Naunyn-Schmiedeberg's Arch Pharmacol (1990) 342:357-361

Naunyn-Schmiedeberg's

Archivesof Pharmacology © Springer-Verlag 1990

Absorption and metabolism of eapsaieinoids following intragastrie administration in rats Josef Donnerer, Rainer Amann, Rufina Schuligoi, and Fred Lembeck Institut f/Jr Experimentelle und Klinische Pharmakologie, Universitfit Graz, Universitfitsplatz 4, A-8010 Graz, Austria Received March 5, 1990/Accepted April 17, 1990

Summary. This study was performed to examine the metabolism and absorption of intragastrically administered capsaicinoids in the anaesthetized rat. [3H]-dihydrocapsaicin ([3H]-DHC) and unlabelled capsaicin were readily absorbed from the gastrointestinal tract but were almost completely metabolized before reaching the general circulation. A certain degree of biotransformation already took place in the intestinal lumen. Unchanged compounds (identified by chromatography) were present in portal vein blood. There seems to be a saturable absorption and degradation process in the gastrointestinal tract and a very effective metabolism in the liver. Less than 5% of the total amount of extracted radioactivity consisted of unchanged [3H]-DHC in trunk blood and brain 15 rain after gastrointestinal application. On the other hand, approximately 50% unchanged [3H]D H C was detected in these tissues 3 rain after i.v. or 90min after s.c. application of the capsaicinoids. Dihydrocapsaicin (DHC) or [3H]-DHC were metabolized when incubated in vitro with liver tissue but not with brain tissue. The metabolic product(s) did not show capsaicin-like biological activity. It can be concluded that rapid hepatic metabolization limits systemic pharmacological effects of enterally absorbed capsaicin.

Key words: Capsaicin - [3H]-dihydrocapsaicin - Gastrointestinal absorption - Degradation - In vitro incubation

Introduction Because of the increasing use of capsaicin in experiments dealing with gastrointestinal protective mechanisms (Holzer and Lippe 1988; Holzer et al. 1989) and the Send offprint requests to J. Donnerer at the above address

general use of capsaicinoid spices in food (Lembeck 1987), information about the absorption and metabolism of capsaicinoids is needed. It is known that capsaicin given directly into the stomach of rats has only minimal excitatory effects visible on immediate blood pressure responses (Lippe et al. 1989) in contrast to intravascular or subcutaneous administration (Donnerer and Lembeck 1983). On the other hand it has been shwon that capsaicin disappears within a rather short time period from the intestinal lumen (Kawada et al. 1984) and should therefore reach the circulatory system. Consequently we focused on determining the extent to which unchanged capsaicinoids can reach systemic circulation and the brain to display systemic pharmacodynamic effects following a gastrointestinal as compared to a parenteral mode of administration. Since biotransformed products of capsaicin are difficult to detect, the use of [3H]-labelled dihydrocapsaicin ([aH]-DHC) allowed us to determine the percentage of unchanged compound in the total extracted radioactivity. Dihydrocapsaicin (DHC) has been shown to display pharmacodynamic and pharmacokinetic properties comparable with those ofcapsaicin (Buck et al. 1982; Kawada et al. 1984). In all of the following in vivo experiments a mixture of [3H]-labelled D H C and unlabelled capsaicin was used. Thus a concentration of the capsaicinoids was reached which corresponded to that used to achieve pharmacodynamic effects following either route of administration, and permitted to compare the absorption and metabolism of the two naturally occurring capsaicinoids. Additional in vitro experiments served to localize the capsaicin-metabolizing activity and to determine possible biological activities of capsaicin and DHC metabolites.

Methods Experimental protocol of i n vivo experiments. Male Sprague-Dawley rats (Himberg, Austria) weighing 300--350 g and fasted overnight were anaesthetized with 50 mg/kg sodium pentobarbital. The tra-

358 chea was cannulated and, if the protocol included i.v. application of drugs, a cannula was placed in the jugular vein. A stock solution of capsaicin (10 mg/ml) was prepared in 10% Tween 80 and 10% ethanol in saline and diluted with saline to the appropriate concentration. [3H]-DHC was supplied in a stock solution in ethanol with a specific activity of 54 Ci/mmol and a radioactivity concentration of 16,3 mCi/ml. This stock solution was diluted to a solution containing 1 ~tCi/~tl (5.6 pg DHC/IxCi) with ethanol and the appropriate amount of this solution was added to the unlabelled capsaicin solution. [3H]-DHC eluted from the HPLC column with more then 99% purity at the position of DHC. For intragastric application a dose of 50 ~tg or 500 Ixgcapsaicin dissolved in 1 ml of solvent was labelled with 12 ~tCi of [3H]-DHC. The drugs were injected into the stomach via a cannula which had been inserted into the oesophagus and had been tightened close to the cardia following a midline abdominal incision. The ligature should prevent reflux of the intragastrically applied material. Care was taken not to obstruct supplying blood vessels. In one set of experiments the pylorus was ligated to prevent passage of the applied drugs into the duodenum whereas in another group the pylorus was left open. Five min after intragastric application, 500 U/kg heparin were injected i.v. and a cannula was inserted into the portal vein with its tip close to the liver. Portal vein blood (approx. 1.5 ml) was collected for 10 min. Thereafter animals were decapitated, trunk blood collected in EDTA-coated tubes and the brain removed and frozen. The stomach or the stomach plus intestine were emptied into vials, the volume was determined and 30 Ixl of it withdrawn for further analysis. A total exposure time of 15 min after intragastric administration of capsaicinoids was chosen because during this period approximately 50% of the administered dose disappears from the intestinal lumen reportedly (Kawada et al. 1984). For i.v. application a dose of 2 mg/kg capsaicin = 0.2 ml of a 1 mg/ml solution per 100 g body weight was labelled with 12 IxCi [3H]-DHC. Following slow (10 s) i.v. injection of this solution, the animals were artificially ventilated for 3 min. Thereafter the rats were decapitated, trunk blood collected and the brain removed as described above. For s.c. application 50 mg/kg capsaicin = 0.5 ml of a 10 mg/ml solution per 100 g body weight was labelled with 20 ItCi [3H]-DHC and injected s.c. in the neck region. The temperature of the animals was kept constant with a heating lamp and after a time period of 90 rain the animals were decapitated, trunk blood collected and the brain removed as described above. At the time points chosen to measure the disposition of capsaicin following i.v. or s.c. application tissue concentrations reach peak levels (Saria et al. 1982).

Extraction and detection of capsaicin and [3 H].DHC. Tissue samples (1 ml of blood, 1 g of brain tissue or 30 I11of gastrointestinal content) or the samples from the in vitro incubation were extracted with 10 vol. ice cold acetone according to Saria et al. (1982). Brain samples were homogenized using a ultrasound tip, blood and gastrointestinal content were mixed with acetone. After centrifugation for 10 min at 3000 × g supernatants were evaporated in a Brinkman sample concentrator. The residue was dissolved in 1 ml eluent (40% acetonitrile in water) and 200 ~tl of it were injected onto a reversed phase HPLC column. Capsaicin and DHC were determined by fluorimetric detection at an excitation wavelength of 270 nm and an emission wavelength of 330 nm according to Saria et al. (1981). Capsaicin and DHC eluted in single peaks. The recovery of capsaicin, DHC and [3H]-DHC by the extraction procedure was 5 0 - 7 0 percent when added to brain, liver or blood samples and 9 0 - 1 0 0 percent when added to gastrointestinal content. When using labelled DHC, 1-min fractions of the outflow were collected in scintillation counting vials, 10 ml of liquid scintillator Ready Solv HP/b, Beckman, Fullerton, USA) added, and radioactivity expressed as disintegrations per min (dpm). Total radioactivity in tissues was determined according to Miller et al. (1983). Briefly, tissue samples were homogenized in 10 vol. Tris-buffer pH 7.4, digested with 1 N sodium hydroxide, neutralized with 1 N perchloric acid and bleached with 3% peroxide.

looBrain

Liver

2_

o D

_L

50-

0

1

30

m~

0

30

Fig.1. Percentage recovery of DHC following the addition of the compound to minced liver or brain tissue. The tissues were extracted immediately (0 min) or after 30 min in vitro incubation at 37 ° C. Means _+ SEM, n = 6, * P < 0.05 compared to all the other conditions

In vitro incubation and determination of biological activity in HPLC fractions. Samples of rat liver or brain were chopped with a McIlwain tissue chopper into 200 ~tm slices in two directions. For the determination of the metabolic activity 100 mg portions of the homogenates were incubated either at 0°C or at 37°C with 1 ~tg DHC or 0.03 ~tCi [3H]-DHC for time periods of 0 to 30 min. At the end of the incubation the compounds were extracted and analyzed as described above. For the determination of biological activity in HPLC fractions 1 g of liver homogenate was incubated either alone or with 100 I~g DHC for 30 rain, extracted and subjected to HPLC analysis as described above. The outflow was collected in 1-min fractions which were concentrated in a Speed Vac Concentrator and redissolved in 0.2 ml Krebs buffer. Capsaicin-like activity was assayed in the following bioassay system: The isolated rat urinary bladder was divided into 2 halves and mounted in two 4 ml organ baths. Contractions were measured isometrically under a preload of 0.5 g. Whereas one preparation was made unresponsive to a further DHC application by a 5-min preexposure to 3 Ixg/ml DHC ("desensitization", cf. Santicioli et al. 1987), the other was left untreated. Now 40 ~tl of the redissolved sample were added to each preparation. A contraction of the untreated, but not of the DHC-preexposed organ, would be indicative for capsaicin-like activity. Substances and statistics. The following substances were used: 8methyl-N-vanillyl-6-nonenamide (capsaicin) from Merck (Darmstadt, FRG), 8-methyl-N-vanillyl-nonanamide (dihydrocapsaicin) from Sigma (St Louis, USA), dihydro-6,7[3H]-capsaicin was synthetized by Internationale Isotope Miinchen (UnterschleiBheim, FRG) and generously provided by Prof. Dr. T. Rozman, Basotherm (Biberach, FRG). Data were analyzed by use of ANOVA and differences between groups were determined by Scheffe's multiple comparisons test. Results

In vitro degradation I n c u b a t i o n o f D H C w i t h liver tissue for 30 m i n at 37 ° C g r e a t l y r e d u c e d the a m o u n t o f the c o m p o u n d r e c o v e r e d a t t h e p o s i t i o n o f D H C o n H P L C ( r e t e n t i o n t i m e 15 rain) w h e n c o m p a r e d to s i m p l y a d d i n g D H C to the tissue a n d e x t r a c t i n g it. W h e n D H C w a s i n c u b a t e d for 30 m i n w i t h b r a i n tissue, t h e m e t a b o l i s m w a s less m a r k e d (Fig. 1). W h e n [ 3 H ] - D H C w a s i n c u b a t e d w i t h liver tissue for

359 100.c: E u3

12pCi [:~-I] DHC + 500~Jg C

% intact [~H]DHC

12pCi [~'-I] DHC + 501.1g C

% intact [st-I]DI-IC

_=

(b .1a

50

+ to '5 z~ ~t

~t

[_a_~j

,,

l

Stomach

I

Stomach + rntestine

Stomach

I

Stomach + Intestine

Fig. 2. Disappearance of capsaicin (C) and [3H]-DHCfrom the gastrointestinal tract after intragastric administration. Stomach: pylorus occluded; stomach + intestine: pylorus open with a sample withdrawn after the whole content of stomach plus intestine had been emptied, Measurements were made 15 min after intragastric application and compared with immediate extraction after application. Percentage [3H]-DHC (open columns) and capsaicin (hatched columns) remaining in the lumen after application of 50 ~tgor 500 ~tg capsaicin. N = 4 for each determination. * P < 0.05 compared to other treatments

30 min, the percentage of radioactivity at the position o f D H C decreased to the same extent as the fluorescencepeak in the experiment using unlabelled DHC. On HPLC, the radioactivity was then found to elute shortly after the solvent front (retention time 1 - 2 min; n = 3, results not shown) - the same phenomenon was observed with the tissue samples in the in vivo experiments described below. Using unlabelled D H C or capsaicin the first peak could not be detected, presumably because it did not fluoresce. Possible biological activity of the early eluting components was assayed in the isolated rat urinary bladder. This was done with extracts from 30 min-incubation experiments with liver tissue using 100 lag D H C starting material (see method section). Whereas the small portion of D H C not metabolized after 30 min incubation (15%) and still eluting at the position o f synthetic D H C induced contractions ( 2 0 - 36% of KC1 maximum) in untreated preparations, but not in D H C (3 lag/ml, 5 min) preexposed tissues, samples obtained from early H P L C fractions containing the large part o f the metabolized compounds or those obtained from liver extracts without D H C added, did not display activity in any of the preparations.

Tissue content of intact [3H]-DHC after intragastric application The amount of unlabelled (intact) capsaicin and labelted (intact) D H C left in the stomach or stomach plus intestine, respectively, 15 rain after application as compared to immediate extraction after administration, is shown in Fig. 2. These amounts correspond to chromatographical-

Fig. 3. Schematic diagram of [3H]-DHC absorption and metabolism

showing the percentage of intact [3H]-DHC in the extracted radioactivity in different regions. Comparison of the effect of adding 500 p.g or 50 pg (values in brackets) unlabelled capsaicin (C). In A the pylorus was occluded so absorption was only possible from the stomach whereas in B the pylorus was left open and absorption could occur from the stomach and small intestine. N = 4 - 6 for each determination. Highest SEM of means was _ 15

ly identified compounds. It can be seen that a lower percentage o f the applied dose o f capsaicin and [3H]D H C is left in the stomach (or stomach plus intestine) when the amount of capsaicin given is reduced from 500 lag to 50 lag. Figure 2 also shows that the disappearance of the labelled D H C correlates with the disappearance of unlabelled capsaicin. However, in the total amount o f tritium-radioactivity extracted from the intestinal content 15 min after administration, there was also already a certain portion of degradation product eluting in the front o f the chromatogram (see also above results from in vitro experiments). This metabolisation or degradation products could also be recovered with the acetone extraction by 100% and may have been formed in the intestinal lumen during the 15 min contact time. The percentages of the unchanged [3H]-DHC in the total extracted radioactivity are shown in Fig. 3 A and 3 B for the gastrointestinal lumen as well as for the further route of drug passage (portal vein blood - trunk blood - brain). Taken togehter, the results from Fig. 2 and Fig. 3 A and 3 B would indicate that within 15 min from administration approximately 3 0 - 50% of capsaicin and D H C are absorbed from the stomach (Fig. 2) with almost no degradation taking place there (Fig. 3A). O f a dose of 50 lag capsaicin (plus tracer amounts of [3H]-DHC) approximately 90% are absorbed from the whole intestine whereas approximately 75% of a 500 lag dose of capsaicin are absorbed (Fig. 2) with

360 100-

-r-

ministration; brain: 3242 __ 796 dpm/g following s.c. versus 2337 + 529 dpm/g following intragastric administration, n = 5 - 6 for each). The recovery of radioactivity in brain and blood samples by the extraction procedure (as compared to total radioacitivty counted in the digested samples - see Methods) was 5 0 - 7 0 % .

Brain

Trunk Blood i

"10 ¢t

_1

50i-

Discussion o

g

0

i.v.s.c.i.g. 3

90 15

Lv. s.c.i.g. 3

90

15 min

Fig. 4. Percentage of unchanged [3H]-DHC in the total amount of extracted radioactivityfrom trunk blood and brain. Determinations were made 3 min after i.v., 90 min after s.c., or 15 rain after intragastric application of the compound. N = 5 for each column. * P < 0.05 compared to other treatments

considerable degradation taking place in the intestine, probably by mucosal enzymes (see Fig. 3 B). The data in Fig. 3 A and 3 B further imply that the percentage of unchanged labelled DHC declined with the route of drug passage (gastrointestinal lumen > portal vein blood > trunk blood and brain). A higher percentage of intact molecules is found in the portal vein blood when absorption occurred only from the stomach as compared with stomach plus small intestine (see Fig. 3 A and 3 B) and it was also higher when 500 ~tg capsaicin were added instead of 50 gg (compare also Fig. 3 A and 3 B). Yet there was no difference in the total amount of radioactivity extracted from portal vein blood in the different sets of experiments (range between 6000 and 10000 dpm/ ml) indicating that degradation or metabolism products of [3H]-DHC were also recovered from blood by the extraction method used (see Methods and above Results).

Disposition of [3H]-DHC in trunk blood and brain following different routes of administration In Fig. 4 the percentage of intact [3H]-DHC in the total extracted radioactivity from brain or trunk blood is compared 3 min after i.v., 90 rain after s.c., or 15 min following intragastric application of[ 3H]labelled DHC together with 50 gg or 500 gg capsaicin. Following either parenteral route of application, approx. 50% unchanged drug was found in trunk blood and in brain; following the intragastric route less than 5% intact molecules were found there. In this context it must be mentioned that, despite the low percentage of intact molecules found in trunk blood and brain after intragastric administration, the total activity extracted was in the same range as the one measured 90 min following s.c. application of the drugs (trunk blood: 5101 _+ 918 dpm/ml following s.c. versus 4776 ___347 dpm/ml following intragastric ad-

The present study clearly shows that, in the rat, capsaicin and DHC are readily absorbed from the gastrointestinal tract, as had been already shown in a different type of experiments (Kawada et al. 1984; Kawada and Iwai 1985). The parallel disappearance of the unlabelled capsaicin and of the labelled DHC from the gastrointestinal lumen points to identical biotransformation or absorption of the two compounds and excludes conversion of one compound to the other. Data on absolute absorption within 15 min after administration could only be calculated and have to consider the metabolism of the capsaicinoids already occurring in the intestinal lumen (Fig. 2 and Fig. 3 A, B). The main conclusion from this study is that the capsaicinoids are absorbed partly in their - chromatographically identified - intact form, but are further metabolized to a great extent in the liver before they reach the general circulation. A higher percentage of intact molecules was absorbed from the stomach than from the duodenum or jejunum. The observation that the percentage of intact [3H]DHC was higher in the intestinal lumen as well as in the portal vein blood when the concentration of simultaneously administered cold capsaicin was higher (Fig. 3 A, B), points to a saturability of the degradation process in the intestinal lumen and in the intestinal wall during absorption. The saturability of the absorption process is also supported by the results which show that more [3H]DHC and capsaicin (as % of total administered dose) are left in the stomach and intestine after 15 min when a high dose of capsaicin is administered (Fig. 2). The radioactivity found and extracted in the brain and trunk blood following intragastric application of [3H]-DHC mainly eluted in the front of the chromatogram. This same fraction is also found after in vitro incubation of [3H]-DHC with liver homogenate indicating that it is probably formed in the liver and transported to the brain. Biotransformation of capsaicinoids can probably also take place in other organs, such as lung or kidney, but is certainly highest in the liver as already shown in a previous report (Kawada and Iwai 1985). An identification of the metabolites or degradation products was not intended, but prevous studies have shown that mainly ring hydroxylation and splitting of the side chain are the important steps (Kawada and Iwai 1985; Miller et al. 1983). From our experiments it can only be stated that the capsaicinoids are transformed to more hydrophilic compounds (deduced from their behaviour on reversed phase HPLC) which do not show fluorescence at wave lengths where capsaicin, DHC, and other capsaicinoids do. A possible pharmacological activity of capsaicin degradation products cannot be excluded but

361 does not seem likely in view of the lack of their in vitro activity on the rat urinary bladder. The low dose of cold capsaicin given into the stomach (50 pg/ml) corresponded to the doses used for various pharmacological experiments in this organ (Holzer and Lippe 1988; Holzer et al. 1989). Assuming the same absorption process for capsaicin as for D H C , it can be argued in this context that p h a r m a c o d y n a m i c effects by capsaicin could take place within the portal vein and the liver, although the concentration of unlabelled capsaicin detected in the portal vein blood with fluorescence in our study was in 7 out of 10 cases below the detection limit of 25 ng/ml and in 3 animals at around 100 ng/ml (0.3 gM). This latter concentration m a y have excitatory effects on the C-fibre system located there (Niijima 1982; Stoppini et al. 1984; A m a n n and Lembeck 1986). When 500pg/ml of capsaicin were given into the stomach, in 6 out of 10 animals portal vein blood concentrations of capsaicin were in the range between 50 and 200 ng/ml. Almost no intact [3H]-DHC was found in the brain after intragastric administration when c o m p a r e d to parenteral administration, although total extractable activity was not significantly different. The most likely explanation is that following parenteral administration only a fraction of the whole blood circulation transits through the liver and thus a greater proportion of intact molecules can reach other organs. Since capsaicin is quickly retained in these tissues (e.g. central nervous system) metabolism in the liver is o f less importance. This finding makes unlikely the role ofextrahepatic tissues as major biotransformers of capsaicinoids. In conclusion, the present results suggest that intragastric D H C and capsaicin are readily absorbed. In contrast to parenteral administration, gastrointestinally absorbed capsaicinoids reach the central nervous system almost exclusively as degradation products which seem to have no capsaicin-like biological activity.

Acknowledgements. The authors wish to thank Dr. P. Holzer for thoughtful comments on the manuscript and Mrs. A. Ebersold for technical assistance. This work was supported by the Austrian Research Funds, grant No. P 6646 M, the Austrian National Bank (grant No. 2811) and the Pain Research Commission of the Austrian Academy of Sciences.

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