XENOBIOTICA,
1991, VOL. 21,
NO.
1, 1-12
The in vitro uptake and metabolism of lignocaine, procainamide and pethidine by tissues of the hindquarters of sheep R. N. UPTON?$, L. E. MATHER and W. B. RUNCIMANS
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Department of Anaesthesia and Intensive Care, The Flinders University of South Australia, Flinders Medical Centre, Bedford Park, S.A. 5042, Australia
Received 13 November 1989; accepted 25 May 1990 1. In vitro studies using tissue slices or tissue homogenates of liver, skeletal muscle, fat skin and blood were conducted to determine whether the uptake of procainamide, lignocaine and pethidine into the hindquarters of sheep was due to distribution or metabolism. Both homogenates and slice preparations of liver showed significant metabolism or uptake, confirming the viability of the preparations.
2. None of the drugs was metabolized in blood and there was minimal uptake of the drugs into the skin. 3. There was metabolism of pethidine in skeletal muscle and substantial uptake of pethidine into fat, indicating that the rapid rate of uptake and prolonged elution of pethidine in the hindquarters was due to both distribution and metabolism. 4. No metabolism of lignocaine in muscle was found, but there was substantial uptake into fat, indicating that the rapid rate of uptake and prolonged elution of lignocaine in the hindquarters was due to its distribution into fat. 5. There was negligible uptake of procainamide into either muscle or fat, presumably due to its relatively low lipophilicity.
Previous studies have shown prolonged uptake of lignocaine (Upton et al. 1988), chlormethiazole, pethidine and minaxolone (Upton et al. 1990) into the hindquarters of sheep during contant-rate infusions of these drugs. T h e hindquarters refers to the hindlimbs and pelvis of sheep, and consists of muscle, fat, bone, and skin, each respectively representing about SO%, 20%, 20% and 10% by weight (Upton 1989). It is therefore representative of the non-visceral tissues which constitute a major part of the total mass of the body. In the previous studies the uptake of lignocaine, chlormethiazole, pethidine and minaxolone into the hindquarters was shown to occur even when the arterial and regional venous blood drug concentrations were essentially constant, and appreciable portions of the doses of the drugs remained uneluted from the hindquarters at the time of cessation of the infusions when the blood concentrations were no longer detectable (Upton et al. 1988, 1990). Three possible mechanisms were suggested to explain these observations: (1) that the uptake was the result of drug metabolism by the hindquarters, (2) that some of the drug left the hindquarters by routes other than the regional venous blood (Upton et al. 1988), and (3) that the uptake was due to slowly reversible drug distribition. In order to distinguish between metabolism (1) and distribution (3), the rates of metabolism by in vitro tissue homogenate
t T o whom correspondence should be addressed.
3 Present address: Department of Anaesthesia and Intensive Care, University of Adelaide, North Terrace, Adelaide, S.A. 5001, Australia. 0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd.
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R. N. Upton et al.
preparations, and the rates of uptake by tissue slice preparations were examined for lignocaine, procainamide and pethidine. In contrast to the other drugs, the distribution of procainamide into the hindquarters is rapidly reversible (Upton et al.
1988).
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Experimental Experimentul design T h e drugs were incubated with tissue homogenates, or slices of tissues in Krebs-~Henseleitoriginal Ringer bicarhonate buffer (Dawson et al. 1974). under an atmosphere of Carbogen (95% oxygen : 5% carbon dioxide, CIG-Medishield, AUS) to maintain relatively physiological p H and gas tensions. liomogenates were assayed directly by solvent extraction to determine the total (bound and unbound) drug conccntrations. Loss of drug from the homogenate was attributed to metabolism if the loss was abolished in control incubations containing perchloric acid and saturated sodium fluoride as a metabolic inhibitor. Uptake into tissue slice preparations was determined by measuring the decrease in drug concentratinn in buffer surrounding the tissue slices. Such uptake, in the absence of uptake by the corresponding homogenate preparation, was attributed to distribution alone. Studies were performed using blood, skeletal muscle, skin and fat tissue. Liver was also studied as a control tissue for metabolism. Tissue collection Blood was collected from the sheep via arterial catheters into heparinized syringes, and then stored in ice. T h e sheep wcrc thrn killed by intravenous administration of saturated KCI solution (30ml) after inducing anaesthesia with thiopentone (2Omg/kg). An area of skin on the upper leg was shaved with electric clippers leaving a layer of wool approximately 1 m m thick. A square of this skin was excised and collected, and the exposed skeletal muscle was dissected free of connective tissue and collected. Pieces of liver and perinepheric fat were removed via an abdominal incision. I’erinepheric fat was used as the source of fat in these studies as tho subcutaneous fat layer of the hindquarters was generally thin, and access to the deeper layers of fat was difficult. All tissues were stored in ice-cold 0.9% saline until needed. Fo u r animals were used per preparation Preparation of homogenates Homogenates were prepared in Krebs-Henseleit original Ringer bicarbonate (Dawson et al. 1974) within 15 min of the death of the animal. Blood ‘homogenate’ was prepared by diluting 5 0 ml of arterial blood with 100ml of buffer. l i v e r and muscle homogenates were prepared by cutting 5 0 g of the tissue into small (2-5 g ) pieces, followed by homogenization with lOOml of buffer (Sorvall Omnimixer Model 17220, USA). During homogenization the vessel of the homogenizer was placed in an ice and water bath, and the pieces were homogenized at maximum speed for periods of 10 s , interspersed with pauses of 5 s, for a total nf 120 s. T h i s procedure produced a good-quality homogenate while keeping the homogenate cool, thereby minimizing the denaturation of protein. I t was not possible to produce a homogenate of fat or skin. Preparation of slices T h e liver and muscle tissues were cu t into cubes weighing approximately 1 g. T h e skin was cut into pieces approximatly 1 cm square. T h e fat was formed into pieces weighing 1 K which were approximately cubc-shaped. Incubation rlrambers ond conditions T h e incubation chambers were 75 ml polypropylene screw-capped containers (sterile container 75I,SC, Disposablc Products, AUS). A l 0 m m hole was made in the centre of the lid of the chambers, which accommodated a rubber bung pierced with an 18 gauge needle. T h e bung allowed access to the homogenate inside the chamber for sampling, and the needle was used to introduce the Carbogen gas, which was distributed to the chambers via a manifold system after passing through a bubble humidifier. A 2 m m hole tin the lid was used to exhaust the Carbogen. Both the homogenates and the slices were incubated in an oscillating water bath at 37°C (water bath model RW1812, Paton Industries, AUS). Incubation of hornogenates For each drug and tissue, one chamber was designated the ‘test’ chamber, the other the ‘control’ chamber. T h e control chambers were used to control for evaporation and for sorption of the drug, and contained perchloric acid (4m1, 3%) saturated with sodium fluoride as a metabolic inhibitor. T h e test chambers contained no inhibitor, but 20Op1 of the inhibitor was added to the sample tubes to stop metabolic activity once the sample was in the tube. T h i s volume of inhibitor was chosen to give the same final dilution of dr u g as in the control chambers. Homogenate ( 2 0 4 was added to both the test and control chambers, and a 20min equilibration period was allowed for t h e chambers to reach stable temperature and gas tensions. After the equilibration period, 1 ml of a 0.2 mg/ml solution of the relevant
In vitro drug disposition in sheep tissues
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drug was added to both the control and test chambers to give a final drug concentration of 9.5 pg/ml. The homogenates were initially mixed for approximately 15 s with a glass stirring rod, and each chamber also contained a 14 mm glass bead to facilitate mixing during incubation. Samples (1 ml) were taken from each chamber with a 1 ml syringe at 1 5 , 30,60,120 and 240 min after the addition of the drug and collected in lOml soda glass tubes.
Incubation of tissue slices The procedure for tissue slice experiments differed from that for the homogenate experiments in the following ways. The test chambers contained 20 ml of Krebs-Henseleit original Ringer bicarbonate and a tissue slice, and the contents of the control chambers were indentical to the test chamber but without the tissue slice. No metabolic inhibitor was required in these studies. The buffer surrounding the slice was sampled (05ml) at 60, 120, 180, 240, 300 and 360min after the addition of the drug, and the sample collected in 1.5 ml plastic tubes. The fat slice experiments were also repeated with fat which had been stored for 2 weeks at 4°C.
Drug assays Samples of homogenate containing procainamide, and samples of buffer containing lignocaine qnd pethidine, were assayed using the methods previously described for blood samples (Upton et al. 1988, 1990). Homogenates containing lignocaine or pethidine were assayed using a double-extraction method similar to that used by Mather and Tucker (1974). T h e samples of buffer containing procainamide were assayed using an external standard method by direct injection into a high-pressure liquid chromatograph (C18 reverse-phase column, methanol : pH 5.5 acetic buffer (35 : 65) and U.V.detection at 280nm). All assays were specific for the unchanged drug concentration.
Data analysis For the homogenate studies the test chamber values were expressed as differences from the mean control concentrations. The total variability was therefore the result of variability in the test values alone. These differences were then used to calculate the mass of drug that had been either taken up or metabolized. This value was then expressed as a percentage of the total mass of drug originally added to the preparations. In slice studies, there was a progressive increase ( C 10%) in drug concentration in the control chambers, presumably due to the evaporation of water. For these studies the results were expressed as the difference between the individual test values and the corresponding individual control values. The approximate half-lives of drug metabolism by liver and muscle were determined from the slope of a straight line through the final three or four time-points of a plot of log drug concentration versus time. This therefore was the time required for a given preparation to decrease the drug concentration in either the homogenate or buffer around the slice by 50%. Because the homogenate preparations contained 20ml of homogenate with 1 part tissue and 2 parts buffer, the total mass of tissue in the homogenates was 6.6 g, compared to 1 g in the tissue slice preparations. The rate of drug metabolism by the liver and muscle was therefore corrected for the mass of tissue present.
Results T h e lignocaine and pethidine concentrations in the control homogenate incubations containing metabolic inhibitor did not change with time, confirming the action of the metabolic inhibitors used. However, the metabolic inhibitors failed to inhibit procainamide metabolism by the liver homogenate (i.e. both the test and control samples has almost undetectable procainamide concentrations) and no data for this are given. T h e absence of decreases in unchanged procainamide concentration in homogenates of muscle indicated no metabolism by this tissue, and therefore precluded the need to examine other metabolic inhibitors for procainamide. T h e time-courses of the percentages of the drugs remaining in the blood ‘homogenates’ are shown in figure 1. It is apparent that there was no significant metabolism of lignocaine, procainamide or pethidine in blood.
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50
O ! 0
50
100
150
200
I 250
Time of incubation (min) Figure I .
'rime-courses of lignocaine and pethidine metabolism by sheep blood homogenates in w i t ~ o .
The time-courses of the percentages of lignocaine (0-0), procainamide ( 0 - - 0 ) and remaining unchanged in the blood homogenate preparations. The data are pethidine (d... ..A) means & SD of four studies.
Figures 2 ( a ) and ( 6 ) show the time-courses of the percentages of the drugs remaining in the liver homogenates and liver slice preparations, respectively. Lignocaine, procainamide and pethidine were consumed b y both preparations and the order of the rate of metabolism was pethidine > lignocaine > procainamide. The time-courses of the percentages of the drugs remaining in the muscle homogenates and muscle slice preparations are shown in figures 3 ( a ) and (b), respectively. In contrast to lignocaine and procainamide, pethidine was metabolized in the muscle homogenates with a half-life of metabolism approximately 40 times greater than that of the liver homogenate (table 1). There was uptake of both lignocaine and pethidine, but not procainamide, into fat. Figure 4 ( a ) shows the time-courses of the percentages of drug remaining in buffer surrounding the fat slice preparations. Figure 4 (b) shows the results from the experiment as shown in figure 4 (a) repeated with the same source of fat after 2 weeks at 4°C. The rates and extents of uptake into this fat were essentially similar to those measured for freshly excised fat. Figure 5 shows the time-courses of the percentages of drug remaining in the skin slice preparation. Uptake of the drugs by skin was detectable in these studies, but was of a much smaller magnitude than uptake into fat or liver. T h e order of the rate of uptake was pethidine > lignocaine > procainamide. The time-courses of the natural logarithms of the percentages of drug remaining in homogenate preparations of liver for lignocaine and pethidine, and homogenate preparations of muscle for pethidine are shown in figure 6 (a).Figure 6 (b) shows the same data for the tissue slice preparations. The half-lives of metabolism corrected for the mass of tissue present are shown in table 1. It is apparent that pethidine metabolism by the tissue slice preparations was approximately 1.9 and 4.7 times faster than in the homogenate preparations for liver and muscle, respectively. For lignocaine, metabolism by the liver slice was 7.8 times faster than in the corresponding liver homogenate.
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In vitro drug disposition in sheep tissues
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Time of incubation (min) (a)
Figure 2.
Time-courses of lignocaine, procainamide and pethidine metabolism and uptake by sheep liver homogenates and slices in vitro.
(0-0) and pethidine ( A . " . - A ) remaining unchanged in the liver homogenate preparations. No procainamide data were available (see text). Data are means+SD of four studies. (b) T h e time-courses of the percentages of lignocaine (0-0). procainamide ( 0 - 4 )and pethidine ( A .....A ) remaining unchanged in the buffer surrounding the liver slice preparations. Data are meansf SD of four studies. ( a ) The time-courses of the percentages of Iignocaine
R. N . Upton et al.
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Time of incubation (rnin) (b) Figure 3.
Time-courses of lignocaine, procainamide and pethidine metabolism and uptake by sheep muscle homogenates and slices in witro.
(a) T h e time-courses of the percentages of lignocaine (0-0). procainamide ( 0 - - 0 )and ...A ) remaining unchanged in the muscle homogenate preparations. Data are pethidine (A,. m c a n s t S U of four studies. (b) The time-courses of the percentages of lignocaine (O-O), procainamide ( 0 0 )and pethidine (A,.. ..A)remaining unchanged in the butfer surrounding the muscle slice preparations. Data are m e a n s k S D of four studies.
Table 1.
Halt-llves of metabolism of Iignocaine, procainamide and pethidine in sheep liver and muscle
hvmogenates and slices ztf vifro. values in h Liver Homogenate Lignocaine Procainamide Pethidine
Slice
Iiomogenate
Slice ##
2.45
##
#
2.37
##
##
2.86
148
139
29.8
19.1
* = N o t done. detectable metabolism.
+*= N o
Muscle
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In vitro drug disposition in sheep tissues
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Time of incubation (min) (4
Time of incubation (min) (6) Time-courses o f Iignocaine, procainamide and pethidine uptake by fresh and aged slices of sheep fat in vitro. ( 0 ) The time-courses of the percentages of lignocaine (0-0).procainamide ( 0 - - 0 ) and pethidine ( A . .. . .A)remaining unchanged in the buffer surrounding the fat slice preparations. Data are m e a n s t S D of four studies. ( b ) T h e time-courses of the percentages of lignocaine (0-0) and pethidine ( A . . . . . A ) remaining unchanged in the buffer surrounding aged fat slice preparations (see text). Data are means+SD of four studies.
Figure 4.
R . N . Upton et al.
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cn 3 0
100
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Time of incubation (min) Figure 5.
'Time-courses of lignocaine, procainarnide and pethidine uptake by slices of sheep skin in oitro.
The time-courses of the percentages of lignocaine (0-0), procainamide ( 0 - - 0 ) and pethidine ( A.. "A) remaining unchanged in the buffer surrounding the skin slice preparations. Data are rneanskSD of four studies.
Discussion Experimental design It is pertinent to discuss the reasons for using both tissue homogenate and tissue slice methods. When a tissue is homogenized, the structure of the tissue is destroyed, leaving small groups of cells or broken cells (Potter 1955). Thus, when a drug is added to a homogenate there is minimal time delay before the drug is in contact with potential sites of binding or metabolism. Furthermore, it would be expected that both unbound and bound drug would be readily extractable from the homogenate during the assay, because of the strong tendency for the drug to partition into the extracting solvent at the basic p H used for the extraction of the drug. Thus, a timedependent decrease in unchanged drug concentration in a homogenate preparation is most likely due to metabolism of the drug. Nevertheless, the only unequivocal evidence of metabolism is when it is possible to mass balance the production of drug metabolites. A disadvantage of using homogenate preparations is that the structure of membranes containing enzymes, substrates and energy production systems are disturbcd, and this may quantitatively and qualitatively affect drug mctabolism (Gillette 1971). The structure of a tissue slice is more complex than that of a homogenate because the majority of structures at the tissue and cellular levels are intact. However, because there is no perfusion, uptake from the medium into the slice is due only to diffusion. In contrast to preparations in which very thin slices are used to minimize diffusion limitations, it was intended to used a preparation in which diffusion had a significant effect in determining the rate of drug uptake into the slice. As for the case in v i v o , uptake may be due to metabolism or distribution because drug bound within the slice cannot be measured by assaying the buffer surrounding the slice. However, if no metabolism occurs, the rate of drug uptake into a sliqe gives an indication of the rate of diffusion of the drug into the tissue in the absence of perfusion. If metabolism
9
In vitro drug disposition in sheep tissues
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-1
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-4
0
100
200
300
I 400
Time of incubation (rnin) (0)
-&T -4
4
0
I 200
100
300
400
Time of incubation (min) (b) Figure 6. Time-courses of the natural logarithms of the percentage of lignocaine and pethidine remaining unchanged in (a) liver and muscle homogenate and (b) liver and muscle incubations. ( a )Lignocaine (0-0) and pethidine ( 0 - - 0 )remaining unchanged in liver homogenates, and pethidine ( A . . . . . A ) in muscle homogenates. Data are meansf SD of four studies. (b) Lignocaine (0-0) and pethidine ( 0 - - 0 ) remaining unchanged in the buffer surrounding liver slice preparations, and pethidine ( A . . . . . A ) for the muscle slice preparations. Data are means* S D of four studies.
does occur, a comparison of the rates of drug disappearance from the homogenate and tissue slice preparations will give an indication of the effect of homogenization on the rate of drug metabolism by the tissue. It is apparent from table 1 that in all cases the rates of metabolism by the tissue slice preparations were approximatly 2-8 times faster than in the homogenate preparations. There are two possible interpretations of this. Firstly, it may be that drug diffusion into the tissue slice was not a major factor limiting the accessiblity of the drugs to the enzymes of metabolism. Secondly, it is possible that the disruption of the tissue caused by homogenization reduced the availability of the substrates and cofactors necessary for drug metabolism. Thus, it is likely that of the two methods, tissue slices were more representative of the in v i m situation.
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In vitro drug metabolism by blood Although this study found no significant metabolism of lignocaine, procainamide or pethidine in blood, Chen et al. (1983) reported that procainamide was metabolized in the blood, particularly during the storage of blood prior to assay. Metabolism by blood has also been documented for drugs susceptible to plasma cholinesterases (Stewart et al. 1977). In vitro drug uptake and metabolism by liver I,iver was included as a reference tissue for drug metabolism, and to demonstrate the sensitivity of the experimental system. I t is of interest to note that the order of the in vitro rate of metabolism by liver tissue reflected the order of the total body clearances of these drugs in oivo (Upton et al. 1988, 1990). In vitro drug uptake and metabolism by muscle The metabolism of pethidine in skeletal muscle reported in this paper is a new finding. There is now extensive evidence that many extrahepatic tissues have the ability to metabolize drugs. Cytochrome P-450 has been found to be widely distributed in the body (Connelly and Bridges 1980), and drug metabolism has been reported in the skin (Pannatier et al. 1978), lungs (Bend et al. 1985, Blitt et al. 1981, James et al. 1977), placenta (Juchau 1980) and other steriodogenic organs (Bend et al. 1978), the kidneys (Acara et al. 1981, Blitt et a f .1981, James et al. 1977), mammary glands (Kitter and Malejka-Ciganti 1982), intestinal mucosa (Curry and Mould 1975) and brain (Oftebro et al. 1979). However, because many of these investigations were primarily directed towards the toxicological implications of extrahepatic metabolism, their pharmacokinetic implications have received less attention. From the evidence available in the literature, it appears that these extrahepatic regions can make significant contributions to the total body clearance of some drugs. For example, in dogs that had their livers surgically bypassed, there was still a measureable clearance of morphine (Hug et al. 1981, Jacqz et al. 1986), lorazepam (Gerkens et ul. 1981) and fentanyl (Hug et a l . 1981). It is probable that pethidine metabolism by the sleletal muscle of sheep was a major component of the uptake of pethidine by the hindquarters of the sheep reported previously (Upton et al. 1990), because of the large mass of muscle in the hindquarters (and the rest of the body) of sheep. Recent investigations have shown that sheep skeletal muscle homogenates can metabolize aspirin by ester hydrolysis (Cossum et al. 1986 a) and that nitroglycerin was metabolized by the hindquarters of the sheep (Cossum et al. 1986 b). Ester hydrolysis is a major route of metabolism of pethidine in some species (Yeh 1984). It is commonly believed that sheep and other herbivores have relatively nonspecific esterases which metabolize a wide range of alkaloids contained in their diet. The few literature reports of comparative drug metabolism between sheep and other species show quantitative differences for the metabolism o f aniline (Kao et al. 1978) and a novel phosphate conjugation pathway for phenol (Kao et al. 1979). More information is needed about the routes and sites of drug metabolism by sheep, particulary in view of the increasing use of these animals for pharmacokinetic research. In vitro drug uptake by skin The difficulty of homogenizing skin meant that only skin slice data were available. Despite the relatively small magnitude of drug uptake into skin compared
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to that in fat or liver, the order of uptake was consistent with the rank order of the lipophilicities of the drugs (Upton et al. 1987)) suggesting a passive partitioning of the drugs into lipophilic sites. Because the incubation medium bathed the entire skin slice, including the exterior surface of the skin, with 1 mm of wool and its attendant wool fat, it is not known whether this small rate of uptake occurs in vivo. However, the metabolism of drugs in the skin of man is well documented (Pannatier et al. 1978), and in view of the large mass of the skin and its relatively high perfusion, it may be a significant site of in vivo metabolism of some drugs in some species. In vitro drug uptake by f a t There was uptake of both lignocaine and pethidine, but not procainamide, into fat. It has been shown that the extent of uptake of a lipophilic drug into fat tissue slices in vitro is proportional to the lipophilicity of the drug (Di Francesco and Bickel 1985). In the present studies the relatively low lipophilicity of procainamide presumably limited its uptake into fat. Di Francesco and Bickel(1985) also showed that the uptake of lipophilic drugs into fat slices was due to the diffusion and partitioning of the drugs into the triglycerides of fat tissue. In the present study it was not possible to homogenize fat and thereby determine if the uptake into fat was due to an active uptake or metabolism process, or was due to such passive distribution. T h e virtually unchanged drug uptake into aged fat, which was presumably less metabolically active than freshly excised fat, indicates that the uptake of lignocaine and pethidine into fat in vitro is due to passive distribution. It is possible that uptake into fat comprises a major component of the hindquarter uptake of lignocaine, and also of pethidine in addition to its metabolism by muscle. However, recent work has shown that while the extent of uptake of some drugs into fat slices in nitro was high, their uptake in vivo was low (Betschart et al. 1988). It has been proposed that the rate and extent of drug uptake into fat in vivo can be influenced by factors other than the lipophilicity of the drug, including fat blood flow and the chemical structure of the drug (Bickel 1984). It is not known whether the blood flow to fat is adequate to account for the hindquarter uptake of lignocaine and pethidine observed in vivo. In the following communication (Upton et al. 1991), the in vivo uptake of lignocaine into the muscle and fat of the hindquarters of sheep will be examined to test the observations made using these in vitro methods.
References ACARA, M . , GESSNER, T., GREIZERSTEIN, H., and TR~JDNOWSKI, R., 1981, Renal N-oxidation of meperidine by the perfused kidney of the rat. Drug Metabolism and Disposition, 9. 75-79. BEND, J . R., SEnABjIT-SINGH,c.J., and PHILPOT, R. M., 1985, The pulmonary uptake, accumulation, and metabolism of xenobiotics. Annual Reviews of Pharmacology and Toxicology, 25, 97-1 25. BEND,J . R . , SMITH, B. R., BALL,L. M., and MUKHTAR, H., 1978, Alkalene and arene oxidemetabolism in hepatic and extrahepatic tissues. Pharmacological and toxicological aspects. In Conjugation Reactions in Drug Biotransformation, edited by A. Aito (Amsterdam: Elsevier), pp. 3-17. BETSCHART, H. R . , JONDORF, W. R., and BICKEL, M. H., 1988, Differences in adipose tissue distribution of basic lipophilic drugs between intraperitoneal and other routes of administration. Xenobiotica, 18, 113-121. BICKEL, M. H., 1984, The role of adipose tissue in the distribution and storage of drugs. Progress in Drug Research, 28, 273-303. BUTT,C. D., GANDOLFI, A. J., SOLTIS, J . J., and BROWN, B. R., 1981, Extrahepatic biotransformationof halothane and enflurane. Anesthesia and Analgesia, 60, 129-1 32. CHEN, M. L., LEE, M. G., and CHIOU, W. L., 1983, Pharrnacokineticsofdrugs in blood. 111. Metabolism of procainamide and storage effects in blood samples. Journal of Pharmaceutical Sciences, 72, 572-574.
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In vitro drug disposition in sheep tissues
CONNELLY, J. C., and BRIDGES,J. W., 1980, The distribution and role of cytochrome P-450 in extrahepatic organs. In Progress in Drug Metabolism, Vol. 5, edited by J. W. Bridges and L. F. Chasseaud (Chichester: John Wiley and Sons), pp. 1-1 1 1 . COSSUM, P. A,, ROBERTS, M. S., KILPATRICK, D., and YONG,A. C., 1986 a, Extrahepatic metabolism and distribution of aspirin in vascular beds of sheep. Journal ofPharmaceutica1 Sciences, 75,731-737. COSSUM, P. A,, ROBERTS, M. S., YONG,A. C., and KILPATRICK, D., 1986 b, Distribution and metabolism of nitroglycerin and its metabolites in vascular beds of sheep. Journal of Pharmacology and Experimental Therapeutics, 237, 959-966. CURRY, S. II., and MOULD,G. P., 1975, A microsomal oxidase system in rat intestinal mucosa. British Journal of Pharmacology, 54, 229P. DAWSON, R. M. C., ELLIOT,D. C., ELLIOT,W. H., and JONES,K. M., 1974, Data for Biochemical Research, 2nd edn (Oxford: Oxford University Press), p. 507. DI FRANCESCO, C., and BICKEL, M. H., 1985, Uptake in witro of lipophilic model compounds into adipose tissue preparations and lipids. Biochemical Pharmacology, 34. 3683-3688. GERKENS, J . F., DFSMOND,P. V., SCHENKER, S., and BRANCH, R. A., 1981, Hepatic and extrahepatic glucuronidation of lorazepam in the dog. HepatoIogy, 1 , 329-335. GILLETTE, J. R., 1971, Techniques for studying drug metabolism in witro. In Fundamentals of Drug Metabolism and Drug Disposition, edited by B. N. La Du, G. H. Mandel and E. L. Way (Baltimore: Williams and Wilkins), pp. 400-416. HUG, C. C. JR, MIJRPHY, M. R., SAMPSON, J. F., TERDLANCHE, J., and AI.DRETE,J. A., 1981, Biotransformation of morphine and fentanyl in anhepatic dogs. Anesthesiology, 55, A261. JACQZ, E., WARD,S.. JOHNSON, R., SCHENKER, S., GERKENS, J., and BRANCH, R. A,, 1986, Extrahepatic glucuronidation of morphine in the dog. Drug Metabolism and Disposition, 14, 627-630. JAMES,M. O., FOUREMAN, G. L., LAW,F. C., and BEND,J. R., 1977, The perinatal development of epoxide-metabolizing enzyme activities in liver and extrahepatic organs of guinea pig and rabbit. Drug Metabolism and Disposition, 5 , 19-28. JUCHAU, M. H.,1980, Drug biotransforrnation in the placenta. Pharmacology and Therapeutics, 8, 501-524. KAO,J., FAULKNER, J., and BRIDGES, J. W., 1978, Metabolism of aniline in rats, pigs and sheep. Drug Metabolism and Disposition, 6, 549-555. KAO,J., BRIDGES, J. W., and FAULKNER, J. K., 1979, Metabolism of [14C]phenol by sheep, pig and rat. Xenobiotica, 9, 141-147. MA.I.HER, L. E., and TUCKER, G. T., 1974, Meperidine and other basic drugs: a general method for their determination in plasma. Journal of Pharmaceutical Sciences, 63, 306-307. OFTEBRO, €I.,STORMER, F. C., and PEDERSEN, J. L., 1979, T h e presence of an adrenodoxin-like ferredoxin and cytochrome P450 in brain mitochondria. Journal of Biological Chemistry, 254, 43314334. PANNATIER, A., JENNER, P., TESTA,B., and ETTER,J. C., 1978, The skin as a drug-metabolizing organ. Drug Metabolism Reviews, 8, 319-343. POTTER, V. R., 1955, Tissue homogenates. Methods in Enzymology, Vol. 1 , edited by S. P. Colowick and N. 0. Kaplan (New York: Academic Press), pp. 10-15. RITTER,C. L., and MAI.EJKA-GIGANTI, D., 1982, Mixed function oxidase in the mammary gland and liver microsomes of lactating rats: effects of 3-methyl cholanthrene and beta naphthoflavone. Biochemical Pharmacology, 31, 239-247. STEWART, D. J., INABA, T., TANG, B. K., and KALOW, W., 1977, Hydrolysis of cocaine in human plasma by choline-esterase. Life Sciences, 20, 1557-1563. UPTON,R. N., 1989, Studies of regional pharmacokinetics in the sheep. Thesis, Flinders LJniversity of South Australia, Redford Park, South Australia. UPTON,R. N., RUNCIMAN, W. B.. and MATHER,L. E., 1987, The relationship between some physicochemical properties of ionizable drugs and their sorption into medical plastics. Australian Journal of Hospital Pharmacy, 17, 267-272. UPTON,R. N . , RUNCIMAN, W. B., MATHER, L. E., CARAPETIS, R. J., and MCLEAN, C. F., 1988, The uptake and elution of lignocaine and procainamide in the hindquarters of the sheep described using mass balance principles. Journal of Pharmacokinetics and Biopharmaceutics, 16, 31-40. UPTON,R. N., MATHER, L.E., RUNCIMAN,W. B., MCLEAN,C. F., and CARAPETIS, R. J., 1990, The uptake and elution of chlormethizole, pethidine and minaxolone in the hindquarters of the sheepimplications for clearance calculations. Journal of Pharmaceutical Sciences (In press). UPTON,R. N., NANCARROW, C., MCLEAN, C. F., MATHER, L. E.,and RUNCIMAN,W. B., 1991, Thein wiwo blood, fat and muscle concentrations of lignocaine and bupivacaine in the hindquarters of sheep. Xenobiotica, 20, 13-22. YEH,S. Y., 1984, Metabolism of meperidine in several animal species. Journal ofPharmoceutica1 Sciences, 73. 1783-1787.
1991, VOL. 21,
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The in vitro uptake and metabolism of lignocaine, procainamide and pethidine by tissues of the hindquarters of sheep R. N. UPTON?$, L. E. MATHER and W. B. RUNCIMANS
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Department of Anaesthesia and Intensive Care, The Flinders University of South Australia, Flinders Medical Centre, Bedford Park, S.A. 5042, Australia
Received 13 November 1989; accepted 25 May 1990 1. In vitro studies using tissue slices or tissue homogenates of liver, skeletal muscle, fat skin and blood were conducted to determine whether the uptake of procainamide, lignocaine and pethidine into the hindquarters of sheep was due to distribution or metabolism. Both homogenates and slice preparations of liver showed significant metabolism or uptake, confirming the viability of the preparations.
2. None of the drugs was metabolized in blood and there was minimal uptake of the drugs into the skin. 3. There was metabolism of pethidine in skeletal muscle and substantial uptake of pethidine into fat, indicating that the rapid rate of uptake and prolonged elution of pethidine in the hindquarters was due to both distribution and metabolism. 4. No metabolism of lignocaine in muscle was found, but there was substantial uptake into fat, indicating that the rapid rate of uptake and prolonged elution of lignocaine in the hindquarters was due to its distribution into fat. 5. There was negligible uptake of procainamide into either muscle or fat, presumably due to its relatively low lipophilicity.
Previous studies have shown prolonged uptake of lignocaine (Upton et al. 1988), chlormethiazole, pethidine and minaxolone (Upton et al. 1990) into the hindquarters of sheep during contant-rate infusions of these drugs. T h e hindquarters refers to the hindlimbs and pelvis of sheep, and consists of muscle, fat, bone, and skin, each respectively representing about SO%, 20%, 20% and 10% by weight (Upton 1989). It is therefore representative of the non-visceral tissues which constitute a major part of the total mass of the body. In the previous studies the uptake of lignocaine, chlormethiazole, pethidine and minaxolone into the hindquarters was shown to occur even when the arterial and regional venous blood drug concentrations were essentially constant, and appreciable portions of the doses of the drugs remained uneluted from the hindquarters at the time of cessation of the infusions when the blood concentrations were no longer detectable (Upton et al. 1988, 1990). Three possible mechanisms were suggested to explain these observations: (1) that the uptake was the result of drug metabolism by the hindquarters, (2) that some of the drug left the hindquarters by routes other than the regional venous blood (Upton et al. 1988), and (3) that the uptake was due to slowly reversible drug distribition. In order to distinguish between metabolism (1) and distribution (3), the rates of metabolism by in vitro tissue homogenate
t T o whom correspondence should be addressed.
3 Present address: Department of Anaesthesia and Intensive Care, University of Adelaide, North Terrace, Adelaide, S.A. 5001, Australia. 0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd.
2
R. N. Upton et al.
preparations, and the rates of uptake by tissue slice preparations were examined for lignocaine, procainamide and pethidine. In contrast to the other drugs, the distribution of procainamide into the hindquarters is rapidly reversible (Upton et al.
1988).
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Experimental Experimentul design T h e drugs were incubated with tissue homogenates, or slices of tissues in Krebs-~Henseleitoriginal Ringer bicarhonate buffer (Dawson et al. 1974). under an atmosphere of Carbogen (95% oxygen : 5% carbon dioxide, CIG-Medishield, AUS) to maintain relatively physiological p H and gas tensions. liomogenates were assayed directly by solvent extraction to determine the total (bound and unbound) drug conccntrations. Loss of drug from the homogenate was attributed to metabolism if the loss was abolished in control incubations containing perchloric acid and saturated sodium fluoride as a metabolic inhibitor. Uptake into tissue slice preparations was determined by measuring the decrease in drug concentratinn in buffer surrounding the tissue slices. Such uptake, in the absence of uptake by the corresponding homogenate preparation, was attributed to distribution alone. Studies were performed using blood, skeletal muscle, skin and fat tissue. Liver was also studied as a control tissue for metabolism. Tissue collection Blood was collected from the sheep via arterial catheters into heparinized syringes, and then stored in ice. T h e sheep wcrc thrn killed by intravenous administration of saturated KCI solution (30ml) after inducing anaesthesia with thiopentone (2Omg/kg). An area of skin on the upper leg was shaved with electric clippers leaving a layer of wool approximately 1 m m thick. A square of this skin was excised and collected, and the exposed skeletal muscle was dissected free of connective tissue and collected. Pieces of liver and perinepheric fat were removed via an abdominal incision. I’erinepheric fat was used as the source of fat in these studies as tho subcutaneous fat layer of the hindquarters was generally thin, and access to the deeper layers of fat was difficult. All tissues were stored in ice-cold 0.9% saline until needed. Fo u r animals were used per preparation Preparation of homogenates Homogenates were prepared in Krebs-Henseleit original Ringer bicarbonate (Dawson et al. 1974) within 15 min of the death of the animal. Blood ‘homogenate’ was prepared by diluting 5 0 ml of arterial blood with 100ml of buffer. l i v e r and muscle homogenates were prepared by cutting 5 0 g of the tissue into small (2-5 g ) pieces, followed by homogenization with lOOml of buffer (Sorvall Omnimixer Model 17220, USA). During homogenization the vessel of the homogenizer was placed in an ice and water bath, and the pieces were homogenized at maximum speed for periods of 10 s , interspersed with pauses of 5 s, for a total nf 120 s. T h i s procedure produced a good-quality homogenate while keeping the homogenate cool, thereby minimizing the denaturation of protein. I t was not possible to produce a homogenate of fat or skin. Preparation of slices T h e liver and muscle tissues were cu t into cubes weighing approximately 1 g. T h e skin was cut into pieces approximatly 1 cm square. T h e fat was formed into pieces weighing 1 K which were approximately cubc-shaped. Incubation rlrambers ond conditions T h e incubation chambers were 75 ml polypropylene screw-capped containers (sterile container 75I,SC, Disposablc Products, AUS). A l 0 m m hole was made in the centre of the lid of the chambers, which accommodated a rubber bung pierced with an 18 gauge needle. T h e bung allowed access to the homogenate inside the chamber for sampling, and the needle was used to introduce the Carbogen gas, which was distributed to the chambers via a manifold system after passing through a bubble humidifier. A 2 m m hole tin the lid was used to exhaust the Carbogen. Both the homogenates and the slices were incubated in an oscillating water bath at 37°C (water bath model RW1812, Paton Industries, AUS). Incubation of hornogenates For each drug and tissue, one chamber was designated the ‘test’ chamber, the other the ‘control’ chamber. T h e control chambers were used to control for evaporation and for sorption of the drug, and contained perchloric acid (4m1, 3%) saturated with sodium fluoride as a metabolic inhibitor. T h e test chambers contained no inhibitor, but 20Op1 of the inhibitor was added to the sample tubes to stop metabolic activity once the sample was in the tube. T h i s volume of inhibitor was chosen to give the same final dilution of dr u g as in the control chambers. Homogenate ( 2 0 4 was added to both the test and control chambers, and a 20min equilibration period was allowed for t h e chambers to reach stable temperature and gas tensions. After the equilibration period, 1 ml of a 0.2 mg/ml solution of the relevant
In vitro drug disposition in sheep tissues
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drug was added to both the control and test chambers to give a final drug concentration of 9.5 pg/ml. The homogenates were initially mixed for approximately 15 s with a glass stirring rod, and each chamber also contained a 14 mm glass bead to facilitate mixing during incubation. Samples (1 ml) were taken from each chamber with a 1 ml syringe at 1 5 , 30,60,120 and 240 min after the addition of the drug and collected in lOml soda glass tubes.
Incubation of tissue slices The procedure for tissue slice experiments differed from that for the homogenate experiments in the following ways. The test chambers contained 20 ml of Krebs-Henseleit original Ringer bicarbonate and a tissue slice, and the contents of the control chambers were indentical to the test chamber but without the tissue slice. No metabolic inhibitor was required in these studies. The buffer surrounding the slice was sampled (05ml) at 60, 120, 180, 240, 300 and 360min after the addition of the drug, and the sample collected in 1.5 ml plastic tubes. The fat slice experiments were also repeated with fat which had been stored for 2 weeks at 4°C.
Drug assays Samples of homogenate containing procainamide, and samples of buffer containing lignocaine qnd pethidine, were assayed using the methods previously described for blood samples (Upton et al. 1988, 1990). Homogenates containing lignocaine or pethidine were assayed using a double-extraction method similar to that used by Mather and Tucker (1974). T h e samples of buffer containing procainamide were assayed using an external standard method by direct injection into a high-pressure liquid chromatograph (C18 reverse-phase column, methanol : pH 5.5 acetic buffer (35 : 65) and U.V.detection at 280nm). All assays were specific for the unchanged drug concentration.
Data analysis For the homogenate studies the test chamber values were expressed as differences from the mean control concentrations. The total variability was therefore the result of variability in the test values alone. These differences were then used to calculate the mass of drug that had been either taken up or metabolized. This value was then expressed as a percentage of the total mass of drug originally added to the preparations. In slice studies, there was a progressive increase ( C 10%) in drug concentration in the control chambers, presumably due to the evaporation of water. For these studies the results were expressed as the difference between the individual test values and the corresponding individual control values. The approximate half-lives of drug metabolism by liver and muscle were determined from the slope of a straight line through the final three or four time-points of a plot of log drug concentration versus time. This therefore was the time required for a given preparation to decrease the drug concentration in either the homogenate or buffer around the slice by 50%. Because the homogenate preparations contained 20ml of homogenate with 1 part tissue and 2 parts buffer, the total mass of tissue in the homogenates was 6.6 g, compared to 1 g in the tissue slice preparations. The rate of drug metabolism by the liver and muscle was therefore corrected for the mass of tissue present.
Results T h e lignocaine and pethidine concentrations in the control homogenate incubations containing metabolic inhibitor did not change with time, confirming the action of the metabolic inhibitors used. However, the metabolic inhibitors failed to inhibit procainamide metabolism by the liver homogenate (i.e. both the test and control samples has almost undetectable procainamide concentrations) and no data for this are given. T h e absence of decreases in unchanged procainamide concentration in homogenates of muscle indicated no metabolism by this tissue, and therefore precluded the need to examine other metabolic inhibitors for procainamide. T h e time-courses of the percentages of the drugs remaining in the blood ‘homogenates’ are shown in figure 1. It is apparent that there was no significant metabolism of lignocaine, procainamide or pethidine in blood.
R. N. Upton et a1
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50
O ! 0
50
100
150
200
I 250
Time of incubation (min) Figure I .
'rime-courses of lignocaine and pethidine metabolism by sheep blood homogenates in w i t ~ o .
The time-courses of the percentages of lignocaine (0-0), procainamide ( 0 - - 0 ) and remaining unchanged in the blood homogenate preparations. The data are pethidine (d... ..A) means & SD of four studies.
Figures 2 ( a ) and ( 6 ) show the time-courses of the percentages of the drugs remaining in the liver homogenates and liver slice preparations, respectively. Lignocaine, procainamide and pethidine were consumed b y both preparations and the order of the rate of metabolism was pethidine > lignocaine > procainamide. The time-courses of the percentages of the drugs remaining in the muscle homogenates and muscle slice preparations are shown in figures 3 ( a ) and (b), respectively. In contrast to lignocaine and procainamide, pethidine was metabolized in the muscle homogenates with a half-life of metabolism approximately 40 times greater than that of the liver homogenate (table 1). There was uptake of both lignocaine and pethidine, but not procainamide, into fat. Figure 4 ( a ) shows the time-courses of the percentages of drug remaining in buffer surrounding the fat slice preparations. Figure 4 (b) shows the results from the experiment as shown in figure 4 (a) repeated with the same source of fat after 2 weeks at 4°C. The rates and extents of uptake into this fat were essentially similar to those measured for freshly excised fat. Figure 5 shows the time-courses of the percentages of drug remaining in the skin slice preparation. Uptake of the drugs by skin was detectable in these studies, but was of a much smaller magnitude than uptake into fat or liver. T h e order of the rate of uptake was pethidine > lignocaine > procainamide. The time-courses of the natural logarithms of the percentages of drug remaining in homogenate preparations of liver for lignocaine and pethidine, and homogenate preparations of muscle for pethidine are shown in figure 6 (a).Figure 6 (b) shows the same data for the tissue slice preparations. The half-lives of metabolism corrected for the mass of tissue present are shown in table 1. It is apparent that pethidine metabolism by the tissue slice preparations was approximately 1.9 and 4.7 times faster than in the homogenate preparations for liver and muscle, respectively. For lignocaine, metabolism by the liver slice was 7.8 times faster than in the corresponding liver homogenate.
5
In vitro drug disposition in sheep tissues
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Figure 2.
Time-courses of lignocaine, procainamide and pethidine metabolism and uptake by sheep liver homogenates and slices in vitro.
(0-0) and pethidine ( A . " . - A ) remaining unchanged in the liver homogenate preparations. No procainamide data were available (see text). Data are means+SD of four studies. (b) T h e time-courses of the percentages of lignocaine (0-0). procainamide ( 0 - 4 )and pethidine ( A .....A ) remaining unchanged in the buffer surrounding the liver slice preparations. Data are meansf SD of four studies. ( a ) The time-courses of the percentages of Iignocaine
R. N . Upton et al.
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Time of incubation (rnin) (b) Figure 3.
Time-courses of lignocaine, procainamide and pethidine metabolism and uptake by sheep muscle homogenates and slices in witro.
(a) T h e time-courses of the percentages of lignocaine (0-0). procainamide ( 0 - - 0 )and ...A ) remaining unchanged in the muscle homogenate preparations. Data are pethidine (A,. m c a n s t S U of four studies. (b) The time-courses of the percentages of lignocaine (O-O), procainamide ( 0 0 )and pethidine (A,.. ..A)remaining unchanged in the butfer surrounding the muscle slice preparations. Data are m e a n s k S D of four studies.
Table 1.
Halt-llves of metabolism of Iignocaine, procainamide and pethidine in sheep liver and muscle
hvmogenates and slices ztf vifro. values in h Liver Homogenate Lignocaine Procainamide Pethidine
Slice
Iiomogenate
Slice ##
2.45
##
#
2.37
##
##
2.86
148
139
29.8
19.1
* = N o t done. detectable metabolism.
+*= N o
Muscle
7
In vitro drug disposition in sheep tissues
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Time of incubation (min) (4
Time of incubation (min) (6) Time-courses o f Iignocaine, procainamide and pethidine uptake by fresh and aged slices of sheep fat in vitro. ( 0 ) The time-courses of the percentages of lignocaine (0-0).procainamide ( 0 - - 0 ) and pethidine ( A . .. . .A)remaining unchanged in the buffer surrounding the fat slice preparations. Data are m e a n s t S D of four studies. ( b ) T h e time-courses of the percentages of lignocaine (0-0) and pethidine ( A . . . . . A ) remaining unchanged in the buffer surrounding aged fat slice preparations (see text). Data are means+SD of four studies.
Figure 4.
R . N . Upton et al.
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Time of incubation (min) Figure 5.
'Time-courses of lignocaine, procainarnide and pethidine uptake by slices of sheep skin in oitro.
The time-courses of the percentages of lignocaine (0-0), procainamide ( 0 - - 0 ) and pethidine ( A.. "A) remaining unchanged in the buffer surrounding the skin slice preparations. Data are rneanskSD of four studies.
Discussion Experimental design It is pertinent to discuss the reasons for using both tissue homogenate and tissue slice methods. When a tissue is homogenized, the structure of the tissue is destroyed, leaving small groups of cells or broken cells (Potter 1955). Thus, when a drug is added to a homogenate there is minimal time delay before the drug is in contact with potential sites of binding or metabolism. Furthermore, it would be expected that both unbound and bound drug would be readily extractable from the homogenate during the assay, because of the strong tendency for the drug to partition into the extracting solvent at the basic p H used for the extraction of the drug. Thus, a timedependent decrease in unchanged drug concentration in a homogenate preparation is most likely due to metabolism of the drug. Nevertheless, the only unequivocal evidence of metabolism is when it is possible to mass balance the production of drug metabolites. A disadvantage of using homogenate preparations is that the structure of membranes containing enzymes, substrates and energy production systems are disturbcd, and this may quantitatively and qualitatively affect drug mctabolism (Gillette 1971). The structure of a tissue slice is more complex than that of a homogenate because the majority of structures at the tissue and cellular levels are intact. However, because there is no perfusion, uptake from the medium into the slice is due only to diffusion. In contrast to preparations in which very thin slices are used to minimize diffusion limitations, it was intended to used a preparation in which diffusion had a significant effect in determining the rate of drug uptake into the slice. As for the case in v i v o , uptake may be due to metabolism or distribution because drug bound within the slice cannot be measured by assaying the buffer surrounding the slice. However, if no metabolism occurs, the rate of drug uptake into a sliqe gives an indication of the rate of diffusion of the drug into the tissue in the absence of perfusion. If metabolism
9
In vitro drug disposition in sheep tissues
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-1
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100
200
300
I 400
Time of incubation (rnin) (0)
-&T -4
4
0
I 200
100
300
400
Time of incubation (min) (b) Figure 6. Time-courses of the natural logarithms of the percentage of lignocaine and pethidine remaining unchanged in (a) liver and muscle homogenate and (b) liver and muscle incubations. ( a )Lignocaine (0-0) and pethidine ( 0 - - 0 )remaining unchanged in liver homogenates, and pethidine ( A . . . . . A ) in muscle homogenates. Data are meansf SD of four studies. (b) Lignocaine (0-0) and pethidine ( 0 - - 0 ) remaining unchanged in the buffer surrounding liver slice preparations, and pethidine ( A . . . . . A ) for the muscle slice preparations. Data are means* S D of four studies.
does occur, a comparison of the rates of drug disappearance from the homogenate and tissue slice preparations will give an indication of the effect of homogenization on the rate of drug metabolism by the tissue. It is apparent from table 1 that in all cases the rates of metabolism by the tissue slice preparations were approximatly 2-8 times faster than in the homogenate preparations. There are two possible interpretations of this. Firstly, it may be that drug diffusion into the tissue slice was not a major factor limiting the accessiblity of the drugs to the enzymes of metabolism. Secondly, it is possible that the disruption of the tissue caused by homogenization reduced the availability of the substrates and cofactors necessary for drug metabolism. Thus, it is likely that of the two methods, tissue slices were more representative of the in v i m situation.
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R. N. Upton et al.
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In vitro drug metabolism by blood Although this study found no significant metabolism of lignocaine, procainamide or pethidine in blood, Chen et al. (1983) reported that procainamide was metabolized in the blood, particularly during the storage of blood prior to assay. Metabolism by blood has also been documented for drugs susceptible to plasma cholinesterases (Stewart et al. 1977). In vitro drug uptake and metabolism by liver I,iver was included as a reference tissue for drug metabolism, and to demonstrate the sensitivity of the experimental system. I t is of interest to note that the order of the in vitro rate of metabolism by liver tissue reflected the order of the total body clearances of these drugs in oivo (Upton et al. 1988, 1990). In vitro drug uptake and metabolism by muscle The metabolism of pethidine in skeletal muscle reported in this paper is a new finding. There is now extensive evidence that many extrahepatic tissues have the ability to metabolize drugs. Cytochrome P-450 has been found to be widely distributed in the body (Connelly and Bridges 1980), and drug metabolism has been reported in the skin (Pannatier et al. 1978), lungs (Bend et al. 1985, Blitt et al. 1981, James et al. 1977), placenta (Juchau 1980) and other steriodogenic organs (Bend et al. 1978), the kidneys (Acara et al. 1981, Blitt et a f .1981, James et al. 1977), mammary glands (Kitter and Malejka-Ciganti 1982), intestinal mucosa (Curry and Mould 1975) and brain (Oftebro et al. 1979). However, because many of these investigations were primarily directed towards the toxicological implications of extrahepatic metabolism, their pharmacokinetic implications have received less attention. From the evidence available in the literature, it appears that these extrahepatic regions can make significant contributions to the total body clearance of some drugs. For example, in dogs that had their livers surgically bypassed, there was still a measureable clearance of morphine (Hug et al. 1981, Jacqz et al. 1986), lorazepam (Gerkens et ul. 1981) and fentanyl (Hug et a l . 1981). It is probable that pethidine metabolism by the sleletal muscle of sheep was a major component of the uptake of pethidine by the hindquarters of the sheep reported previously (Upton et al. 1990), because of the large mass of muscle in the hindquarters (and the rest of the body) of sheep. Recent investigations have shown that sheep skeletal muscle homogenates can metabolize aspirin by ester hydrolysis (Cossum et al. 1986 a) and that nitroglycerin was metabolized by the hindquarters of the sheep (Cossum et al. 1986 b). Ester hydrolysis is a major route of metabolism of pethidine in some species (Yeh 1984). It is commonly believed that sheep and other herbivores have relatively nonspecific esterases which metabolize a wide range of alkaloids contained in their diet. The few literature reports of comparative drug metabolism between sheep and other species show quantitative differences for the metabolism o f aniline (Kao et al. 1978) and a novel phosphate conjugation pathway for phenol (Kao et al. 1979). More information is needed about the routes and sites of drug metabolism by sheep, particulary in view of the increasing use of these animals for pharmacokinetic research. In vitro drug uptake by skin The difficulty of homogenizing skin meant that only skin slice data were available. Despite the relatively small magnitude of drug uptake into skin compared
In vitro drug disposition in sheep tissues
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to that in fat or liver, the order of uptake was consistent with the rank order of the lipophilicities of the drugs (Upton et al. 1987)) suggesting a passive partitioning of the drugs into lipophilic sites. Because the incubation medium bathed the entire skin slice, including the exterior surface of the skin, with 1 mm of wool and its attendant wool fat, it is not known whether this small rate of uptake occurs in vivo. However, the metabolism of drugs in the skin of man is well documented (Pannatier et al. 1978), and in view of the large mass of the skin and its relatively high perfusion, it may be a significant site of in vivo metabolism of some drugs in some species. In vitro drug uptake by f a t There was uptake of both lignocaine and pethidine, but not procainamide, into fat. It has been shown that the extent of uptake of a lipophilic drug into fat tissue slices in vitro is proportional to the lipophilicity of the drug (Di Francesco and Bickel 1985). In the present studies the relatively low lipophilicity of procainamide presumably limited its uptake into fat. Di Francesco and Bickel(1985) also showed that the uptake of lipophilic drugs into fat slices was due to the diffusion and partitioning of the drugs into the triglycerides of fat tissue. In the present study it was not possible to homogenize fat and thereby determine if the uptake into fat was due to an active uptake or metabolism process, or was due to such passive distribution. T h e virtually unchanged drug uptake into aged fat, which was presumably less metabolically active than freshly excised fat, indicates that the uptake of lignocaine and pethidine into fat in vitro is due to passive distribution. It is possible that uptake into fat comprises a major component of the hindquarter uptake of lignocaine, and also of pethidine in addition to its metabolism by muscle. However, recent work has shown that while the extent of uptake of some drugs into fat slices in nitro was high, their uptake in vivo was low (Betschart et al. 1988). It has been proposed that the rate and extent of drug uptake into fat in vivo can be influenced by factors other than the lipophilicity of the drug, including fat blood flow and the chemical structure of the drug (Bickel 1984). It is not known whether the blood flow to fat is adequate to account for the hindquarter uptake of lignocaine and pethidine observed in vivo. In the following communication (Upton et al. 1991), the in vivo uptake of lignocaine into the muscle and fat of the hindquarters of sheep will be examined to test the observations made using these in vitro methods.
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In vitro drug disposition in sheep tissues
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