metabolism, distribution and excretion of flupenthixol decanoate in dogs and rats

Acta pharmacol. et toxicol. 1971, 29, 339-358.

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

Metabolism, Distribution and Excretion of Flupenthixol Decanoate in Dogs and Rats

BY

A. Jnrgensen, K. Fredricson Overn and V. Hansen (Received October 13, 1970)

Abstract: The metabolism of flupenthixol decanoate administered in viscoleo@J by intramuscular or subcutaneous injection has been studied by thin-layer chromatographic techniques after extraction of urine, faeces and organs from dogs and rats. Since only minute amounts of the compound itself were detected in the organs and a high hydrolytic activity of blood and organs was demonstrated by in vitro experiments, it was concluded that the compound is hydrolyzed in the organism. The flupenthixol thus formed was found to be metabolized by sulphoxidation and by dealkylation in the side chain following the same pattern as that previously shown for flupenthixol. The neuroleptic effect seen in the pharmacological studies is presumably due to flupenthixol, since this is quanti- tatively by far the most important substance found in brain extracts. In order to study the depot effect of the preparation, blood levels and excretion were followed after administration of the 3H-labelled compound. These studies indicated, that a sustained release of drug from a depot was obtained. The presence of a relatively limited depot was demonstrated.

Key-words: Neuroleptic - sustained release preparation - kinetics - chromatography, thin-layer - radioisotopes.

The thioxanthene flupenthixol (fluanxoP) is well established in psychiatry as a neuroleptic drug without any sedative properties. A depot preparation (fluanxoP depot) suitable for intramuscular injection in oil has been obtained by the esterification of flupenthixol with decanoic acid. A prolonged neuroleptic effect of this preparation as compared to orally administered flupenthixol has been shown in pharmacological studies by MPILLER NIELSEN et al. (unpublished results) and FRANCK (1970) and in clinical trials by REMVIG et al. (1968), GJESTLAND (1970), ENERHETM et al. (1970) and BEDE i& WD- SEN (1970).

The present publication describes studies performed in order to compare the pharmacological studies with the biochemical data and to determine the distribution, metabolism and excretion pattern of the depot preparation.

340 A. JIZIRGENSEN, K. FREDRICSON OVER0 AND V. HANSEN

Materials and methods A. Studies with unlabelled compound. 1. Substance.

In flupenthixol (for formula see fig. 1) the side chain is attached to the ring structure with a double bond. Since the ring structure is asymmetrical the compound exists in two isomeric forms named a and p. In the depot preparation the pure a-isomer has been used, since this form has been shown to possess the highest biological activity.

2. Animals and dosage. Adult pure bred Beagle dogs of either sex from our own stock were given 6 mg/kg

of a-flupenthixol decanoate in viscoleo@ (0.3 ml/kg) by injection in the posterior musculature of the thigh once a week for 6 months.

Rats, SPF, [WistarJAflHanlMo (Hann. 67)] were given a subcutaneous injection of 10-14 mg/kg a-flupenthixol decanoate in viscoleo@ (0.1 ml/rat) in the nape of the neck twice a week for 7 weeks.

3. Samples. Twenty four hour samples of urine and of faeces were collected separately from the

dogs after 10 days and after administration for 3 months. Samples of the liver, lungs, kidneys, brain, muscle (quadriceps femoris), fat and bile were obtained 9 days after the administration of the last dose. Urine and faeces were collected from the rats during the first 24 hours, after 4 weeks and also at the end of the study. Samples of organs were obtained 2 days after the last dose.

4. Preparation of extracts. Samples of faeces and organs were homogenized 1 t 4 (weightlweight) in water

before extraction. Simple extracts were prepared by extracting three times with the double volume

of dichloroethane (DCE) at pH 10. Separation was achieved by centrifugation. The extracts were filtered through anhydrous sodium sulphate and evaporated to dryness under vacuum at 40". The residue was dissolved in 1.0 ml of chloroform and used for thin-layer chromatography (TLC). After exhaustive extraction, hydrolysis was undertaken with p-glucuronidase, bacterial (Sigma), after which the samples were extracted as before.

More purified extracts were obtained by introducing rinsing steps as illustrated in fig. 2. Following basic extraction with DCE, the organic extract was evaporated and the residue dissolved in 4 ml of 0.05 M acetic acid. The acid phase was extracted with petroleum benzene, which was subsequently divided into two portions: one was prepared for TLC directly (c) - the other was evaporated and the residue redissolved in 5 ml of 5 M sodium hydroxide and placed in a boiling waterbath for 30 minutes for hydrolysis. The hydrolyzed sample was then extracted with DCE and prepared for TLC (b). The remaining acetic acid phase was adjusted to pH 10 and extracted with DCE. The extracts were prepared for TLC as above (a). All the extractions were made by shaking three times with a double volume of organic solvent. 50 ml of urine and 5-20 grams of organ or faeces were used for extraction. The residues were dissolved in 500 PI of chloroform of which 50-250 p1 were spotted for TLC.

5 . Thin-layer chromatography (TLC) . TLC was carried out on glass plates (20 X 20 cm) coated with a 250 p thick layer of

Silica Gel G. according to Stahl (Merck). After coating, the plates were activated at 110" for 30 min. and stored.

STUDIES ON FLUPENTHIXOL DECANOATE 341

S zCH-CH2-Ch-N q - C H 2 - CH2-0-CO 4 C b Ia-CH 3 W

Flupenthixol decanoate (FPT-dec.)

w Flupenthixol (FPT)

S g = C H - C t - $ - C H 2 - N A NH

W LU 5-051 (HN-FPT)

n - C H y C H 2 0 H W

Flupenthixol-sulphoxide (FPT-SO)

S

Trifluormethylthioxanthone ( T FT) Fig. 1. Reference substances.

342 A. JIZIRGENSEN, K. FREDRICSON OVER0 AND V. HANSEN

The following solvent systems were used:

1 Isopentyl alcohol Acetic acid Water

I1 Propyl alcohol (n) Water

111 Propyl alcohol (n) Water

IV Acetone Heptane Diethy lamine

Benzene V Methanol

80 ml 20 ml 20 ml 85 ml 15 ml 95 ml

5 ml 60 ml 40 ml 10 ml 39.1 g 60.9 g

In some instances two-dimensional chromatography was carried out using rinsing systems such as chloroform : ether (85 : 15) or benzene in the first direction and one of the systems mentioned above in the second. The plate was thoroughly dried between the runs.

Concentrated sulphuric acid was used as the spray reagent. The plates were observed in UV-light.

The following substances were used as references (for formulas see fig. 1): a-flupenthixol decanoate (a-FPT-dec.), flupenthixol (FPT) (pure a and mixture of a and fl), flupenthixol sulphoxide (FPT-SO), Lu 5-051 (HN-FPT) and trifluormethylthioxanthone (TFT). Rf-values for the reference substances are shown in table 1.

The sulphoxides of FPT-dec. and HN-FPT, which were not available as reference substances, were formed directly on the TLC-plate by applying 2 drops of 10 % H2Oz on top of a sample of FPT-dec. and HN-FPT, respectively.

Fl 1 OO°C. 30 min

Fig. 2. Extraction procedure.

STUDIES ON FLUPENTHIXOL DECANOATE

Solvent system

I

I1

111

IV

V

343

Reference substances

FPT-dec. I FPT I FPT-SO 1 HN-FPT

0.36 0.19 0.31 0.27 0.33

0.50 0.27 0.11 0.41 0.21 0.05

0.74 0.20 0.10 0.04

0.35 0.26

0.27

0.67

0.86

0.95 0.60 0.50

0.66 0.61

0.94 0.76

B. Studies on labelled compounds. 1. Labelled compounds.

2-trifluormethylthioxanthone (TFT) was tritium (T) labelled by catalytic exchange by New England Nuclear.

The specific activity of the raw product was 93.6 mci/mg. Thin-layer chromatography (TLC) revealed that not more than 62.5 % was TFT. At least 7 labelled impurities were found to be present.

TFT was purified by several recrystallizations from acetic acid and diluted 5 times with unlabelled substance. TLC showed that 91.5 % of the radioactivity followed TFT.

From TFT flupenthixol was synthesized in 3 steps and flupenthixol decanoate in a further step as shown in fig. 3. The first step consisted of a reaction with dimethyla- minopropylmagnesiumchloride (DMAPMgCI) to give N 7057, which was purified to 98 % (radiochemical purity) by recrystallization from petroleum ether.

N 7057 was dehydrated in a mixture of acetic acid and hydrochloric acid to give N 796, which in turn was converted to FPT by amine exchange with hydroxyethyl- piperazine (HEPPZ). FPT was purified by recrystallization as base from petrol ether. By this procedure pure a-isomer was obtained. The radiochemical purity of this compound was > 90 %.

a-FPT-dec. was synthesized by reaction of a-FPT with decanoylchloride (DECI). a-FPT-dec. was purified by recrystallization as an oxalate and controlled by TLC in several systems. Two lots were made:

Lot no. 1: 3H-a-Flupenthixol decanoate:

Specific activity: 578 @/mg Radiochemical purity: > 85 % (About 8.5 % of TFT present).

Table 1. Rf-values in TLC.

I

TFT

0.93

0.83

0.87

0.87

0.94

344 A. J0RGENSEN, K. FREDRICSON OVER0 AND V. HANSEN gr D M A P M g C l .’$- CH 3 HAc,

T

CY - C t i 2 - CH2 - N( CH3 HCI

1

T FT N 7057

gCF3 -

T B=cH S - C k - n

.CH2-Nd-c+-

a - F P T - d e c . Fig. 3. Synthesis of tritium labelled FPT and FPT-dec.

STUDIES ON FLUPENTHLXOL DECANOATE 345

Lot no. 2: 3H-a-Flupenthixol: Specific activity: 560 pci/mg Radiochemical purity: > 90%

3H-a-Flupenthixol decanoate: Specific activity: 378 yci/mg Radiochemical purity: > 95 %

3H-a-FPT-dec. was prepared for intramuscular injection by dissolution in viscoleo@ (20 mglml).

2. Animal studies. a. Rat study on blood levels and excretion. Male Wistar/Af/Han/Mo (Han 67) rats,

SPF, (115-130 g) were given an intramuscular injection of 5 mg 3H-a-FPT-dec. (lot no. l)/kg body weight into the left musculus extensor quadriceps femoris. At %, 2 and 8 hours, and 1, 3, 6, 10, 16, 21 and 28 days after injection, two rats from each of the groups were killed under ether anaesthesia and blood samples were collected in tubes containing EDTA to prevent clotting. From the four rats killed 21 and 28 days after injection, 24 hour samples of urine and faeces were collected separately during the entire experimental period.

b. Rat study on distribution. Male Wistar/Af/Han/Mo (Han 67) rats, SPF, (205-250 g) were given an intramuscular injection of 4 mg 3H-a-FPT-dec. (lot no. 2)/kg body weight in the left musculus extensor quadriceps femoris. At 6 hours, and 1, 4, 7 and 14 days after the injection, 2 animals were killed by exsanguination under ether anaesthesia. Blood samples were collected in tubes containing EDTA to prevent clotting and the brain, lungs, liver, heart, kidneys, spleen, epididymal fat and the left leg were removed. From the two rats killed on the 14th day after the injection blood samples were drawn from the orbital vein plexus at s, 2, 6 hours, and 1, 2, 4, 7, 10 and 14 days after administration of the drug.

c. Dog study on blood levels and excretion. Six adult pure bred Beagle dogs of either sex (b. w. 9.4-11.5 kg) from our own stock, divided into three groups of one male and one female each, were used in the study. Two groups were given 2 and 6 mg 3H-a-FPT-dec. (lot no. 2)/kg respectively by intramuscular injection in the posterior musculature of the thigh, while the third group, for comparison, was given an oral dose of 1 mg sH-a-FPT/kg as pure substance in a capsule. Blood samples were drawn by vein puncture at different times after administration of the drug. Urine and faeces samples were collected in the periods 0-1, 1-2, 3-4, 6-7, 10-11, 15-16 and 24-25 days respectively after administration of the drug.

3. In vitro study on hydrolysis. Two male Wistar/Af/Han/Mo (Han 67) rats, SPF, were bled under ether anaesthesia.

The blood was collected in heparinized tubes and diluted with 4 volumes of 0.1 M phosphate buffer, pH 7.4. Brain, liver, lungs and kidneys were removed and homogenized in 4 volumes of the same buffer. To 12.5 ml of buffer, diluted blood and organ homogenates were added 26.5 yg of 3H-a-FPT-dec. and the samples then incu- bated with shaking at 37". Five ml samples were removed for analysis after 1 and 2 hours respectively.

4. Preparation of samples for liquid scintillation counting. The radioactive samples were measured in a Beckman Liquid Scintillation Counter

with automatic external standardization. The scintillators used were toluene: triton X-100 (2 : 1) containing 6 g/l PPO and 0.3 g/l dimethyl-POPOP for whole blood samples

22'

346 A. J0RGENSEN, K. FREDRICSON OVER0 AND V. HANSEN

and for all other samples the dioxane-methanol-toluene-naphthalene mixture (diotol) described by HERBERC (1960) and modified by replacing POPOP with dimethyl- POPOP, was used. 10 ml portions of the scintillators were used.

a. Blood. To 0.1 ml of whole blood were added 1 ml of concentrated NH:3-solution and 0.1 ml of 30 % HSOpsolution. After gentle shaking the sample was kept at room temperature for one hour and heated for another hour in an oven at 60". After cooling the scintillator was added. 0.5 ml of serum was counted directly in diotol scintillator.

b. Tissues. Homogenates containing 1 part by weight in 4 volumes of water were made of the brain, lungs, liver and kidneys. A 0.5 ml aliquot was added to the scintillator. The heart, epididymal fat, left leg and spleen were heated in 4 volumes of 0.5 N-NaOH solution in sealed tubes on a water bath at 90-95" for 30 min. with occasional shaking. Aliquots of 0.5 ml were taken for counting. The remainder of the animal body, named carcass, was refluxed in 2 volumes of ethanolic 0.5 M-KOH solution for one hour. After cooling and centrifugation 0.2 ml of the supernatant was counted.

c. Urine and faeces. 0.2 or 0.5 ml of urine was counted directly. The samples of rat faeces were ground in a mortar to give a homogenous mixture. Faeces from the dog wee homogenized in 2 or 3 volumes of water. 0.5 g oi rat faeces and 2 g of homogenate of dog faeces were heated for an hour in sealed tubes in an oven at 70-80" with 1 ml of 60 % HC104-solution and 2 ml of 30 % H~O2-solution. After cooling the solution was neutralized with solid NaOH and mixed with 40 ml of cellosolve. 1 ml of this mixture was then counted.

5. Extraction and chromatography. Dog urine (2-4 ml), homogenates of dog faeces (corresponding to 1-2 g of faeces)

and homogenates of rat organs (3-5 ml) were adjusted to pH 10 and extracted three times with 20-30 ml of DCE. The pooled extracts were concentrated to 3-5 ml at low pressure. One aliquot of the concentrate was counted and another subjected to TLC. The samples from the in vitro study were shaken with 20 ml of DCE following addition of 0.5 ml 2 N-NaOH solution. Aliquots of the DCE phases were chromatographed. The TLC was run on 5 X 20 cm glass plates prepared as described above. Solvent systems I, I11 and IV were used. After development of the plates the silica gel was scraped off automatically for each half centimeter into separate counting vials and the radioactivity determined.

Results A. Studies with unlabelled compound. Comments on the procedures.

When studying blind samples to which reference substances had been added, it was observed that partial decomposition of the substances occurs during the extraction and TLC-procedures. Thus minute amounts of FPT are formed from FPT-dec. The formation of negligible amounts of sulphoxides, TFT and some unidentified products was also observed. In addition some isomerisation of a-FPT occurs relatively easily during extraction and also during TLC, as demonstrated by two-dimensional TLC, using the same


solvent system in both directions. These facts have been kept in mind when assessing the dosed samples used.

By the simple extraction technique, nearly complete extraction of the added standards was achieved as judged by TLC. By the more complicated technique small amounts of the substances were lost. However, considerable amounts of impurities were eliminated, thus providing an extract much more suitable for TLC. In addition partial separation of FPT-dec. from the olther compounds was accomplished by extracting the acetic acid phase with petroleum benzene (see fig. 2). In this step FPT-dec. is completely extracted together with minute amounts of FPT. The main part of FPT and all of the other metabolites, however, remain in the acid phase and appear in extract a, while FPT-dec. appears in extracts b (hydrolyzed to FPT) and c. The use of other methods for obtaining more pure extracts, as e. g. gel filtration was complicated by absorption of the compounds to the gel and also by the instability of the compounds.

Dog studies. TLC of simple extracts of urine revealed the presence of yellow fluorescent

spots corresponding to the reference substance FPT, and orange fluorescent spots corresponding to FPT-SO. The two metabolites were present in approximately equal amounts (about 40 m pglrnl). No other substances could be detected. Neither was there any further release of substances observed after hydrolysis. The same results were obtained at TLC of purified extracts.

In extracts of faeces, distinct yellow spots corresponding to FPT and HN- FPT were detected in approximately equal concentrations (about 0.5 pg/g). No other substances were observed.

Useful results with TLC on organs were obtained only by using purified extracts and two-dimensional TLC. In the a-extract of liver (see fig. 2) small amounts of FPT and very small amounts of HN-FPT and FPT-SO were observed. The c-extracts contained small amounts of FPT-dec. and extremely small amounts of FPT. When the contents of the latter extract had been submitted to hydrolysis only FPT was detected in the extract (b-extract). Thus the presence in the liver of FPT-dec., FPT, NH-FPT and possibly FPT- SO was demonstrated. The main compounds, FPT-dec. and FPT, were judged to be of the same order of concentration (approximately 25-100 m pg/g).

The same pattern of drug and metabolites was detected in the lungs, the amounts being of the same order as in the liver. In the kidneys, however, the amounts were very low. In the brain and muscle neither drug nor metabolites could be detected.

The bile was found to contain some drug material, the identification of which was impossible because of interference by the impurities.

The FPT detected by TLC in the samples mentioned above appeared as

348 A. JORGENSEN, K. FREDRICSON OVER0 AND V. HANSEN

two spots corresponding to the two isomeric forms of FPT, a and p, in solvent systems I and 11. The two spots were of approximately equal size, thus indicating a larger degree of isomerisation than that normally occurring during the procedures. This seems to indicate that a certain degree of iso- mensation takes place in the animal body.

Rat studies. In extracts from rat urine FPT-SO was found to be the main compound.

The concentration was estimated to be about 0.5 pg/ml. In addition FPT (approximately 0.3 pg/ml) and HN-FPT-SO were detected. By hydrolysis with p-glucuronidase further amounts especially of FPT, but also of FPT-SO, were released. Extracts of faeces were found to contain FPT and HN-FPT as the main metabolites. In addition the presence of very small amounts of FPT-SO was indicated.

60- a

days

%-dose

2-

1- n 3

2 L 6 8 10 12 16 16 18 20 22 2L 26 28

Fig. 4. a. Blood level of radioactivity in rats killed at different times after administration of 5 mg FPT-dec./kg intramuscularly. n = 2. Ordinate: Radioactivity expressed as mpg

FPT-dec./ml. b. Total excretion of radioactivity in 24 hour samples of urine and faeces from rats

given 5 mg FPT-dec./kg intramuscularly.


Studies on extracts from organs revealed only the presence of very small

The contents of FPT detected by TLC in these samples were divided into amounts of FPT in the liver.

two spots of about the same size, as was also the case in the dog material.

B. Studies on labelled compounds. Rat study on blood levels and excretion. The radioactivity in rat blood

expressed as mpg FPT-dec./ml blood following intramuscular injection of FPT-dec. is shown in fig. 4a. The curve shows a peak value 8 hours after injection and a rather slow decrease after this time. In fig. 4b the total excretion of radioactivity in the urine and faeces is shown. The exponential excretion curve estimated by a regression analysis using the method of least squares on the excretion data is also shown. The regression analysis gave an elimination half-life of 8 days for the total excretion of radioactivity. The total excretion within 28 days was 71.4 %, indicating that part of the radioactive substance was still in the animal body at the end of the study. The faecal excretion was highest being about seven times greater than the urinary excretion.

Rat study on distribution. The concentration of radioactivity in rat blood drawn from the orbital vein plexus is shown in fig. 5. The curve is very similar to that obtained from the above mentioned study in the rat (fig. 4a), with a peak value at 6 hours after injection. The tissue concentrations of radioactivity expressed as pg FPT-dec./g tissue are given in table 2. Apart

70-

2 4 6 0 10 12 14 days 0‘

Fig. 5. Concentration of radioactivity in rat blood drawn from the orbital vein plexus (n = 2). Dose: 4 mg FPT-dec./kg intramuscularly. Ordinate: Radioactivity expressed as

mpg FPT-dec./ml.

W

VI

0 ?

Tabl

e 2.

Ch

mm

hn

Of

radi

oact

ivity

in r

at t

issue

s af

ter

4 m

g FP

T-d

ec./k

g in

tram

uscu

larl

y (E

xpre

ssed

as

pg F

PT-d

ec./g

and

% o

f do

se)

(n =

2).

&I m 2

pg/g

%

-dos

e w

glg

%-d

ose

wg/

g %

-dos

e Fg

/g

%-d

ose

pg/g

%

-dos

e T:

6 ho

urs

1 d

ay

4 da

ys

7 da

ys

14 d

ays

“Z

-

-

........

........

.....

0.03

B

lood

-

-

0.10

-

0.02

-

Bra

in

........

........

.....

0.11

0.

0 0.

09

0.0

0.04

0.

0 0.

04

0.0

0.03

Heart

........

........

.....

0.15

0.

0 0.

12

0.0

0.04

0.

0 0.

04

0.0

0.03

L

ungs

....

........

........

. 1.

3 0.

2 1.

5 0.

3 0.

2 0.

0 0.

2 0.

0 0.

1 L

iver

......

........

........

.. 1.

6 1.

7 1.

5 1.

6 0.

3 0.

3 0.

3 0.

4 0.

2 K

idne

ys

......

......

......

0.

6 0.

1 0.6

0.

1 0.

1 0.

0 0.

2 0.

0 0.

1 Sp

leen

....

........

........

. 0.

5 0.

0 0.

4 0.

0 0.

1 0.

0 0.

1 0.

0 0.

1 E

pid.

fat

....

......

......

.. 0.

1 0.0

0.1

0.1

0.1

0.0

0.1

0.0

0.0

Lef

t le

g ...

......

......

...

37.8

58

.9

10.9

19

.2

17.7

34

.0

8.3

16.6

7.

0 “C

arca

ss” ...

......

......

...

20.9

14

.2

1.8

2.3

u

0.0

0.0

8 0.

3 z 0

0.0

w 0.0

0.0

m > 2:

12.7

s

1.4

U c

Tot

al

81.8

35

.5

36.1

19

.3

14.4

z

........

........

.....

> 3 z


from the left leg in which the injection was performed, the highest concentrations were seen in the liver, the lungs, the kidneys and the spleen, while the concentrations in the brain and the blood were considerably lower. A maximum concentration was obtained 6 hours after injection in all the tissues ex- cept the lungs, in which a maximum concentration was found 24 hours after injection. As the decrease of radioactivity in the tissues was not exponential no half-life for the radioactivity in the tissues could be estimated. However, it should be stressed, that the concentration of radioactivity in the brain was still measurable 14 days after injection, when it was one third of the concentration found at 24 hours.

Relatively large variations were seen in the extractability of the radioactivity from the organs. This ranged from 11-59 Yo for the brain, 24-69 yo for the liver, 67-92 'yo for the kidneys and 38-77 yo for the lungs. The per-

% 20- FPT

Fig. 6. Thin-layer chromatograms of brain extract from a rat killed 7 days after administration of 4 mg FPT-dec./kg intramuscularly. Abscissa: Distance from the starting point. Ordinate: Amounts of radioactivity in per cent of total radioactivity on

the plate. Upper: Solvent system I. Lower: Solvent system 111.

352 A. JPIRGENSEN, K. FREDRICSON OVER0 AND V. HANSEN

centage of extractable radioactivity for all organs decreased with time after administration.

In the chromatograms of the extracts of the brain (see fig. 6) a large peak corresponding to FPT was seen. This peak mounted to 52-83 yo of the extractable radioactivity in the first week, decreasing to 32-63 yo at 2 weeks after administration. The presence of small amounts of FPT-dec. and some metabolites was also indicated. In addition the presence of TFT was indicated. This substance is probably not a metabolite, as it is formed spontaneously during the handling of samples.

The chromatograms of extracts of the liver, lungs and kidneys had the same general appearance. The peak corresponding to FPT was the largest, but it was less marked than in the brain. In addition to FPT the presence of FPT-dec., FPT-dec.-SO and FPT-SO together with very small amounts of other metabolites was indicated. A peak corresponding to TFT was always seen.

Dog study on blood levels and excretion. The concentration of radioactivity expressed as mpg parent substance/ml serum in the serum from dogs given FPT-dec. or FPT is shown in fig. 7. Following FPT-dec. peak concentrations are seen at 7 days after administration, while in the two dogs given FPT the peak concentrations are seen at 2 and 24 hours respectively.

Fig. 7. Concentration of radioactivity in serum from dogs given FPT orally (1 mglkg) or FPT-dec. by intramuscular injection (2 or 6 mg/kg) expressed as mpg of parent

substance per ml serum.

Tabl

e 3.

E

xcre

tion

of r

adio

activ

ity i

n ur

ine

and

faec

es f

rom

dog

s.

1 m

g FP

T/k

g p.

0.

%-d

ose/

%

-dos

e %

-dos

e/

%-d

ose

ml X

103

m

l X 1

03

Uri

ne

2 m

g FP

T-d

ec./k

g i.

m.

% -d

ose/

%

-dos

e %

-dos

e/

% -d

ose

ml X

103

m

l X 1

03

6 m

g FP

T-de

c./k

g i.

m.

% -d

ose/

%

-dos

e %

-dos

e/

%-d

ose

ml X

103

m

l X 1

03

0- 1

day

28

.5

5.0

79.4

11

.9

1-

2 da

ys

13.0

2.

0 8.

1 2.0

3-

4 da

ys

2.0

0.3

2.8

0.8

6- 7

day

s 1.

2 0.

2 1.

2 0.

3 10

-11

days

0.6

0.

1 0.

7 0.

2 15

-16

days

0.

4 0.

1 0.

3 0.

1 24

-25

days

0.2

0.

01

1.1

0.2

1.4

0.1

1.4

0.1

1.6

0.2

2.0

0.2

2.8

0.5

4.7

1.0

3.7

0.4

5.4

0.9

1.7

0.4

1.6

0.3

1.1

0.2

1.1

0.1

0.7

0.1

0.6

0.04

Faec

es

0-

1 d

ay

1- 2

day

s 3-

4

days

6- 7

day

s 10

-11

days

15

-16

days

24

-25

days

%-d

ose/

g

x1

03

360.

3 15

4.6

11.1

1.

7 1.

5 0.

7 0.

3

%-d

ose

8.9

14.2

1.

6 0.

2 0.

1 0.

02

0.02

%-d

ose/

g

x1

03

625.

6

11.4

2.

2 0.

9 0.

6 0.

4

%-d

ose

53.4

0.8

0.1

0.03

0.

04

0.03

-

% -d

ose/

gX

103

12.9

11

.0

34.7

25

.1

9.3

8.4

1.8

%-d

ose

% -d

ose/

g

x1

03

%

-dos

e

0.5

0.5

1.9

1.1

0.1

0.2

0.03

3.5

57.0

36

.5

7.9

28.9

1.

0

0.3

3.6

1.2

0.3

0.4

0.03

1.4

0.2

2.6

0.2

0.3

1.9

0.6

0.9

2.2

0.6

4.1

1 .o

4.8

0.6

8.1

2.2

1.5

0.5

2.2

0.5

0.2

1.4

0.2

1.2

0.1

0.8

0.1

0.9

% -d

ose/

%

-dos

e %

-dos

e/

%-d

ose

gx

10

3

gx

10

3

20.8

1.

6 6.

1 0.

6 -

- 41

.0

4.6

56.8

5.

7 34

.7

1.0

120.

6 2.5

16

.9

1.2

7.6

0.3

1.7

0.1

7.2

0.3

5.8

0.2

2.0

0.1

-

-

w

VI

w


%-dos./ml

x 1 ~ 3

. im.

I 4 a 12 16 20 24 28 ~a~~

Fig. 8. Excretion of radioactivity in urine from dogs given FPT orally (2 dogs) or FPT-dec. intramuscularly (4 dogs).

The excretion of radioactivity in the urine and faeces is shown in table 3. Since large variations were seen in the amount of urine and faeces obtained from different dogs as well as in the same dog at different periods, the samples can not be considered as 24 hours collections. Therefore the percentage dose/ mi urine and percentage dose/g faeces respectively have been considered instead of the percentage dose/time interval. The mean values for excretion in the urine (expressed as %-dose/ml) after administration of FPT-dec. (4 dogs) and FPT (2 dogs) are shown in a semilogarithmic plot in fig. 8. The curves show the same picture for excretion in the urine as that obtained for the concentration of radioactivity in the serum (fig. 7). The high value seen in the interval 24-25 days after oral administration of flupenthixol is ex- plained by a high concentration in one of the two urine samples from this interval, possibly due to contamination. Roughly the same pattern as for the urinary excretion was seen for faecal excretion (table 3), but in this case the


1 hour 2 hours Buffer

maximum excretion in the interval 6-7 days was only seen in one of the dogs given FPT-dec., while the other three dogs showed maximum excretion at 3-4 days after injectioln.

Seven-37 yo of the radioactivity could be extracted from the urine and 21-54 % from the faeces at an alkaline pH. The TLC of extracts of urine from dogs given FPT shows that FPT-SO was the major excretion product in the urine. In addition, FPT, HN-FPT and HN-FPT-SO were seen. The TLC extracts of faeces was to some extent interfered with by impurities, but there was indication of the presence of FPT and the sulphoxide of this compound.

TLC of urine and faeces extracts from dogs given FPT-dec. indicated that the sulphoxides of FPT and HN-FPT were the major excretion products followed by HN-FPT. The presence of FPT-dec., FPT-dec.-SO and FPT in very small amounts was also indicated.

In vitro study on hydrolysis. As shown in table 4 the amount of radioactivity extractable in the hydrolysis experiment ranged from about 100 per cent for the pure buffer to 36 per cent for the one hour sample of kidney homogenate. The relative amounts of FPT-dec. and FPT show that the highest hydrolytic activity was found in the brain homogenate and diluted blood, while the homogenates of liver, lungs and kidneys had less activity. A

% -Radioactivity Distribution of radioactivity in DCE-phase on FPT-dec. and FPT

% -FPT-dec. % -FPT

102 82 5 93 77 6

Table 4.

Extraction and chromatographic analysis of in vitro sampIes.

~~~ ~

72 3 70 4 85

1 hour 2 hours Brain

1 hour 60 20 67 12 78

1 hour 50 25 64

2 hours

16 75

1 hour 36 20 64

2 hours

22 67 2 hours

1 I 74

I 55

Liver

Lungs

Kidneys

356 A. JORGENSEN, K. FREDRICSON OVER0 AND V. HANSEN

small spontaneous degradation was observed in the buffer samples. In fig. 9 the chromatograms of the extracts of the 2 hour samples are shown. The FPT-peak shows a tendency to separate into two peaks, indicating the formation of the @-isomer of FPT. The degradation product, TFT, was seen on all the chromatograms.

a

b

d 91. 'I.

LO- L o -

30-

20-

10-

C

30-

r rv-dec. TFT 10- FPT-dec TFT

0 2 I 6 8 10 12 16 ~ r , ~ m 0 2 L 6 E 10 12 1L 1 6 c m

-1. a

L O - FPT

30-

20-

10- I \ FPT-drc

-1. LO-

30- F P I

f

I ! 10- / \

Fig. 9. Thin-layer chromatograms of extracts of 2 hour samples from the in vitro hydrolysis study. a: Buffer, b: Brain homogenate, c: Diluted blood, d: Liver horno-

genate, e: Lung homogenate, f Kidney homogenate. Solvent system I.


Discussion The presence of a relatively limited depot of flupenthixol decanoate at the

site of injection is indicated by the large amount of radioactivity maintained in the leg, where the injection was given. The detection in the organs of unchanged flupenthixol decanoate as well as the high hydrolytic activity of the blood and the organs as demonstrated by in vitro experiments, suggests the release of unhydrolyzed ester from the depot and subsequent hydrolysis. Since flupenthixol is capable of passing through the blood brain barrier (JBRGENSEN et al. 1969), it seems unlikely that the more lipophilic flupenthixol decanoate should be unable to do so. Thus the flupenthixol found in the brain might originate from flupenthixol decanoate hydrolyzed both in the brain and elsewhere. This is not in agreement with the results on the fluphenazine ester, fluphenazine enanthate, by EBERT & HESS (1965), who sug- gested, that the fluphenazine found in the brain, originated solely from the ester hydrolyzed outside the brain.

The neuroleptic effect seen in rats after the administration of flupenthixol decanoate in the pharmacological studies of M0LLER NIELSEN et al. (unpublished) and FRANCK (1970) is presumably due to the presence of flupenthixol in the brain as this is the main substance found in brain extracts.

The distribution of radioactivity following the administration of flupenthixol decanoate to rats is identical to that found after oral administration of flupenthixol (JBRGENSEN et al. 1969) and very similar to that seen after the administration of other psychotropic drugs e. g. fluphenazine enanthate (EBERT & HESS 1965) and prathiadene (dosulepinum NFN) (HDRE~OVSKG et al. 1967). The substances detected in organs from dogs given unlabelled flupenthixol decanoate indicate a distribution pattern similar to that found in rats. However, when blood levels are considered, a difference is seen between rats and dogs, as rats show a maximum within the first 24 hours, while a maximum blood level is obtained in the dog 7 days after administration. The reason for this species difference is obscure, but is probably to be sought in differences in the rate of release of the drug from the depot.

A comparison between the amounts of radioactivity in blood and organs obtained after administration of flupenthixol decanoate and flupenthixol (JBRGENSEN et al. 1969) respectively, reveals that higher levels are maintained for a longer period of time when flupenthixol decanoate is administered, indicating that injection of the decanoic acid ester in oil causes a sustained release of drug from the depot. This is further supported by the elimination data, The elimination half-life in rats was estimated as 8 days following flupenthixol decanoate as compared to one day after flupenthixol (JBRGENSEN et al. 1969). Thus the biochemical findings correlate well with the prolonged effect seen in the pharmacological studies with flupenthixol decanoate (MPILLER NIELSEN


et al. unpublished; FRANCK 1970). Apart from possible traces of unchanged flupenthixol decanoate and its sulphoxide, the metabolites found in the urine and faeces are the same as those found after flupenthixol (J0RGENSEN et al. 1969), showing that flupenthixol decanoate is largely converted to flupenthi- xoil and that the pathway for biotransformation of flupenthixol does not change.

The presence of both isomers of flupenthixol after the administration of a-flupenthixol decanoate has been demonstrated in these studies. Since, however, the spontaneous formation otf the @-isomer during the procedures has been shown, the degree of isomerisation within the organism can not be estimated.

A c k n o w l e d g e m e n t s

assistance. The authors wish to thank their laboratory staff for skilful technical

R E F E R E N C E S

Bilde, Annelise & Anna Madsen: Flupenthixoldepotbehandling af schizophrene. Nordisk Psykiatrisk Tidsskrift 1970, XXIV, 245-249.

Ebert, A. G . d S. M. Hess: The distribution and metabolism of fluphenazine enanthate. J . Pharmacol. Exp. Therap. 1965,148, 412-21.

Enerheim, Birgitta, C. G. Gottfries & Catherine SundCn: Kliniska forsok med flupenthixol-depi-behandling vid lingvariga schizofrena psykoser. Nordisk Psykiatrisk Tidsskrift 1970, XXIV, 239-244.

Franck, K. F.: The prolonged inhibition of the conditioned avoidance response in rats and mice after depot injection of flupenthixol decanoate in viscoleo@’. Collegium Internationale Neuro-Psychopharmacologicum. VII International Congress, August 11-15, 1970, Prague, Czechoslovakia.

Gjestland, Arnulf: Flupenthixol i depotform - Nytt hjelpemiddel i psykiatrisk ettervern. T . norske Legeforen. 1970,90, 475-77.

Herberg, R. J.: Determination of carbon-14 and tritium in blood and other whole tissues. Anal. Chem. 1960, 32, 42-46.

HoreSovsk9, O., Franc & P. Krans: Biochemistry of drug IX. The metabolic fate of a new psychotropic drug ll-(3-dimethylaminopropylidene)-6,ll-dihydrodibenz-(b, c)-thiepine (prothiadene). Biochern. Pharmacol. 1967,16, 2421-29.

Jgjrgensen, Aksel, Villy Hansen, Ulla Dahl Larsen & A. Rauf Khan: Metabolism, distribution and excretion of flupenthixol. Acta pharmacol. et toxicol. 1969, 27, 301-13.

Remvig, Jgjrgen, Martin Lotz I& Knud Odgaard: Flupenthixol-depot-behandling ved kronisk skizofreni. Nordisk Psykiatrisk Tidsskrift 1968, XXII, 392-400.

metabolism, distribution and excretion of flupenthixol decanoate in dogs and rats

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