Br. J. exp. Path. (1977) 58, 28
TISSUE LEVELS OF (3-14C) COUMARIN IN THE RAT: DISTRIBUTION AND EXCRETION N. B. PILLER
Fronm the Ellectron Microscope Unit, Department of Zoology, University of Adelaide, G.P.O. Box 498, Adelaide, South Australia, 5001 Received for publication Augw3t 3, 1976
Summary.-The benzo-pyrones (including coumarin) are a very effective therapy for mild thermal oedema and cases of acute and chronic lymphoedema. In this preliminary report the distribution of a single injected dose of coumarin was followed in normal tissues of rats for 100 hours. Comparisons are to be made later with drug levels in thermally injured and lymphoedematous tissues. The results show 7.4%0 of the injected dose to remain in the tissues after 100 h. During this time 309%0 was excreted in the faeces and approximately 47%0 excreted in the urine. At any given time most of the dose was present in the gut, muscular tissues, skin and liver. For the gut tissues this was 33%0, for the muscular tissues 28%, for the skin 18% and for the liver 16%. The highest concentrations per gram of tissue were however in the kidney and liver, representing the two organs of metabolism and excretion of the coumarin. THERE are only a few reports on the metabolism of coumarin in animals (Furuya, 1958; Mead, Smith and Williams, 1958; Booth et al., 1959; Kaighen and Williams, 1961; Van Sumere and Teuchy, 1971) and even fewer in man (Shilling, Crampton and Longland, 1969). Only in the study of Kaighen and Williams (1961) was radio-labelled coumarin used. All of these reports show great variation in results. Some are undoubtedlv due to the animal species used (Kaighen and Williams, 1961), the various dose levels and the modes of administration. Coumarin (a member of the benzopyrone group) has been shown to be a very effective therapy against most forms of high protein oedemas (Casley-Smith, 1976).
Alone and in combination with other benzo-pyrones (viz. sodium rutin sulphate, troxerutin and rutin), it has been shown to be a particularly effective therapy for mild thermal oedema (Piller, 1975a and b; 1976a, b and c), and lymphoedema, (FoldiBorcsok and Foldi, 1972; Casley-Smith
and Piller, 1974; Clodius, Deak and Piller, 1976; Piller and Clodius, 1976a and b)). Used as a therapy for both acute and chronic lymphoedema, it has produced some dramatic results (Clodius, 1976; Clodius et al., 1976; Piller and Clodius, 1976a and b). Despite its ever increasing application there are still many aspects of its mode of action which have yet to be researched or are obscure. To date it is known that upon administration, coumarin and all related compounds bind to serum proteins (Garten and Wosilait, 1971; O'Reilly, 1971; BauerStaeb and Niebes, 1976). Thus, wherever the protein goes, so too does the drug. Its main action is to enhance the normal lysis of the abnormally accumulated proteins (Piller and Casley-Smith, 1975; CasleySmith, 1976; Piller, 1976b and d), although it is certainly a drug of many actions (Casley-Smith, 1976; Piller, 1976e). It is also a stimulator of the reticulo-endothelial system (Kovach et al., 1976; Piller, 1976f). In thermal oedema, coumarin enhances the protein lysing ability of the macro-
29
DISTRIBUTION AND EXCRETION OF COUMARIN
phages (Piller, 1976g), while in lymphoedema the fibroblasts and neutrophils are believed to be the main cells involved (Piller, 1976h). The removal of the excess protein prevents further formation of fibrotic tissue (Casley-Smith, 1976; Piller, 1976i). In addition coumarin and the benzopyrones, like other anti-inflammatory drugs (Houck and Sharma, 1968) seem to cause the removal of existing fibrotic tissue by causing its lysis (Clodius et al., 1976; Piller and Clodius, 1976b). However, in the search for a more effective dose level it was realized that the bonding of the drug to the serum proteins effectively reduces the amount of free drug available at any time. Thus it is useful to know how much of the drug is in this state. Secondly it has been shown that upon coumarin administration more protein enters the tissues (Piller, 1976j); since this entry of protein-drug complex into the tissues (especially the target tissues) is an integral part of the mode of the drug's action, it is very useful to know how much actually enters the tissues and to know how much leaves and how much remains over various time intervals. In this preliminary trial only normal animals were used, to avoid complications in distribution kinetics. Later studies will deal with distribution in lymphoedematous and thermally injured tissues. In addition, for simplicity of analysis, only a single dose of drug was administered.
chloroform (50 : 50) gave results of 99%, 99% and 98% respectively. An analysis of chemical purity showed the infra-red absorption spectrum to be identical to that of coumarin reference material. (Information supplied by The Radiochemical Centre.) Coumarin for intraperitoneal injection was prepared as follows. Two hundred and fifty milligrammes coumarin (9.375 mCi) was first dissolved in 4 ml A.R. ethanol, then gently warmed to 500. Small quantities of double distilled water (5 ml) held at the same temperature were then carefully added. The preparation was allowed to cool to body temperature and then injected. Each animal received 25 mg/kg as a single injection. At 2, 6, 10, 24, 48, 72 and 100 h after injection, samples were taken from the blood, brain (cerebral hemisphere), heart, kidney, liver, lung, thigh muscle, skin (lateral aspect of thigh), small intestine and spleen. Faeces weight during the time intervals were recorded and average radio-activity noted. The total weights of all other tissues were also recorded. In each case 100 mg (wet weight) of the tissues or 0-1 m blood were removed and placed in glass scintillation vials. To each, 2 ml of Soluene (Packard Chemical Co.) was added, followed by incubation and frequent agitation at 550 for 3 h to facilitate tissue digestion. Ten millilitres of Insta Gel (Packard Chemical Co.) was then added, followed by 10 ml of 30% w/v H202 to bleach any colouration. Counting was performed in a Packard multi-channel scintillation counter. The preparation of a series of standards gave a linear relationship between counts and jug coumarin. All data was thus expressed as jug14C-coumarin/g (wet weight) of tissue. Knowing the average total weight of the various organs and tissues the total amount of 14Ccoumarin in each could be calculated and expressed in terms of the original dose. Although it is well known that residual blood 4 ,l/g for volumes in the tissues vary from 220 ul/g for the lung skeletal muscle to (Altman, 1924), these differences were ignored since they were insignificant compared to the total coumarin in the tissues. ,
,
METHODS Seventy female albino rats average weight 198-16 ± 8-53 g of the S.P.F. strain were fed on Lab Chow (Charlick Pty.) and water ad libiturm. On the day before drug administration all food was withdrawn. Coumarin was obtained as 3-14C coumarin from The Radiochemical Centre (Amersham, England). Its specific activity was 5-5 mCi/ mmol (37-5 ,uCi/mg). An analysis of radiochemical purity ascertained by thin layer chromatography on silica gel in (a) benzene: ethanol (90: 10), (b) toluene : ethyl formate: formic acid (50 : 40: 10), and (c) benzene:
RESULTS
Tabulation of the various organ and tissue weights in rats with an average weight of 198 g (Table I) shows muscle (91 g), skin (32 g), and the gut (27 g) to be the main bulk of the weight. Other organs such as the kidney and liver while
30
N. B. PILLER
of low weight, often contained high levels of 3-14C-coumarin. Two hours after the injection of 5000 jtg of 3-14C-coumarin a total of 4608 jtg were accounted for on the various tissues. This value may be slightly overestimated because of the levels in the gut. This represents almost 50% of the amount remaining. Since only the small intestine was examined and that this region is important for the reabsorption of coumarin it is likely that the average level distributed over the entire gut tissue may be slightly lower. Although the skeletal structure which 17 g was not included in amounts to the analysis for 3-14C-coumarin, the levels should be very low. Taken as it stands the 4608 ,tg of coumarin accounted for is 92.2% of the injected dose (Table I). Six hours after the injection of 3-14C_
coumarin 35.2% of the dose was accounted for in the body tissues; of this 39 % was present in the muscular tissues, 21.5% in the gut tissues, 20.4% in the liver and 10.2% in the skin. Ten hours after the injection of 3-14C-coumarin 23.3% of the injected dose remained in the body tissues; of this, 36% was present in the muscular tissues, 23.5% in the liver, 19% in the skin and 14% in the gut. Twenty-four hours after the injection of 3-14C-coumarin 38% of the injected dose remained in the tissues; this is 499% more than at 10 h! The percentage in the gut tissues increased to 34%, while the muscular tissues contained 29.6%, the skin 21 *8% and the liver 11%. The larger proportion present in the gut tissues may be a consequence of the sampling technique whereby only small intestine was examined. Since this
TABLE I.-Total Coumarin Tissue Levels 2h
Total weight g
,
dug14C-cou-
marin/g Total Tissue (± s.e.) 40 4 (5.5) Blood 10 (0 52) 210 Kidney 1-5 (0-061) 140 (8.9) 7 Brain 1-0 (0 003) 7-0 (0 88) 8 0-7 (0-017) 11-2 (1 9) Heart 3 Spleen 0 4 (0-018) 7 - 6 (0 9) 504 7-2 (0-211) 70 (7 3) Liver 1-2 (0.055) 15 (0.96) 18 Lung 10 (1-45) 910 Muscle 91 (3 8) 27 (1-27) 84 (20- 9) 2268 Gut 32 (1- 86) 20 (1- 79) 640 Skin 4608 Total pg 92 -2 % of original dose remaining
lOh
6h A-
A
,1g 4C-cou-
ug14C-coumarin/g Total 2-7 52 3-6 4- 6 5-2 50 10 7-5 14 5-6
48h A Total weight g pg1 4CTissue Total (± s.e.) coumarin/g 7 -5 10 (0 52) Blood 0.75 (1-3) 15 L0 (0-6) Kidney 1-5 (0-061) 1-6 (0 11) 1 -6 1.0 (0.003) Brain 1 9 (0-21) 1 -3 0 7 (0-017) Heart 2-6 (0.24) 0 4 (0-018) 1*0 Spleen 2 '2 158 7-2 (0-211) (0- 94) Liver 1-2 (0-055) 3 - 6 (0-2) 4-3 Lung 2-7 (0-21) 246 Muscle 91 (3 8) 27 (1-27) 6-5 (0 30) 176 Gut 32 (1-86) 3-0 (1-0) 416 Skin 1027 Total ,ug O/o of original dose remaining 20-5
(82) 27 (9 75) 78 (0 33) 36 3 (0 4) 2 (0-54) (2 6) 360 12 (O 9) (0 9]) 683 (0 85) 378 (0-51) 179 1758 35-2
24h -
pg 14C_-cou-
marin/g Total 1-6 (1-96) 16 30 3 5-5 4 38 10 4- 6 6-1 7 0
(2 3) (0 3) (0- 66) (0-31) (10-6) (0-71) (0 77) (0 26) (1 - 88)
marin/g Total (1-08) 10 (1-48) 30
1 20 3 5-4 4-4 30 9
45 3 3-8 1-6 274
(0.36)
3 3-8 (0- 38) 1-8 (0 59) 217 (1-05) 10-7 6-2 (0 36) 564 24 (0 30) 648 13 (1-48) 416 1904
11-9 419 165 224 1163 23 -3
(0.86)
38-1
72h
100 h
A
jug1 4Ccoumarin/g
0-55 (0-11) 4-5 (0.48) 1-3 (0-12) 1-0 (0.07) 1-4 (0-21) 13-0 (0.43) 2-0 (0.03) 2 -1 (0-23) 2-8 (0.28) 2 -5 (0-16)
Total 5.5 6-8
1-3 0 7 0-6 94 2 -4 191 76 80 458 9 2
Pg94C-
coumarin/g 0-4 (0-10) 1-7 1-25 0-8 0.9 11-5 1-3 2 -1 1-4 1-5
(0.36) (0-28) (0.05) (0-01) (0.56) (0.08) (0-20) (0.06)
(0-03)
Total 4 2-6 1-25 0-56 0-36 82-8 1-56 191 37-8 48 370 7 -4
DISTRIBUTION AND EXCRETION OF COUMARIN
is the site of re-absorption of substances from the gut and especially since it appears coumarin compounds are actively re-absorbed (Schaper and Brummer, 1976), these levels may be an over-estimate of the general gut levels. Forty-eight hours after the administration of 3-14C-coumarin 20.5% of the injected dose remained in the tissues; of this, 40.5% was present in the skin, 23.9% in the muscular tissues, 17% in the gut tissues and 15.3% in the liver. At 72 h 9.2% of the injected dose remained in the tissues; of this, that of the muscle was 41.7%, the liver 20.5%, the skin 17.4% and the gut 16-5 0//Q. At 100 h 7.4% of the injected dose remained in the tissues; of this, 51.6% was in the muscular tissues, 22.3% in the liver, 12.9% in the skin and 10-1% in the gut. Table II shows the total amount of faeces produced over the time intervals 0-2 h, 2-6 h, etc., and gives an estimate of the total activity in these time intervals. During the first 2 h after injection, only 1-1 % of the dose was excreted via the faeces, from 0-6 h 3.53% was excreted, from 0-10h 7-14%, from 0-24h 17.9%, from 0-48 h 27%, from 0-72 h 30% and from 0-100 h 30.88%. During the 100 h of observation, 30.88% of the total injected dose amounting to 1544 mg was excreted via the faeces. Over this time interval it represents 1-30 g/24 h.
31
The cumulative percent of the injected dose which was voided in the urine was estimated using data of Kaighen and Williams (1961). In this respect 1.67% of the injected dose was excreted in the urine in the first 2 h, while in the first 6 h 5-35% was excreted, for 10 h it woS 10.83%, for 24 h 27.2%, for 48 h 40.980/0' for 72 h 45.55% and for 100 h 46.86%. Thus at the end of 100 h 7.4% of the injected dose remained in the tissues, 30.88% were excreted in the faeces and 46.86% in the urine, accounting altogether for 85% of the injected dose. DISCUSSION
The first work on the metabolism of coumarin was by Mead et al. (1958), using 100 mg of coumarin/kg and rabbits as an experimental species. The coumarin was given by a gastric tube. Urine and faeces were collected for 5 days. During this time 25% of the dose was recovered as urinary metabolites, however they found no evidence of ring fission. Later in that year Furuya (1958) also using rabbits given 200 mg of coumarin/kg via a stomach tube, showed that 43.3% of the injected dose could be recovered as urinary metabolites after 48 h of observation. Of this amount 28.2% was recovered as 3 hydroxy coumarin. In 1961 Kaighen and Williams were the first to study the metabolism of coumarin using a radio-labelled molecule
(3-14C-coumarin.)
TABLE II. Amount of Faeces Produced and Content of Radio Tracer Cumulative Cumulative Average Cumulative Cumulative % injected % injected Time faeces Total ,zg faeces dose in interval dose in ,ug* weight /C4g faeces coumarin urinet (h) weight coumarin/g coumarin (g) 55-8 1.10 0-372 0-2 150 55-8 1-67 0-372 284 120-7 176-5 0-797 3-53 2-6 0-425 5.357 356-9 470 7-38 6-10 1.181 180-4 10.83 0*384 620 2-049 17-90 538-2 895-1 27-2 10-24 0-868 3.349 350 455-0 1350-0 27-00 24-48 1-300 40 98 150-6 1506-6 4-554 30 01 125 45.55 48-72 1-205 5-442 30 88 49 43-5 1544d1 0 888 46-86 72-100 * Coumarin here is taken to mean total coumarins including metabolic products of the originally injected 3-14C-coumarin. t This value has been estimated from the earlier results of Kaighen and Williams (1961) about the excretion ratios of 3-14C-coumarin in urine and faeces.
3
32
W. B. PILLER
They compared excretion in rabbits and rats. For the rabbit they gave 50 mg/kg orally. After a maximum of 4 days' observation they found 92.4% of the dose to be excreted via the urine with only small quantities in the faeces. Three main kinds of metabolites were found: (1) acid hlbile precursors (15%); (2) the hydroxy coumarins (39.9%); and (3) o-hydroxyphenyl acids (23%). Thus, in the rabbit about 70% of the radioactivity was in the form of carbon compounds containing an intact ring structure while in about 30% the heterocyclic ring had been opened. Kaighen and Williams (1961) also studied the fate of 3-14C-coumarin in rats. Using a dose level of 100 ma/kg orally they found on average that 55.4% of the injected dose was excreted in the urine and that 36-5% was excreted in the faeces over an average of 5-15 days. The main urinary metabolite was o-hydroxyphenyl acetic acid (20%) suggesting that coumarin is more extensively metabolized via opening of the heterocyclic ring. Similar evidence of opening of the heterocyclic ring was found by Booth et al. (1959). Only an average of 1.5% of the injected dose remained in the tissues after an average of 4.4 days. This
result corresponds fairly well with the results obtained here where 7.4% of the injected dose remained in the tissues after 4-16 days. Using small oral doses of 10 mg/kg in rats, Kaighen and Williams (1961) reported a 50-50 distribution in the excretion of coumarin in the faeces and urine. The mode of excretion thus certainly seems dose dependent. In 1971 Van Sumere and Teuchy used 2-14C-coumarin in a metabolism study in rats. Using 195 mg/kg they found that only little of the tracer was incorporated into the organs and tissues of the animals 24 h after the drug's injection. The highest levels of activity were 8.76% of the injected dose in the caecum, 13.4% in the faeces, 24-8% in the respiratory gases and 37-5% in the urine.
If the data from Table I are examined at the 24 h observation period, 38% of the injected dose remains. That of the gut is 12.96% (a rough approximation to that in the caecum); from Table II 17.9% of the total dose has been excreted as faeces. Respiratory gases and urine were not measured. However if the total percent of the dose remaining in the tissues is added to the percent of the dose excreted in the faeces, only 56% of the total dose can be accounted for. If a similar distribution of metabolic products occurs here as Kaighen and Williams (1961) observed, then about 27.2% of the dose would be expected in the urine. Of the total dose injected 83% can now be accounted for. The remainder may be excreted via the respiratorv gases as Van Sumere and Teuchy (1971) suggest. In attempting to account for all of the injected dose at other times there are a few problems. Two hours after the injection of 3-14C-coumarin 92-2% can be accounted for in the tissues, while 1.1% has been excreted via the faeces. Again using the assumption of Kaighen and Williams regarding the ratios of excretion of metabolic products in faeces and urine, 1 67% would have been excreted via the latter route. Six hours after injection only 35.2% of the injected dose could be accounted for in the tissues, 3.53% in the faeces and 5.4% in the urine; a total of only 44.4%. Similarly 10 h after injection the tissues accounted for 23.3%, the faeces 7X14% and the urine 10X8%; a total of 41.3%. At 24 h after injection, as mentioned above 83% of the dose could be accounted for. Likewise at 48, 72 and 100 h respectively after injection 88-5%, 84-7% and 85 1% of the total injected dose could be
accounted for. What of the extremely low levels at 6 and 10 h? Since at later observation times up to 85% of the injected dose could be accounted for it means that the dose did not leave the animal, but merely remained in sonme tissue whose levels were not
DISTRIBUTION AND EXCRETION OF COUMARIN
recorded. In this respect the lower regions of the small intestine may warrant further examination. This is especially important since there is some evidence of the active transport of coumarin across membranes (Schaper and Brummer, 1976). As the small intestine is important in re-absorption some of the coumarin as well as its metabolic products may be in transit in the intestinal tissues, since the sample tissue for monitoring 3-14C-coumarin levels was from the proximal region of the small intestine large elevations in lower regions may have been missed. This study on rats with uninjured tissues shows that when coumarin is administered to animals at the optimal dose levels necessary to promote maximal resolution of thermally induced oedema and lymphoedema (Piller and CasleySmith, 1975; Piller, 1976k and 1) it remains in the tissues for a considerable length of time. If we consider the levels remaining at the end of 24 h (the optimum interval of drug administration-Piller, 1976k), 38% of the dose remains in the tissues. At this time the blood concentration is 1P0 ,tg of total coumarins/ml (Table I). Of course, there is much biological variation between different animal species when it comes to metabolism of coumarin, the rate of excretion and residual tissue levels (Furuya, 1958; Mead et al., 1958; Booth et al., 1959; Kaighen and Williams, 1969; Van Sumere and Teuchy, 1971). As well as this, different dose levels within the same species can also have some influence. Likewise, man (Shilling et al., 1969) differs from animals with respect to metabolism and excretion. Shilling et al. (1969) found 79% of an orally administered dose of coumarin (200 mg) to be excreted in the urine in 24 h mainly as 7-hydroxy-coumarin. The rats which I observed excreted only 45% of the injected dose in this time, of which about 55% was excreted in the urine and 37% in the faeces. Kaighen and Williams
33
(1961) found only about 1% of 7-hydroxycoumarin. Certainly then metabolism in man and rats is vastly different, with more extensive ring opening of the heterocyclic ring occurring. This work is, however, useful to assess tissue distribution of the coulmarin. Comparison with thermally injured and lymphoedematous tissues may give much useful information regarding variations in metabolism and distribution in these pathologically altered cases and perhaps give us more insight as to why the benzopyrones are a very effective therapy for these conditions. I am most grateful to Schaper and Brummer Pharmaceutical Co. for their generous supply of 3-14C-coumarin. Also thanks to the Australian University Research Grants Commission and to the Department of Further Education for their support. REFERENCES ALTMAN, P. L. (1924) Blood and Other Body Fluids. Ed. D. S. Dittmer. Biological Handbook Series No. 7. BAUER-STAEB, G. & NIEBES, P. (1976) The Binding of Polyphenols (Rutin and Some of its 0-flHydroxyethyl Derivatives) to Human Serum Proteins. Experientia, 32, 367. BOOTH, A. N., MASRI, M. S., ROBBINS, D. J., EMERSON, O. H., JONEs, F. T. & DEEDS, F. (1959) Urinary Metabolites of Coumarin and O-Coumaric Acid. J. biol. Chem., 234, 946. CASLEY-SMITH, J. R. (1976) The Actions of the Benzo-pyrones on the Blood-Tissue-Lymph System. Folia angiol., 22, 9. CASLEY-SMITH, J. R. & PILLER, N. B. (1974) The Pathogenesis of Oedemas and the Therapeutic Action of Coumarin and other Compounds. Folia angiol., 33, Suppl. 3. CLODIUS, L. (1976) Secondary Arm Lymphoedema InLymphoedema: Ed. L. Clodius. Stuttgart: Thieme. CLODIUS, L., DEAK, L. & PILLER, N. B. (1976) A New Instrument for the Evaluation of Tissue Tonicity in Lymphoedema. Lymphology, 9, 1. FOLDI-BORCS6K, E. & F6LDI, M. (1972) Effect of External Lymph Drainage and of Coumarin Treatment on Dextran Oedema. Angiologica, 9, 99. FURUYA, T. (1958) Studies on the Metabolism of Naturally Occurring Coumarin. V. Urinary Metabolites of Coumarin and Di-hydro coumarin. Chem. pharm. Bull., Tokyo, 6, 701. GARTEN, S. & WOSILAIT, W. D. (1971) Comparative Study of the Binding of Coumarin Anti-coagulants and Serum Albumins. Biochem. Pharmac., 20, 1661.
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N. B. PILLER
HOUCK, T. C. & SHARMA, U. K. (1968) Induction of Collagenolytic and Proteolytic Activity in Rat and Human Fibroblasts by Anti-inflammatory Drugs. Science, N.Y., 161, 1361. KAIGHEN, M. & WILLIAMS, R. T. (1961) The Metabolism of 3-'4C-coumarin. J. Med. Pharm. Chem. 3, 25. KOVACH, A. G. B., FOLDI, M., SZLAMKA, I., ECKER, A. & HAMURI, M. (1965) The Effect of Esberriven 9 -a Melilotus Preparation-on the Activity of the Reticulo-endothelial System. ArzneimittelFor8ch., 19, 610. MEAD, I. A., SMITH, J. N. & WILLIAMS, R. T. (1958) Studies in Detoxication, 72: The Metabolism of Coumarin and of O-Coumaric Acid. Biochem. J., 68, 67. O'REILLY, R. H. (1971) Interaction of Several Coumarin Compounds with Human and Canine Plasma Albumin. Molec. Pharmac., 7, 209. PILLER, N. B. (1975a) The Resolution of Thermal Oedema at Various Temperatures under Coumarin Treatment. Br. J. exp. Path., 56, 83. PILLER, N. B. (1975b) A Comparison of the Effectiveness of Some Anti-inflammatory Drugs on Thermal Oedema. Br. J. exp. Path., 56, 554. PILLER, N. B. (1976a) A Comparison of the Effect of Benzo-pyrones and other Drugs with Antiinflammatory Properties on Acid and Neutral Protease Activity Levels in Various Tissues after Thermal Injury. Br. J. exp. Path., 57, 411. PILLER, N. B. (1976b) Drug Induced Proteolysis: A Correlation with Oedema Reducing Ability. Br. J. exp. Path., 57, 266. PILLER, N. B. (1976c) A Conservative Means of Reducing the Intensity and Duration of Thermally induced Oedema. Burn8, 2, 143. PILLER, N. B. (1976d) Further Evidence for the Induction of Proteolysis by Coumarin in Rats with Various High Protein Oedemas. ArzrneimittelFor8ch. (in press). PILLER, N. B. (1976e) An Integration of the Many Modes of Action of Coumarin: An Explanation of its Effectiveness as a Therapy for Thermally Injured Tissue. Arzneimittel-For8ch. (in press). PILLER, N. B. (1976f) The Effect of Coumarin on the Liver Weight of Thermally Injured Rats. Re8. exp. Med. (in press).
PILLER, N. B. (1976g) The Ineffectiveness of Coumarin Treatment of Thermal Oedema of Macrophage-Free Rats. Br. J. exp. Path., 57, 170. PILLER, N. B. (1976h) The Action of the Benzopyrones on an experimental model of Lymphoedema. A Contribution to their Mode of Action. Br. J. exp. Path., 57, 713. PILLER, N. B. (1976i) Conservative Treatment of Acute and Chronic Lymphoedema with the Benzo-pyrones. Lymphology, 9, 132. PILLER, N. B. (1976j) The Influence of Coumarin on Tissue Levels of Radio-labelled Plasma Protein and Povidone (polyvinylpyrrolidine) in Normal and Thermally Injured Rats. Clin. exper. Pharmac. Phy8iol., 3, 523. PILLER, N. B. (1976k) The Effect of Benzopyrones on the Maximal Swelling Volume and Resolution of Thermally Induced Oedema in the Rat: A Determination of Their Optimal Frequency of Administration. Folia angiol., 24, 160. PILLER, N. B. (19761) Benzo-pyrone Treatment of Mild Thermal Oedema: Determination of the Most Effective Dose. Arzneimittel-For8ch (in press). PILLER, N. B. & CASLEY-SMITH, J. R. (1975) The Effect of Coumarin on Protein and PVP Clearance from Rat Legs with Various High Protein Oedemas. Br. J. exp. Path., 56, 439. PILLER, N. B. & CLODIUS, L. (1967a) Clinical Effectiveness of Venalot g on Primary and Secondary Lymphoedema. Eur. J. clin. Inve8t. (submitted for publication). PILLER, N. B. & CLODIUS, L. (1967b) The Use of a Tissue Tonometer as a Diagnostic Aid in Lymphoedema: A Determination of its Conservative Treatment with Benzopyrones. Lymphology, 9, 127. SCHAFER & BRUMMER (1976) The 'Company's Compilation of Documents on Venalot ® Depot. SHILLING, W. N., CRAMPTON, R. F. & LoNGLAND, R. C. (1969) The Metabolism of Coumarin in Man. Nature, Lond., 221, 665. VAN SUMERE, C. F. & TEUCHY, H. (1971) The Metabolism of (2-14C) Coumarin and (2-'4C)-hydroxycoumarin in the Rat. Arche int. Physiol. Biochim., 79. 665.
TISSUE LEVELS OF (3-14C) COUMARIN IN THE RAT: DISTRIBUTION AND EXCRETION N. B. PILLER
Fronm the Ellectron Microscope Unit, Department of Zoology, University of Adelaide, G.P.O. Box 498, Adelaide, South Australia, 5001 Received for publication Augw3t 3, 1976
Summary.-The benzo-pyrones (including coumarin) are a very effective therapy for mild thermal oedema and cases of acute and chronic lymphoedema. In this preliminary report the distribution of a single injected dose of coumarin was followed in normal tissues of rats for 100 hours. Comparisons are to be made later with drug levels in thermally injured and lymphoedematous tissues. The results show 7.4%0 of the injected dose to remain in the tissues after 100 h. During this time 309%0 was excreted in the faeces and approximately 47%0 excreted in the urine. At any given time most of the dose was present in the gut, muscular tissues, skin and liver. For the gut tissues this was 33%0, for the muscular tissues 28%, for the skin 18% and for the liver 16%. The highest concentrations per gram of tissue were however in the kidney and liver, representing the two organs of metabolism and excretion of the coumarin. THERE are only a few reports on the metabolism of coumarin in animals (Furuya, 1958; Mead, Smith and Williams, 1958; Booth et al., 1959; Kaighen and Williams, 1961; Van Sumere and Teuchy, 1971) and even fewer in man (Shilling, Crampton and Longland, 1969). Only in the study of Kaighen and Williams (1961) was radio-labelled coumarin used. All of these reports show great variation in results. Some are undoubtedlv due to the animal species used (Kaighen and Williams, 1961), the various dose levels and the modes of administration. Coumarin (a member of the benzopyrone group) has been shown to be a very effective therapy against most forms of high protein oedemas (Casley-Smith, 1976).
Alone and in combination with other benzo-pyrones (viz. sodium rutin sulphate, troxerutin and rutin), it has been shown to be a particularly effective therapy for mild thermal oedema (Piller, 1975a and b; 1976a, b and c), and lymphoedema, (FoldiBorcsok and Foldi, 1972; Casley-Smith
and Piller, 1974; Clodius, Deak and Piller, 1976; Piller and Clodius, 1976a and b)). Used as a therapy for both acute and chronic lymphoedema, it has produced some dramatic results (Clodius, 1976; Clodius et al., 1976; Piller and Clodius, 1976a and b). Despite its ever increasing application there are still many aspects of its mode of action which have yet to be researched or are obscure. To date it is known that upon administration, coumarin and all related compounds bind to serum proteins (Garten and Wosilait, 1971; O'Reilly, 1971; BauerStaeb and Niebes, 1976). Thus, wherever the protein goes, so too does the drug. Its main action is to enhance the normal lysis of the abnormally accumulated proteins (Piller and Casley-Smith, 1975; CasleySmith, 1976; Piller, 1976b and d), although it is certainly a drug of many actions (Casley-Smith, 1976; Piller, 1976e). It is also a stimulator of the reticulo-endothelial system (Kovach et al., 1976; Piller, 1976f). In thermal oedema, coumarin enhances the protein lysing ability of the macro-
29
DISTRIBUTION AND EXCRETION OF COUMARIN
phages (Piller, 1976g), while in lymphoedema the fibroblasts and neutrophils are believed to be the main cells involved (Piller, 1976h). The removal of the excess protein prevents further formation of fibrotic tissue (Casley-Smith, 1976; Piller, 1976i). In addition coumarin and the benzopyrones, like other anti-inflammatory drugs (Houck and Sharma, 1968) seem to cause the removal of existing fibrotic tissue by causing its lysis (Clodius et al., 1976; Piller and Clodius, 1976b). However, in the search for a more effective dose level it was realized that the bonding of the drug to the serum proteins effectively reduces the amount of free drug available at any time. Thus it is useful to know how much of the drug is in this state. Secondly it has been shown that upon coumarin administration more protein enters the tissues (Piller, 1976j); since this entry of protein-drug complex into the tissues (especially the target tissues) is an integral part of the mode of the drug's action, it is very useful to know how much actually enters the tissues and to know how much leaves and how much remains over various time intervals. In this preliminary trial only normal animals were used, to avoid complications in distribution kinetics. Later studies will deal with distribution in lymphoedematous and thermally injured tissues. In addition, for simplicity of analysis, only a single dose of drug was administered.
chloroform (50 : 50) gave results of 99%, 99% and 98% respectively. An analysis of chemical purity showed the infra-red absorption spectrum to be identical to that of coumarin reference material. (Information supplied by The Radiochemical Centre.) Coumarin for intraperitoneal injection was prepared as follows. Two hundred and fifty milligrammes coumarin (9.375 mCi) was first dissolved in 4 ml A.R. ethanol, then gently warmed to 500. Small quantities of double distilled water (5 ml) held at the same temperature were then carefully added. The preparation was allowed to cool to body temperature and then injected. Each animal received 25 mg/kg as a single injection. At 2, 6, 10, 24, 48, 72 and 100 h after injection, samples were taken from the blood, brain (cerebral hemisphere), heart, kidney, liver, lung, thigh muscle, skin (lateral aspect of thigh), small intestine and spleen. Faeces weight during the time intervals were recorded and average radio-activity noted. The total weights of all other tissues were also recorded. In each case 100 mg (wet weight) of the tissues or 0-1 m blood were removed and placed in glass scintillation vials. To each, 2 ml of Soluene (Packard Chemical Co.) was added, followed by incubation and frequent agitation at 550 for 3 h to facilitate tissue digestion. Ten millilitres of Insta Gel (Packard Chemical Co.) was then added, followed by 10 ml of 30% w/v H202 to bleach any colouration. Counting was performed in a Packard multi-channel scintillation counter. The preparation of a series of standards gave a linear relationship between counts and jug coumarin. All data was thus expressed as jug14C-coumarin/g (wet weight) of tissue. Knowing the average total weight of the various organs and tissues the total amount of 14Ccoumarin in each could be calculated and expressed in terms of the original dose. Although it is well known that residual blood 4 ,l/g for volumes in the tissues vary from 220 ul/g for the lung skeletal muscle to (Altman, 1924), these differences were ignored since they were insignificant compared to the total coumarin in the tissues. ,
,
METHODS Seventy female albino rats average weight 198-16 ± 8-53 g of the S.P.F. strain were fed on Lab Chow (Charlick Pty.) and water ad libiturm. On the day before drug administration all food was withdrawn. Coumarin was obtained as 3-14C coumarin from The Radiochemical Centre (Amersham, England). Its specific activity was 5-5 mCi/ mmol (37-5 ,uCi/mg). An analysis of radiochemical purity ascertained by thin layer chromatography on silica gel in (a) benzene: ethanol (90: 10), (b) toluene : ethyl formate: formic acid (50 : 40: 10), and (c) benzene:
RESULTS
Tabulation of the various organ and tissue weights in rats with an average weight of 198 g (Table I) shows muscle (91 g), skin (32 g), and the gut (27 g) to be the main bulk of the weight. Other organs such as the kidney and liver while
30
N. B. PILLER
of low weight, often contained high levels of 3-14C-coumarin. Two hours after the injection of 5000 jtg of 3-14C-coumarin a total of 4608 jtg were accounted for on the various tissues. This value may be slightly overestimated because of the levels in the gut. This represents almost 50% of the amount remaining. Since only the small intestine was examined and that this region is important for the reabsorption of coumarin it is likely that the average level distributed over the entire gut tissue may be slightly lower. Although the skeletal structure which 17 g was not included in amounts to the analysis for 3-14C-coumarin, the levels should be very low. Taken as it stands the 4608 ,tg of coumarin accounted for is 92.2% of the injected dose (Table I). Six hours after the injection of 3-14C_
coumarin 35.2% of the dose was accounted for in the body tissues; of this 39 % was present in the muscular tissues, 21.5% in the gut tissues, 20.4% in the liver and 10.2% in the skin. Ten hours after the injection of 3-14C-coumarin 23.3% of the injected dose remained in the body tissues; of this, 36% was present in the muscular tissues, 23.5% in the liver, 19% in the skin and 14% in the gut. Twenty-four hours after the injection of 3-14C-coumarin 38% of the injected dose remained in the tissues; this is 499% more than at 10 h! The percentage in the gut tissues increased to 34%, while the muscular tissues contained 29.6%, the skin 21 *8% and the liver 11%. The larger proportion present in the gut tissues may be a consequence of the sampling technique whereby only small intestine was examined. Since this
TABLE I.-Total Coumarin Tissue Levels 2h
Total weight g
,
dug14C-cou-
marin/g Total Tissue (± s.e.) 40 4 (5.5) Blood 10 (0 52) 210 Kidney 1-5 (0-061) 140 (8.9) 7 Brain 1-0 (0 003) 7-0 (0 88) 8 0-7 (0-017) 11-2 (1 9) Heart 3 Spleen 0 4 (0-018) 7 - 6 (0 9) 504 7-2 (0-211) 70 (7 3) Liver 1-2 (0.055) 15 (0.96) 18 Lung 10 (1-45) 910 Muscle 91 (3 8) 27 (1-27) 84 (20- 9) 2268 Gut 32 (1- 86) 20 (1- 79) 640 Skin 4608 Total pg 92 -2 % of original dose remaining
lOh
6h A-
A
,1g 4C-cou-
ug14C-coumarin/g Total 2-7 52 3-6 4- 6 5-2 50 10 7-5 14 5-6
48h A Total weight g pg1 4CTissue Total (± s.e.) coumarin/g 7 -5 10 (0 52) Blood 0.75 (1-3) 15 L0 (0-6) Kidney 1-5 (0-061) 1-6 (0 11) 1 -6 1.0 (0.003) Brain 1 9 (0-21) 1 -3 0 7 (0-017) Heart 2-6 (0.24) 0 4 (0-018) 1*0 Spleen 2 '2 158 7-2 (0-211) (0- 94) Liver 1-2 (0-055) 3 - 6 (0-2) 4-3 Lung 2-7 (0-21) 246 Muscle 91 (3 8) 27 (1-27) 6-5 (0 30) 176 Gut 32 (1-86) 3-0 (1-0) 416 Skin 1027 Total ,ug O/o of original dose remaining 20-5
(82) 27 (9 75) 78 (0 33) 36 3 (0 4) 2 (0-54) (2 6) 360 12 (O 9) (0 9]) 683 (0 85) 378 (0-51) 179 1758 35-2
24h -
pg 14C_-cou-
marin/g Total 1-6 (1-96) 16 30 3 5-5 4 38 10 4- 6 6-1 7 0
(2 3) (0 3) (0- 66) (0-31) (10-6) (0-71) (0 77) (0 26) (1 - 88)
marin/g Total (1-08) 10 (1-48) 30
1 20 3 5-4 4-4 30 9
45 3 3-8 1-6 274
(0.36)
3 3-8 (0- 38) 1-8 (0 59) 217 (1-05) 10-7 6-2 (0 36) 564 24 (0 30) 648 13 (1-48) 416 1904
11-9 419 165 224 1163 23 -3
(0.86)
38-1
72h
100 h
A
jug1 4Ccoumarin/g
0-55 (0-11) 4-5 (0.48) 1-3 (0-12) 1-0 (0.07) 1-4 (0-21) 13-0 (0.43) 2-0 (0.03) 2 -1 (0-23) 2-8 (0.28) 2 -5 (0-16)
Total 5.5 6-8
1-3 0 7 0-6 94 2 -4 191 76 80 458 9 2
Pg94C-
coumarin/g 0-4 (0-10) 1-7 1-25 0-8 0.9 11-5 1-3 2 -1 1-4 1-5
(0.36) (0-28) (0.05) (0-01) (0.56) (0.08) (0-20) (0.06)
(0-03)
Total 4 2-6 1-25 0-56 0-36 82-8 1-56 191 37-8 48 370 7 -4
DISTRIBUTION AND EXCRETION OF COUMARIN
is the site of re-absorption of substances from the gut and especially since it appears coumarin compounds are actively re-absorbed (Schaper and Brummer, 1976), these levels may be an over-estimate of the general gut levels. Forty-eight hours after the administration of 3-14C-coumarin 20.5% of the injected dose remained in the tissues; of this, 40.5% was present in the skin, 23.9% in the muscular tissues, 17% in the gut tissues and 15.3% in the liver. At 72 h 9.2% of the injected dose remained in the tissues; of this, that of the muscle was 41.7%, the liver 20.5%, the skin 17.4% and the gut 16-5 0//Q. At 100 h 7.4% of the injected dose remained in the tissues; of this, 51.6% was in the muscular tissues, 22.3% in the liver, 12.9% in the skin and 10-1% in the gut. Table II shows the total amount of faeces produced over the time intervals 0-2 h, 2-6 h, etc., and gives an estimate of the total activity in these time intervals. During the first 2 h after injection, only 1-1 % of the dose was excreted via the faeces, from 0-6 h 3.53% was excreted, from 0-10h 7-14%, from 0-24h 17.9%, from 0-48 h 27%, from 0-72 h 30% and from 0-100 h 30.88%. During the 100 h of observation, 30.88% of the total injected dose amounting to 1544 mg was excreted via the faeces. Over this time interval it represents 1-30 g/24 h.
31
The cumulative percent of the injected dose which was voided in the urine was estimated using data of Kaighen and Williams (1961). In this respect 1.67% of the injected dose was excreted in the urine in the first 2 h, while in the first 6 h 5-35% was excreted, for 10 h it woS 10.83%, for 24 h 27.2%, for 48 h 40.980/0' for 72 h 45.55% and for 100 h 46.86%. Thus at the end of 100 h 7.4% of the injected dose remained in the tissues, 30.88% were excreted in the faeces and 46.86% in the urine, accounting altogether for 85% of the injected dose. DISCUSSION
The first work on the metabolism of coumarin was by Mead et al. (1958), using 100 mg of coumarin/kg and rabbits as an experimental species. The coumarin was given by a gastric tube. Urine and faeces were collected for 5 days. During this time 25% of the dose was recovered as urinary metabolites, however they found no evidence of ring fission. Later in that year Furuya (1958) also using rabbits given 200 mg of coumarin/kg via a stomach tube, showed that 43.3% of the injected dose could be recovered as urinary metabolites after 48 h of observation. Of this amount 28.2% was recovered as 3 hydroxy coumarin. In 1961 Kaighen and Williams were the first to study the metabolism of coumarin using a radio-labelled molecule
(3-14C-coumarin.)
TABLE II. Amount of Faeces Produced and Content of Radio Tracer Cumulative Cumulative Average Cumulative Cumulative % injected % injected Time faeces Total ,zg faeces dose in interval dose in ,ug* weight /C4g faeces coumarin urinet (h) weight coumarin/g coumarin (g) 55-8 1.10 0-372 0-2 150 55-8 1-67 0-372 284 120-7 176-5 0-797 3-53 2-6 0-425 5.357 356-9 470 7-38 6-10 1.181 180-4 10.83 0*384 620 2-049 17-90 538-2 895-1 27-2 10-24 0-868 3.349 350 455-0 1350-0 27-00 24-48 1-300 40 98 150-6 1506-6 4-554 30 01 125 45.55 48-72 1-205 5-442 30 88 49 43-5 1544d1 0 888 46-86 72-100 * Coumarin here is taken to mean total coumarins including metabolic products of the originally injected 3-14C-coumarin. t This value has been estimated from the earlier results of Kaighen and Williams (1961) about the excretion ratios of 3-14C-coumarin in urine and faeces.
3
32
W. B. PILLER
They compared excretion in rabbits and rats. For the rabbit they gave 50 mg/kg orally. After a maximum of 4 days' observation they found 92.4% of the dose to be excreted via the urine with only small quantities in the faeces. Three main kinds of metabolites were found: (1) acid hlbile precursors (15%); (2) the hydroxy coumarins (39.9%); and (3) o-hydroxyphenyl acids (23%). Thus, in the rabbit about 70% of the radioactivity was in the form of carbon compounds containing an intact ring structure while in about 30% the heterocyclic ring had been opened. Kaighen and Williams (1961) also studied the fate of 3-14C-coumarin in rats. Using a dose level of 100 ma/kg orally they found on average that 55.4% of the injected dose was excreted in the urine and that 36-5% was excreted in the faeces over an average of 5-15 days. The main urinary metabolite was o-hydroxyphenyl acetic acid (20%) suggesting that coumarin is more extensively metabolized via opening of the heterocyclic ring. Similar evidence of opening of the heterocyclic ring was found by Booth et al. (1959). Only an average of 1.5% of the injected dose remained in the tissues after an average of 4.4 days. This
result corresponds fairly well with the results obtained here where 7.4% of the injected dose remained in the tissues after 4-16 days. Using small oral doses of 10 mg/kg in rats, Kaighen and Williams (1961) reported a 50-50 distribution in the excretion of coumarin in the faeces and urine. The mode of excretion thus certainly seems dose dependent. In 1971 Van Sumere and Teuchy used 2-14C-coumarin in a metabolism study in rats. Using 195 mg/kg they found that only little of the tracer was incorporated into the organs and tissues of the animals 24 h after the drug's injection. The highest levels of activity were 8.76% of the injected dose in the caecum, 13.4% in the faeces, 24-8% in the respiratory gases and 37-5% in the urine.
If the data from Table I are examined at the 24 h observation period, 38% of the injected dose remains. That of the gut is 12.96% (a rough approximation to that in the caecum); from Table II 17.9% of the total dose has been excreted as faeces. Respiratory gases and urine were not measured. However if the total percent of the dose remaining in the tissues is added to the percent of the dose excreted in the faeces, only 56% of the total dose can be accounted for. If a similar distribution of metabolic products occurs here as Kaighen and Williams (1961) observed, then about 27.2% of the dose would be expected in the urine. Of the total dose injected 83% can now be accounted for. The remainder may be excreted via the respiratorv gases as Van Sumere and Teuchy (1971) suggest. In attempting to account for all of the injected dose at other times there are a few problems. Two hours after the injection of 3-14C-coumarin 92-2% can be accounted for in the tissues, while 1.1% has been excreted via the faeces. Again using the assumption of Kaighen and Williams regarding the ratios of excretion of metabolic products in faeces and urine, 1 67% would have been excreted via the latter route. Six hours after injection only 35.2% of the injected dose could be accounted for in the tissues, 3.53% in the faeces and 5.4% in the urine; a total of only 44.4%. Similarly 10 h after injection the tissues accounted for 23.3%, the faeces 7X14% and the urine 10X8%; a total of 41.3%. At 24 h after injection, as mentioned above 83% of the dose could be accounted for. Likewise at 48, 72 and 100 h respectively after injection 88-5%, 84-7% and 85 1% of the total injected dose could be
accounted for. What of the extremely low levels at 6 and 10 h? Since at later observation times up to 85% of the injected dose could be accounted for it means that the dose did not leave the animal, but merely remained in sonme tissue whose levels were not
DISTRIBUTION AND EXCRETION OF COUMARIN
recorded. In this respect the lower regions of the small intestine may warrant further examination. This is especially important since there is some evidence of the active transport of coumarin across membranes (Schaper and Brummer, 1976). As the small intestine is important in re-absorption some of the coumarin as well as its metabolic products may be in transit in the intestinal tissues, since the sample tissue for monitoring 3-14C-coumarin levels was from the proximal region of the small intestine large elevations in lower regions may have been missed. This study on rats with uninjured tissues shows that when coumarin is administered to animals at the optimal dose levels necessary to promote maximal resolution of thermally induced oedema and lymphoedema (Piller and CasleySmith, 1975; Piller, 1976k and 1) it remains in the tissues for a considerable length of time. If we consider the levels remaining at the end of 24 h (the optimum interval of drug administration-Piller, 1976k), 38% of the dose remains in the tissues. At this time the blood concentration is 1P0 ,tg of total coumarins/ml (Table I). Of course, there is much biological variation between different animal species when it comes to metabolism of coumarin, the rate of excretion and residual tissue levels (Furuya, 1958; Mead et al., 1958; Booth et al., 1959; Kaighen and Williams, 1969; Van Sumere and Teuchy, 1971). As well as this, different dose levels within the same species can also have some influence. Likewise, man (Shilling et al., 1969) differs from animals with respect to metabolism and excretion. Shilling et al. (1969) found 79% of an orally administered dose of coumarin (200 mg) to be excreted in the urine in 24 h mainly as 7-hydroxy-coumarin. The rats which I observed excreted only 45% of the injected dose in this time, of which about 55% was excreted in the urine and 37% in the faeces. Kaighen and Williams
33
(1961) found only about 1% of 7-hydroxycoumarin. Certainly then metabolism in man and rats is vastly different, with more extensive ring opening of the heterocyclic ring occurring. This work is, however, useful to assess tissue distribution of the coulmarin. Comparison with thermally injured and lymphoedematous tissues may give much useful information regarding variations in metabolism and distribution in these pathologically altered cases and perhaps give us more insight as to why the benzopyrones are a very effective therapy for these conditions. I am most grateful to Schaper and Brummer Pharmaceutical Co. for their generous supply of 3-14C-coumarin. Also thanks to the Australian University Research Grants Commission and to the Department of Further Education for their support. REFERENCES ALTMAN, P. L. (1924) Blood and Other Body Fluids. Ed. D. S. Dittmer. Biological Handbook Series No. 7. BAUER-STAEB, G. & NIEBES, P. (1976) The Binding of Polyphenols (Rutin and Some of its 0-flHydroxyethyl Derivatives) to Human Serum Proteins. Experientia, 32, 367. BOOTH, A. N., MASRI, M. S., ROBBINS, D. J., EMERSON, O. H., JONEs, F. T. & DEEDS, F. (1959) Urinary Metabolites of Coumarin and O-Coumaric Acid. J. biol. Chem., 234, 946. CASLEY-SMITH, J. R. (1976) The Actions of the Benzo-pyrones on the Blood-Tissue-Lymph System. Folia angiol., 22, 9. CASLEY-SMITH, J. R. & PILLER, N. B. (1974) The Pathogenesis of Oedemas and the Therapeutic Action of Coumarin and other Compounds. Folia angiol., 33, Suppl. 3. CLODIUS, L. (1976) Secondary Arm Lymphoedema InLymphoedema: Ed. L. Clodius. Stuttgart: Thieme. CLODIUS, L., DEAK, L. & PILLER, N. B. (1976) A New Instrument for the Evaluation of Tissue Tonicity in Lymphoedema. Lymphology, 9, 1. FOLDI-BORCS6K, E. & F6LDI, M. (1972) Effect of External Lymph Drainage and of Coumarin Treatment on Dextran Oedema. Angiologica, 9, 99. FURUYA, T. (1958) Studies on the Metabolism of Naturally Occurring Coumarin. V. Urinary Metabolites of Coumarin and Di-hydro coumarin. Chem. pharm. Bull., Tokyo, 6, 701. GARTEN, S. & WOSILAIT, W. D. (1971) Comparative Study of the Binding of Coumarin Anti-coagulants and Serum Albumins. Biochem. Pharmac., 20, 1661.
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N. B. PILLER
HOUCK, T. C. & SHARMA, U. K. (1968) Induction of Collagenolytic and Proteolytic Activity in Rat and Human Fibroblasts by Anti-inflammatory Drugs. Science, N.Y., 161, 1361. KAIGHEN, M. & WILLIAMS, R. T. (1961) The Metabolism of 3-'4C-coumarin. J. Med. Pharm. Chem. 3, 25. KOVACH, A. G. B., FOLDI, M., SZLAMKA, I., ECKER, A. & HAMURI, M. (1965) The Effect of Esberriven 9 -a Melilotus Preparation-on the Activity of the Reticulo-endothelial System. ArzneimittelFor8ch., 19, 610. MEAD, I. A., SMITH, J. N. & WILLIAMS, R. T. (1958) Studies in Detoxication, 72: The Metabolism of Coumarin and of O-Coumaric Acid. Biochem. J., 68, 67. O'REILLY, R. H. (1971) Interaction of Several Coumarin Compounds with Human and Canine Plasma Albumin. Molec. Pharmac., 7, 209. PILLER, N. B. (1975a) The Resolution of Thermal Oedema at Various Temperatures under Coumarin Treatment. Br. J. exp. Path., 56, 83. PILLER, N. B. (1975b) A Comparison of the Effectiveness of Some Anti-inflammatory Drugs on Thermal Oedema. Br. J. exp. Path., 56, 554. PILLER, N. B. (1976a) A Comparison of the Effect of Benzo-pyrones and other Drugs with Antiinflammatory Properties on Acid and Neutral Protease Activity Levels in Various Tissues after Thermal Injury. Br. J. exp. Path., 57, 411. PILLER, N. B. (1976b) Drug Induced Proteolysis: A Correlation with Oedema Reducing Ability. Br. J. exp. Path., 57, 266. PILLER, N. B. (1976c) A Conservative Means of Reducing the Intensity and Duration of Thermally induced Oedema. Burn8, 2, 143. PILLER, N. B. (1976d) Further Evidence for the Induction of Proteolysis by Coumarin in Rats with Various High Protein Oedemas. ArzrneimittelFor8ch. (in press). PILLER, N. B. (1976e) An Integration of the Many Modes of Action of Coumarin: An Explanation of its Effectiveness as a Therapy for Thermally Injured Tissue. Arzneimittel-For8ch. (in press). PILLER, N. B. (1976f) The Effect of Coumarin on the Liver Weight of Thermally Injured Rats. Re8. exp. Med. (in press).
PILLER, N. B. (1976g) The Ineffectiveness of Coumarin Treatment of Thermal Oedema of Macrophage-Free Rats. Br. J. exp. Path., 57, 170. PILLER, N. B. (1976h) The Action of the Benzopyrones on an experimental model of Lymphoedema. A Contribution to their Mode of Action. Br. J. exp. Path., 57, 713. PILLER, N. B. (1976i) Conservative Treatment of Acute and Chronic Lymphoedema with the Benzo-pyrones. Lymphology, 9, 132. PILLER, N. B. (1976j) The Influence of Coumarin on Tissue Levels of Radio-labelled Plasma Protein and Povidone (polyvinylpyrrolidine) in Normal and Thermally Injured Rats. Clin. exper. Pharmac. Phy8iol., 3, 523. PILLER, N. B. (1976k) The Effect of Benzopyrones on the Maximal Swelling Volume and Resolution of Thermally Induced Oedema in the Rat: A Determination of Their Optimal Frequency of Administration. Folia angiol., 24, 160. PILLER, N. B. (19761) Benzo-pyrone Treatment of Mild Thermal Oedema: Determination of the Most Effective Dose. Arzneimittel-For8ch (in press). PILLER, N. B. & CASLEY-SMITH, J. R. (1975) The Effect of Coumarin on Protein and PVP Clearance from Rat Legs with Various High Protein Oedemas. Br. J. exp. Path., 56, 439. PILLER, N. B. & CLODIUS, L. (1967a) Clinical Effectiveness of Venalot g on Primary and Secondary Lymphoedema. Eur. J. clin. Inve8t. (submitted for publication). PILLER, N. B. & CLODIUS, L. (1967b) The Use of a Tissue Tonometer as a Diagnostic Aid in Lymphoedema: A Determination of its Conservative Treatment with Benzopyrones. Lymphology, 9, 127. SCHAFER & BRUMMER (1976) The 'Company's Compilation of Documents on Venalot ® Depot. SHILLING, W. N., CRAMPTON, R. F. & LoNGLAND, R. C. (1969) The Metabolism of Coumarin in Man. Nature, Lond., 221, 665. VAN SUMERE, C. F. & TEUCHY, H. (1971) The Metabolism of (2-14C) Coumarin and (2-'4C)-hydroxycoumarin in the Rat. Arche int. Physiol. Biochim., 79. 665.
