Curare-like agents: Relation between lipid solubility and transport into bile in perfused rat liver

EUROPEAN JOURNAL OF PHARMACOLOGY 10 (1970) 283-289. NORTH-HOLLAND PUBLISHING COMPANY

CURARE-LIKE

AGENTS: RELATION BETWEEN LIPID SOLUBILITY

AND TRANSPORT

INTO BILE IN PERFUSED

RAT LIVER

D.K.F. MEIJER and J.G. WEITERING Department of Pharmacology, University o f Groningen, Bloemsingel 1, Groningen, The Netherlands

Received 30 January 1970

Accepted 11 February 1970

D.K.F. MEIJER and J.G. WELTERING,Curare-like agents: relation between lipid solubility and transport fllto bile in perfused rat liver, European J. Pharmacol. 10 (1970) 283-289. The biliary excretion of six bis-quaternary ammonium compounds, alcuronium (Alloferine(~), dimethyltubocurarine, gallamine, hexafiuorenium (MylaxenR(~, pancuronium (Pavulon(-~) and d-tubocurarine was studied in the isolated perfused rat liver. Thin-layer chromatography of bile samples showed great differences in transport into bile. Hexafluorenium and d-tubocurarine are excreted to a large extent but only very small amounts of the other compounds appeared in bile. The lipophilic character of the compounds was estimated by shaking various aqueous solutions with octan-l-ol. Passage into the organic solvent of d-tubocurarine but not of the other agents was influenced by the pH of the aqueous phase. Only hexafluorenium and d-tubocurarine could be extracted into the organic solvent to a significant degree, suggesting that lipid solubility is an important factor in the transport of bis-quaternary ammonium compounds from plasma to bile. Lipid solubility Biliary excretion of curare-like agents

1. INTRODUCTION There is much evidence of quantitative differences in the excretion of curare-like drugs in the bile. While biliary excretion of d-tubocurarine is important especially in the absence of renal function (Ebbinghaus, 1954; Robelet et al., 1964; Cohen et al., 1967) this is not true for other curare-like agents in spite of similarity in chemical structure (fig. 1). Marsh (1952) found that dimethyl-curare is excreted in bile in only very small amounts; renal excretion is probably far more important for this compound (Collier et al., 1948; Dal Santo, 1964). Mushin et al. (1949) presented evidence that 100 percent of the injected dose of gallamine could be recovered from the urine of man and mammals, while Feldman et al. (1969) recently showed that gallamine H3 is not significantly excreted in dog bile. Two hours after injection of alloferine in cats 10 percent was found in bile (Liithi, 1967). Very little is known about the excretion ofhexafluor-

Isolated perfused rat liver Bis-quaternary ammonium compounds

enium and pancuronium. The possible involvement of an active transport process in the biliary excretion of d-tubocurarine was suggested by Cohen (1967) and Meijer and Scaf (1968). We have now studied the biliary excretion o f six curare-like drugs in the isolated perfused rat liver, and have attempted to relate differences in biliary excretion of the compounds to their lipophilic character.

2. METHODS AND MATERIALS 2.1. Isolated perfused rat liver The perfusion technique was modified from the method of Miller et al. (1951) and Brauer et al. (1951). The surgical procedure and perfusion apparatus have been described in detail by Northrop and Parks (1964). The perfusion fluid was equilibrated with oxygen containing 5 percent CO2 and was supplied to the completely isolated rat liver by a

284

D.K.b:Meijer, J. G. Weitering, Biliary excretion o f curare-like agents

CH30~+ HO~ . ~ . ~

C H 3 0 ~

N(CH3}2

(CH3)2~1~

cH312~~

OH

~ O C H

N(CH3)2

OCH3

J~"~OC

3

H3

DimethyLtubocurarine

d-Tubocurarine

IJ ~ - - ' N ~ _

-(CH2)2 -N(C2H5)3 O-(CH2)2 -~(C2H5)3 + O-(CH2)2 -N(C2H5)3

CH

HOHoC-CH

~+ CH2- CH=CH2

CH-CH20H

CH N

CH2=CH-CH2-

Gattamine

+

ALcurOnium

OOC.CH3 OH3

c,H3

HexafLuorenium

CH3CO0 Poncuronium

Fig. 1. Structural formulae of curare-likeagents.

portal vein cannula and drained from a cannula in the thoracic vena cava. Bile was collected from a bile duct catheter. Livers were taken from fed male wistar rats weighing 2 5 0 - 3 5 0 g, anesthetized with pentobarbital sodium. The interval between occluding the vena porta and connecting the liver to the perfusion system was no longer than two minutes. Perfusion temperature

was 37.8 + 0.1°C. Hydrostatic perfusion pressure was 13-15 cm H20, providing a blood flow through the organ of about 1.7 ml/min/g liver wet weight. The isolated liver was placed in a chamber in an inverted position against a hemispherical support. The perfusion fluid consisted of equal parts of Krebs-bicarbonate-albumine solution (4% Bovine albumin) and defibrinated rat blood collected from rats under light

D.K.F.Meijer,J.G.Weitering,Biliaryexcretion of curare-likeagents ether anesthesia. The blood was defibrinated to avoid the use of heparin which can strongly bind quaternary ammonium compounds (Cheymol et al., 1955). The volume of perfusate was 150-170 ml. Collection of donor blood and the operative procedure were carried out under strictly aseptic conditions. The entire apparatus with the exception of the perspex parts was thoroughly cleaned and autoclaved. The perpex parts were disinfected with 5 percent formaldehyde solution and rinsed with sterile saline. Ampicillin 10/ag/ml/perfusate was added to inhibit bacterial growth. The pH of the perfusion medium was monitored on a Knick pH meter during the whole experiment by connecting a hydrogen-ion flow electrode (Ingold) to the system. We found that the pH of the perfusate (7.35-7.40 under standard conditions) was a reliable indicator of the functional state of the perfusion. When oxygen lack or bacterial contamination occurred the pH immediately fell to values of 7.30-7.25. To check the functional state of the perfused livers, clearance tests with bromsulfthaleine and procainamide-ethobromide were performed, and the amount and composition of the bile (total bile acids, total bilirubin) were measured. After connecting the liver to the perfusion system one hour was allowed for equilibration of bile flow, blood flow, glucose concentration and pH of the perfusate. Three experiments were performed with each drug. 400/ag in saline was given in the first 40 min period and 400 #g in the second 40 min period. Bile was collected for 30 min periods 10 min after each administration of drugs. 2.2. Histochemistry of perfused livers In order to indicate functional normality, the content and distribution of enzymes was studied in specimens of livers after four hours perfusion, fresh livers and livers which were kept for four hours at 37°C in oxygen containing 5% COz. After fixation, histochemical staining reactions (Koudstaal, Hardonk and Hadders, 1966) were performed for the following enzymes: phosphorylase, alkaline phosphatase, acid phosphatase, acid-5-nucleotidase, adenosine triphosphatase, indoxylesterase, naphtol-as-d-esterase, c~naphtyl esterase, acetylcholinesterase, aminopeptidase, succinic dehydrogenase, lactic dehydrogenase, isocitric dehydrogenase, p-hydroxybutyric dehydrogen-

285

ase, glucose-6-phosphate dehydrogenase, NAD reductase and NADH reductase. Generally enzyme activities and distribution pattern of livers perfused for four hours agreed very well with those of control livers. Mitochondrial enzymes and phosphorylase activity were unchanged as compared with fresh livers. The activity of ATPase, 5-nucleotidase and "lysosomal" enzymes were slightly increased with a sharper localisation of acid phosphatase at the biliary pole of the hepatic parenchym cell. The reaction of alkaline phosphatase was irregular in perfused and control livers while acetylcholinesterase and aminopeptidase showed practically no activity in both the control and perfused livers. The histological appearance of tissue, taken at the end of the perfusion was normal except for a little edema in the portal triads. Livers kept for four hours in carbogen gas at 37.5°C showed decreased enzyme activities and necrosis. So it was concluded that perfused rat livers maintained their histochemical integrity during a perfusion of four hours. 2.3. Determination of the curare-like compounds by thin-layer chromatography Thin-layer chromatography was used to obtain semiquantitative data on the amounts of curare-like drugs excreted in bile and to identify the excreted compounds. Bile samples of 0.01 ml or 0.02 ml were applied to siligacel-G-plates which were developed with acetone-HC1 1 N (1:4) and isopropanoI-HCl 1 N (1:1). The position of the compounds was visualized by spraying with Jodplateat reagent (Hexachloroplatin reagent Merck). Standard solutions of the curare-like agents in bile were applied to the same plates in various concentrations. Semiquantitative data were obtained by visual comparison of the coloured spots. It appeared that using this standardized procedure 25/ag/ml bile of all the curare-like drugs studied was always detectable. The accuracy of the method was checked by applying standard solutions of dimethyltubocurarine, hexafluorenium and d-tubocurarine to siligacel plates (n = 4) together with bile samples of various concentrations which were unknown to the observer. In all cases the estimated values obtained by visual comparison with the reference spots differed no more than 20 percent from the real values.

286

D.K.b:Mei/er. J.G. Weitering, Biliary excretion of curare-like agents

2.4. Determination o f the solubility in organic' solven ts The lipid solubility of the curare-like agents was determined in triplicate by partition between equal volumes of an aqueous phase and octan-l-ol shaken for 15 min at room temperature. The layers were separated by centrifugation. Concentrations in the aqueous phase and in octanol were determined by measuring the optical density on a Zeiss spectrophotometer (Alcuronium max = 293 mm, dimethyl-tubocurarine max = 280 mm, gallamine max = 267 ram, hexafluorenium max = 275 ram, d-tubocurarine max = 280 ram). Samples were always read against the corresponding phase o f a blank extraction. Pancuronium was determined fluorimetrically after reextraction from the organic solvent with 0.0l N HC1 and formation of a fluorescent complex with Rose Bengal as described for d-tubocurarine (Cohen 1963). Modified Krebs-Ringer solutions, containing NaC1, KC1, CaC12, KH2PO4 and MgSO4 in physiological concentrations (pH = 5.0) were used as aqueous phase. CO2 and NaHCO3 were omitted because CO2 is rapidly removed into the organic solvent. The pH was brought to 6,6, 7.2, 7.4 and 8.1 by adding small amounts of

NaOH 0.2N. Extractions were performed under nitrogen gas to prevent contamination with COz. pH of the aqueous phase before and after the extractions were checked with a micro pH electrode (Electrofact). Phosphate buffers pH 7.4 and 8.3 were made from 0.6 M Na2 HPO4 and KHz PO4 solutions.

3. RESULTS Very small amounts of alkaloids can be detected in bile using thin-layer chromatography and spraying with Jodplateat reagent. Visual comparison with known amounts of reference compounds in bile offered a sensitive and relatively simple method for studying biliary excretion of quaternary ammonium compounds. The results for six curare-like agents are shown in table 1. When alcuronium, gallamine and pancuronium were added to the perfusate in two subsequent doses, the presence of very weak spots on the developed thin-layer plates indicated that biliary excretion in both periods must be very low. Small but significant amounts of probably unchanged dimethyltubocurarine appeared in bile. Hexafluorenium and

Table 1 Biliary excretion of curare-like agents in the isolated perfused rat liver. Compound

Dose in ug

Amount of the drug excreted in t~g/ml bile experiment 1

experiment 2

experiment 3

Alcuronium

400 + 400

~ 25 ~ 25

~ 25 "( 25

~ 25 ~ 25

Dimethyltubocurarine

400 + 40

20-30 40-60

20-30 40-60

20-30 60-90

Gallamine

400 + 400

~ 25 ~25

~ 25 ~25

~ 25 ~25

Hexafluorenium

400 + 400

120-180 240-360

80-120 240-360

100-150 240-360

Pancuronium

400 + 400

~ 25 <25

~ 25 ~25

~ 25 <25

d-Tubocurarine

400 + 400

120-180 200-300

100-150 200-300

100-150 240-360

Biliary excretion of curare-like agents was studied with the isolated perfused rat liver in three experiments for each compound. The drug was added to the perfusion fluid in two doses, l0 min after each administration of the drug bile was collected for a 30 min period. Bile production was 0.35-0.40 ml per 30 min. Semiquantitative data on concentration in bile were obtained with thin-layer chromatography. (See Methods.)

287

D.K.F.Meijer, J. G. lCeitering, Biliary excretion o f curare-like agents

d-tubocurarine appear in bile in much larger amounts. No metabolites of the two agents were detected in bile; thin-layer chromatography with different solvent systems revealed only one spot with Rf values identical with that of the authentic drugs. Bile-plasma ratios of d-tubocurarine were estimated to be 100 or more (Meijer and Scaf, 1968) from experiments in which bile and plasma levels were determined fluorimetrically. Partition between octan-l-ol and an aqueous phase is a good model for studying the penetration of drugs through cellular membranes, due to the polar characteristics of the particular organic solvent (Hansch and Fujita, 1962). Extractions were carried out with modified KrebsRinger solutions with an electrolyte composition resembling that of rat plasma. To rule out possible influences of varying CO2 levels in both phases no CO2 and NaHCO3 were added. Percentages of the compounds in octanol after 15 min shaking are shown in table 2. Insignificant amounts of alcuronium, dimethyl-tubocurarine and pancuronium were extracted in octanol while a low percentage of gallamine appeared in the organic phase. Only hexafluorenium and d-tubocurarine could be extracted from Krebs-Ringer solutions pH 7.4 to a large degree indicating lipid solubility is high despite the polar nature of the drugs. Distribution of d-tubocurarine between the two phases was influenced by the pH of the aqueous solutions (fig. 2). The concentration in octanol was very low at

60-

-6O

50-

-50

-cA ~0m

~

-~o 8

iI

<

z iI

30-

• 30

2o-

-20

10-

-10

° w

LLI

!

5,0

6.0

70 "~

8.0 pH

9.10

10.0

Fig. 2. Extraction of d-tubocurarine into octan-l-ol at various pH values of the aqueous phase. Percentages of the drugs in the organic phase are indicated with O. The broken line represents the theoretical dissociation of a phenolic -OH group with a pKa value of 8.1. Ionisation of the second -OH group of the d-tubocurarine molecule (pKa = 9.1) is disregarded. pH 5.0 and increased when the pH was raised, pK a values of the phenolic-OH groups in the d-tubocurarine molecule have been estimated to be 8.1 and 9.1 (Kalow, 1954). It was of interest to find that extraction percentages increased parallel with the theoretical dissociation of a -OH group with a pK a of 8.1 (fig. 2), suggesting that the forming of zwitterions promoted the passage into the octan-l-ol. This is supported by the fact that extraction of dimethyltubocurarine was very low at pH 5.0 and 7.4. The composition of the aqueous phase proved to be very

Table 2 Extraction of curare-like agents from various aqueous solutions into octanol. Compound

Krebs 8.1

Alcuronium Dimethyl-Tc Gallamine Hexafluorenium Pancuronium d-Tubocurarine

Krebs 7.4

Krebs 5.0

Phosphate 8.3

Phosphate 7.4

( 1%

~ 1%

< 1%

< 1%

( 1%

< 1%

( 1%

< 1%

( 1%

< 1%

3%

3%

3%

3%

3%

51%

50%

46%

3%

6%

( 1%

( 1%

( 1%

( 1%

( 1%

30%

15%

( 1%

10%

5%

Results are given as percentages of the drugs detected in the organic phase after shaking for 15 min. Each value is a mean of three extractions.

2 88

D.K. i': Meijer, J. G. Weitering, Biliary excretion o f curare-like agents

important for passage(s) into the organic solvent of d-tubocurarine and hexafluorenium. Extraction percentages were considerably lower when the partitioning was performed with phosphate buffers instead of the more physiological Krebs solution. This was most striking in the case of hexafluorenium. We cannot at present offer an explanation for this phenomenon but it illustrates the point that various aqueous solutions should be used in partition studies.

4. DISCUSSION The biliary excretion of curare-like drugs by the rat liver is not uniform in spite of their related chemical structures (fig. 1). The thin-layer chromatography method we have used in this study can only give semi-quantitative data. However, the differences we have found with this method between the excretion of the various compounds were very marked. In our opinion it can therefore be concluded that transport to bile in the perfused rat liver is substantial only in the case of two bis-quaternary compounds: hexafluorenium and d-tubocurarine. In preliminary excretion studies in our laboratory the same biliary excretion pattern for curare-like agents was found in the intact rat, pointing to the existence of an important alternative route of elimination in addition to the urinary pathway for the two drugs mentioned. The findings for d-tubocurarine and gallamine are in accord with experimental data from dogs (Feldman et al., 1969) while our result on dimethyl tubocurarine strengthens the suggestion made by Collier et al. (1948) that the rat with the kidneys eliminated has some mechanism for rapidly removing d-tubocurarine from the circulation but that this mechanism does not operate or operates very slowly for the dimethylether of d-tubocurarine. It seems likely that the striking difference in biliary excretion between the compounds is based on the formation of zwitterions due to ionized phenolic -OH groups at pH 7.4 in the d-tubocurarine molecule. Formation of such zwitterions could facilitate passage into organic solvents and through cell membranes (Kalow, 1959 b). This is supported by our own data revealing that extraction of d-tubocurarine into octanol is influenced by varying the pH of the aqueous phase in the range where

ionisation of the phenolic -OH group occurs. It is tempting to suggest that variations in pH of the body fluids results in alteration of the whole distribution and excretion pattern of this particular quaternary compound due to changes in lipid solubility. It was suggested by Cavallito et al. (1956)that hexafluorenium has a relatively lipophilic nature because the large fluorene rings could mask the highly polar onium groups in the molecule. We have shown that this compound can be readily extracted in octanol and this indicates its high lipid solubility. Differences in biliary excretion of chemical closely related drugs might be due to differences in biotransformation, protein binding which could limit entrance into the liver, or lipid solubility. Lipophilic nature may be important for the penetration of the drugs into the liver cells (Kurz, 1964) or the biliary excretion process (Schanker, 1965 ; Meyer-Brunot, 1968). Because data from the literature indicated that biotransformation of alcuronium, dimethylcurare, gallamine and d-tubocurarine is unimportant (Liithi, 1966: Dal Santo, 1964; Feldman et al., 1969; Cohen et al., 1967) and the barrier between blood and the liver cells for plasma proteins is relative rather than absolute, it seemed worth looking for differences in lipid solubility. The two curare-like compounds which are excreted largely in bile are much more lipid soluble than the other four agents. Although additional factors could be involved, these results strongly suggest that the lipophilic character of the bisquaternary nitrogen compounds is important for the actual transport process into the bile canaliculi and/or the penetration from plasma to the transport sites in the liver.

ACKNOWLEDGEMENTS We thank Mallinckrodt Chemical Works (St. Louis, USA) for supplying hexafluorenium (Mylaxen@) through the mediation of Nourypharma (Oss, the Netherlands), Verenigde Pharmaceutische Fabrieken N.V. (Apeldoorn, The Netherlands) for dimethyl-tubocurarine and N.V. Organon (Oss, The Netherlands) for pancuronium (Pavulon(g)). We are indebted to Dr. Hardonk and Dr. Koudstaal from the Laboratory of Pathological Anatomy, University of Groningen for performing the histochemical examinations.

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