucuronidase or hydro- chloric acid made another 5-1 I % of the radioactivity extractable.

Table 4. Per cent radioactivity extractable

at pH 9.

Time after administration % extractable

radioactivity

I 2

: : - 1 8 ~~

Brain. . . . , .

I 24 Lungs.. . . . . 4

2 Liver. . . . . .

91.6 ~ 86.1

14.5 ' 37.2 1 90.4 87. I ! 87.4

91 .8

~~

310 A. JBRGENSEN, V. HANSEN, U. D. LARSEN A N D A. R. KHAN

% o f dosr

70 t F t c e r

Uflrlc

I L 7 10 Days

F t c e r 6ot f 0

30 / Uflrlc : : k =

I L 7 10 Days

Fig. 6. Cumulative excretion of radioactivity following oral administration of JH-flupenthixol.

Chromatography of the extracts showed that the main excretion product in urine was FPT-SO. Smaller quantitites of FPT and HN- FPT-SO were found. After hydrolysis using P-glucuronidase or hydro- chloric acid more FPT and HN-FPT could be detected indicating a conjugation of these two compounds with glucuronic acid.

About 54 % of the radioactivity present could be extracted in the faeces obtained between 8 and 24 hours. From faeces obtained at later periods relatively small quantities could be extracted. Acid- and P-glucuronidase hydrolysis gave slightly more extractable radioactivity, but in the latter case, it was not a specific effect of the P-glucuronidase, since equal quantities could be extracted from a control sample treated in the same way without the addition of P-glucuronidase. Many inpurities extracted from faeces interfered with the chromatographic analysis. The main

30 Lot /

I 60 Fects

5 0 .

LO -

.

30 -

2 0 .

10.

* Ufint

I 2 3 L 5 Days I 2 3 L 5 Days

Fig. 7. Cumulative excretion of radioactivity following intravenous injection of 3H-flupenthixol.

FATE OF FLUPENTHIXOL 31 1

excretion product in the faeces was unchanged FPT. Small quantities of HN-FPT were also seen.

b. Intravenous administrution. The cumulative excretion of radioactivity following intravenous injection of -1H-flupenthixol is shown in fig. 7. The total excretion in the faeces and urine was 59.3% and 16.4% of the dose respectively. 14.4% remained in the body at the end of the five days period.

The excretion patterns following oral and intravenous administration were very similar. The only difference seen was in the urinary excretion during the period 0-4 hours. This difference is most likely due to a more rapid distribution after intravenous injection than after oral administration.

In the oral and intravenous excretion studies 83 % and 90% respectively of the administered dose could be accounted for. It should be noted, however, that small quantities of radioactivity were expired as 3H2O. This most likely originated from the radioactivity situated in the ethanolic group, which was oxidized to CO2 and water, after this group had been split off and HN-flupenthixol had been formed. In a separate study, the expired water collected for a short period of time did in fact contain some radioactivity. It is difficult, however, to make long lasting studies of this kind and in consequence it was not possible to obtain any quantitative data.

Bile collection study.

Bile collection from four rats during the first five hours following an intravenous dose of 2 mg/kg flupenthixol yielded 6.5 ml of bile containing 12.6% of the dose given.

Discussion The biotransformation pattern of flupenthixol suggested in fig. 4,

indicates that sulphoxidation and dealkylation in the side chain are the main routes of degradation. This is in good agreement with that found for the other thiaxanthenes, chlorprothixene (Huus & KHAN 1967 ; RAAFLAUB 1967) and clopenthixol (KHAN 1968). It is remarkable that no phenolic metabolite could be detected, since other tricyclic psychotropic drugs e.g. chlorpromazine (HUANG 1967) and imipramine (CRAMMER & SCOTT 1966) form phenolic metabolites in considerable amounts.

The distribution of flupenthixol does not differ substantially from that of other similar neuroleptic drugs e.g. fluphenazine (EBERT & HESS 1965). It is remarkable that the lowest concentration seen in any organ was in

312 A. JQRGENSEN, V. HANSEN, U. D. LARSEN AND A. R. KHAN

the brain. Peak concentrations in the brain and most other organs were seen, four hours after oral administration.

The distribution study indicated a biological half-life of about 16 hours. From the excretion studies, half-lives of about 22 hours and about 27 hours could be estimated, following oral and intravenous administration respectively. The substantial faecal excretion seen after oral administration might be thought to indicate incomplete absorption. However, since the same excretion pattern was seen following intravenous injection, this possibility is not likely. The large biliary excretion indicates a considerable enterohepatic circulation and the substance from this circulation, escaping reabsorption, could account for the excreted substance found in faeces. However, excretion by the salivary glands, the fundus glands and the pancreas should also be considered. The nature of the non-extractable radioactivity cannot be accounted for, but this radioactivity seems to be bound by a strong and rather unspecific binding, as this phenomenon is also described for other substances (EBERT & HESS 1965; G. PLYM FORSHELL et ul.).

The excretion pattern showing excretion via the faeces to be 4-5 times the urinary excretion is in good agreement with results published on phenothiazines with a piperazine side chain (SYMCHOWICZ et ul. 1962; FLANAGAN et al. 1962). The major faecal excretion seen with these compounds is possibly a consequence of the structure of the side chain, as the urinary excretion of chlorpromazine and chlorprothixene, which have no piperazine-group in the side chain, is almost equal to the faecal excretion.

R E F E R E N C E S

Bergstrom, S. & S. Lindstedt: A Note of the Tritiation of Organic Compounds in Tritium

Crammer, 1. L. & B. Scott: New Metabolites of Imipramine. Psychopharrriacologia 1966,

Ebert, A. G. & S. M. Hess: The Distribution and Metabolism of Fluphenazine Enanthate. J . Pharmacol. Exp. Ther. 1965, 148, 412-21.

Flanagan, T. L., J. H. Newman, A. R. Maass, & E. J. von Loon: Excretion Patterns of Phenothiazine-S35 Compounds i n Rats. Effect of Structure on Metabolism. J. Pharm. Sci. 1962, 51, 996-99.

Forshell, G. Plym, P. Schaurnan, V. Hansen, U. Dahl Larsen, A. Jergensen, & K. Overe: Distribution and Metabolism of 3,3-Dimethyl-l-(3-Methyl-aminopropyl)-l-Phenyl- Phtalane (Lu 3-OlO), a Bicyclic Compound with Thymoleptic Properties. Acra Pharm- acol. et Toxicol. 1968, 26, 507-520.

Herberg, R. I.: Determination of Carbon-14 and Tritium in Blood and other wholeTissues. Anal. Chem. 1960, 32, 42-46.

Huang, C. L. : Isolation and Identification of Urinary Chlorpromazine Metabolites in Man. Int . J. Neuropharmacol. 1967, 6 , 1-13.

Gas. Acta Chem. Scand. 1957, 11, 1275.

8, 461-68.

FATE O F FLUPENTHIXOL 313

Huus, I . & A. Rauf Khan: Studies on the Metabolism of Chlorprothixene (Truxal 8) in

Khan, A. Rauf: Some Aspects of Clopenthixol (Sordinol @) Metabolism in Rats and

Meller Nielsen, I. : Farmakologisk karakteristik af flupenthixol. Nord. Psychiatrisk Tids-

Petersen, P. V. & 1. Msller Nielsen: Thiaxanthene Derivatives. Psychopharmacological

Raaflaub, J.: Zum Metabolismus des Chlorprothixen. Arzneim-Forsch. 1967,17, 1391 - 1 395. Smith, I . : Chromatographic and Electrophoretic Techniques 1 , Heinemann, London 1960. Stahl, E. : Diinnschicht-Chromatographie. Springer-Verlag. 1967. Symchowicz, S., W. D. Peckhan, M. Eisler, & P. L. Perlman: The Distribution and Ex-

cretion of Radioactivity after Administration of 3sS-labelled Perphenazine (Trilafon). Biorh. Pharmacol. 1962, 11, 417-422.

Rats and Dogs. Acru Pharnrucol.

Humans. Acta Pharmacol. el To.ricol. 1969, 27, 202-21 I .

skriff 1967, XXI, 424-430.

Agents 1964, 1, 301-324. Academic Press Inc., New York.

Toxicol. 1967, 25, 377-404.

metabolism, distribution and excretion of flupenthixol

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