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Study of clofibric acid distribution, metabolism and model, in crab pachygrapsus marmoratus (decapoda, brachyura)
Comp. Btochem. Physiol.,
1977. Vol, 58C, pp. 173 to 175. Pergamon
Press. Printed in Great Britain
STUDY OF CLOFIBRIC ACID DISTRIBUTION, METABOLISM AND MODEL, IN CRAB P A C H Y G R A P S U S M A R M O R A T U S (DECAPODA, BRACHYURA) JACQUES LAUTIER 1, JEAN-LOuIS CHANAL 2, JACQUES GUIBERT 2 and JEAN-G. LAGARRIGUE 1 1Laboratoire de Physiologie des Invert~brts, U.S.T.L., Place E. BataiUon, 34060 Montpellier Cedex, France and 2Laboratoire de Physique, Facult6 de Pharmacie, Avenue C. Flahault, 34000 Montpellier, France
(Received 9 February 1977) Abstract--I, The DL50 of clofibric acid which is injected intravenously in crab Pachygrapsus marmoratus is 173.5 mg/kg. 2. Hypolipidemic drug distribution is distinct accdding to the organ. The fixation power of stomach and hepatopancreas is high; on the other hand, crab muscle fixes less clofibric acid than a mammal's; ovary radioactivity is higher than the testicular one. 3. The difference between males and females seems to consist in higher urinary excretion of labelled compound in males. 4. Metabolisation which begins after 8 hr increases slowly and is more important in males. 5. Pharmacokinetic model indicates that clofibric acid is distinctly distributed according to organs and sex.
INTRODUCTION
In consideration of their therapeutic importance, there are numerous hypolipidemic drugs. They belong to various chemical groups and their efficiency in pharmacology in mammals has been proved but their action is not yet known. In Invertebrates, these lipid depressants have been little studied. As far as we know, only Levinson & Levinson (1973) studied ethyl-p-chlorophenoxyisobutyrate (ECPIB) in Dermestes maculatus; these authors established that this hypolipidemic drug retarded the growth of these insects and suppressed their fecundity. In our study on lipid metabolism in the crab Pachy9rapsus marmoratus, we made a pharmacokinetic study of clofibric acid. MATERIALS AND METHODS
Pachygrapsus marmoratus were collected from the Mediterranean shore at Frontignan-Plage and acclimatised to aquarium sea water (salinity 37 + lYoo;temperature: 20°C) for at least 1 week before use. 1. Determination of urinary excretion and clofibric acid distribution in hemolymph and different organs The pharmacokinetic study is founded on utilisation of labelled clofibric acid (carboxyl t4C) with a specific activity of 20 mCi/mmole. The drug is administered by intravenous injection in proportion of 50 mg/kg dissolved in 10Yo ethanol at the rate of 10 mg/ml. This injection is slowly made in a thoracic metamer through the articular membrane of a crab leg; these animals are in C4 intermolt stage. Immediately after the injection, the crab is kept in 200 ml of sea water during a time which corresponds to hemolymph coagulation at the injection place. So, we can fix by difference the radioactivity which has really been injected in the animal. The crabs which are individually put into another series of boxes containing 200 ml of sea water, are then killed at different times. For each animal, the radioactivity in hemolymph and organs previously weighed and dissolved in toluene (hepatopancreas, stomach, gonads and muscle) is determined with a Packard Tricarb liquid 173
scintillation counter model 3320. In a parallel way, the urinary radioactivity is measured with an Instagel scintillation solution.
2. Investigation of urinary metabolite Urine, which is directly taken out of the urinary pore at times 6, 10, 24, 48 hr after labelled clofibric acid injection, is put on plates of silica gel (Merck, Kieselgel 60 F 254). Three solvent systems are used: solvent I--chloroform, acetic acid (95/5) (v/v); solvent II--toluene, acetone (50/50) (v/v); solvent III--benzene, dioxan, acetic acid (90/25/4)
(v/v/v).
The thin-layer plates are put into contact with Kodirex sensitized plates allowing localisation of labelled spots after development. RESULTS AND DISCUSSION
1. Determination of DL5o Toxicity is studied in C,, male and female crabs whose weight is between 4.5 and 5.5 g. Clofibric acid injection at increasing quantities in a thoracic metamer allows a calculation of DLso according to the Behrens and Karber formula. The value which is found (173.5 mg/kg) is obviously lower than the one determined in the mouse. 2. Determination of urinary excretion and clofibric acid distribution in hemolymph and different organs Results (Table I) are expressed for hemolymph as a percentage of radioactivity per organ in proportion to initial dose and for urines in cumulative percentages. The radioactivity decrease in hemolymph is slow up to 8 hr, significant between 8 and 24 hr and lower between 24 and 48 hr. In males, the rates are always higher. In the organs, the radioactivity maximum is reached faster in females (5 hr) than in males (8 or 24 hr) and hepatopancreas has highest rates. Distribution in other organs is smaller with differences between the sexes in the gonads where ovary radioac-
JACQUES LAUTIER
174
et
al.
Table 1. Distribution of clofibric acid in the crab Pachyorapsus marmoratus in proportion with initial dose
Urine Hemolymph Hepatopancreas Stomach Testicles or ovaries
1 or 2 hr
5 hr
8 hr
24 hr
2.91 4- 1.57" 6.32 4- 3.25t 39.72 4- 10.23 34.71 4- 3.36 5.59 4- 0.36 4.18 4- 0.39 1.92 4- 0.02 1.91 4- 0.05 0.30 4- 0.10 1.60 4- 0.58
6.63 + 3.14 14.94 4- 8.10 34.54 4- 10.89 27.57 4- 12.46 5.22 4- 0.32 4.48 4- 0.63 2.30 4- 0.27 2.10 4- 0.05 0.32 4- 0.02 1.48 _ 0.14
23.15 4- 5.50 15.32 4- 4.11 23.63 4- 7.58 23.39 4- 6.37 6.17 4- 0.44 3.86 4- 0.56 1.84 4- 0.17 1.44 4- 0.13 0.38 4- 0.07 1.00 4- 0.09
41.61 4- 6.75 34.70 4- 7.72 11.44 + 2.41 6.66 4- 4.25 7.29 _+ 0.22 3.60 4- 0.42 2.99 4- 0.06 1.55 4- 0.25 0.32 4- 0.02 1.63 4- 0.68
48 hr
53.49 37.92 5.21 4.17 6.94 7.47 2.88 4.97 0.31 6.93
4- 6.96 4- 8.24 4- 2.64 4- 2.08 4- 0.79 4- 3.68 4- 0.26 4- 2.73 _+ 0.05 _+ 3.26
* Males. t Females. tivity of l g of the organ (Table 2) indicates the fixation power of the drug and its metabolites. In hepatopancreas, elofibric acid accumulation is pronounced from the first hour after injection. In females, this mechanism is much slower. After 8 hr, this proportion increases considerably in both sexes, thus proving in hepatopancreas an important accumulation of doffbric acid and its metabolites. The stomach and gonads show the same radioactivity, small at first and increasing later on. However, the fixation power of the stomach is higher than that of the hepatopancreas. The ovary fixes more clofibric acid than the testicle. It is practically impossible to determine exactly the muscular mass of crab. This proportion is lower than one during the first 8 hr. So the clofibric acid injected is not fixed at this time; this takes place after the 24th hr but the fixation power is relatively slow contrary to what happens in mammals (Langer & Levy, 1968). The urinary elimination of clofibric acid and its metabolites is different in both sexes. After 8 hr, it is higher in males than in females. This difference appears at the 48th hour.
Table 2. Proportion study: radioactivity of 1 g of organ relative to radioactivity of one millilitre hemolymph in the crab Pachyorapsus marmoratus 1 or
Hepatopancreas/ Hemolymph Stomach/ Hemolymph Testicles or ovaries/ Hemolymph Muscle/ Hemolymph
2hr
5 hr
8 hr 24hr
8 hr
1.15" 0.53t 0.89 0.70 0.59 0.59 0.70 0.66
1.78 1.50 2.76 1.70 0.92 0.74 0.65 0.89
2.91 0.92 2.74 1.08 1.24 1.12 0.78 0.73
28.14 29.17 32.13 38.57 12.90 17.08 3.51 7.26
6.22 8.24 6.29 9.36 2.93 1.68 2.76 3.03
3. Investigation of urinary metabolites Study of chromatograms shows an absence of metabolism, 6 hr after clofibric acid injection. There is only one spot corresponding to the unaltered drug J R : = 0.52 (solvent I) 0.25 (solvent II) 0.30 (solvent
liD]. After 8 hr, metabolism begins. At that time, chromatograms show two spots: that of the unaltered drug and that of a metabolite [R I = 0.09 (solvent I) 0.05 (solvent II) 0.07 (solvent III)]; with these solvents, there is only one urinary metabolite of clofibric acid in crab. By chromatographic spots elution it is possible to estimate the percentage of clofibric acid urinary metabolite (Table 3). Metabolism increases slowly. It is more important in male than in female; this might explain the total urinary elimination in males 48 hr after injection. 4. Pharmacokinetic model determination This determination is based on hemolymph data; urinary data, which are less clear for precise results, will serve as a control for the proposed model according to the hemolymph graph. A dialysis experiment, the protocol of which has been described by Dubreuil (1974) and Blanchard (1975), shows that clofibric acid binding to hemolymph proteins is about 19%. Martin 0965) showed that this protein binding did not disturb the drug distribution between different tissues and direct elimination or metabolism. It is possible to develop a pharmacokinetic model including two compartments distinguished by their interchange kinetics: a central
k~2
* Males. 1" Females. k21
p21
Table 3. Percentage of clofibric acid urinary metabolite in the crab Pachygrapsus marmoratus 6 hr
10 hr
24 hr
48 hr
0% 0%
12.2% 6.2%
26.2% 17.9%
87.9% 31.6%
Rel
Males Females
Fig. 1. Model with two clofibric acid compartments in the crab Pachygrapsus marmoratus.
175
Study of clofibric acid distribution compartment 1 or C constituted by hemolymph and a peripheral compartment 2 or P by hepatopancreas, stomach and gonads. In referring to the model, the clofibric acid concentration in the central compartment follows the relation:
dC/dt
=
-kl2C
-
k~lC + k2xP.
The integration of this equation by Laplace conversion gives the general relation: C = Ae - ~ + Be -a~ in which A, B, ~ and fl are complicated constants including distribution and elimination stages of drug from the central compartment. F r o m graphic and mathematic analysis, we may evaluate the different parameters ct, fl, A, B and the interchange constants in females and males. Females: C = 1.09e-°'°2t + 1.58 e -°'°6t
k12 = 0.01 h r - 1 ; k21 = 0.036hr-1; k~ = 0.033 h r - 1 Males: C = 1.43e -°'°16t + 1.15e -°'°~t kl2 = 0.008 h r - 1 ;
k21 = 0.034 h r - 1., kel = 0.040 h r - 1. These calculated values were put on an analogue computer, a Telemecanique Alpam S100 calculator connected with a XY Bryans 26000 recorder.
In females, the simulated graphs and experimental points agree for the peripheral compartment. In males, correspondence is also reached but only up to 8 hr; later, the central compartment and urinary elimination graphs came below experimental points in consequence of a higher clofibric acid metabolism. The pharmacokinetic model of clofibric acid in Pachygrapsus marmoratus agrees well that this drug is distributed differently according to organs and sex. The fixation in the peripheral compartment from 8 hr after hypolipidemic drug injection corresponds to the metabolite and unaltered drug accumulation.
REFERENCES
BLANCHARDM. S. (1975) Etude du module pharmacocin~tique d'un sulfamide fi propri~t~s diur~tiques. Th~se Doctorat de Pharmacie, Universit~ de Montpellier, 476, 1-135. DUBREUIL J. (1974) Etude pharmacocin~tique de la bro-. mocresotinamide et d'analogues structuraux. Th~se Doctorat de Pharmacie, Universit6 de Montpellier, 482, 1-174. LANGERT. & LEVY R. I. (1968) Acute muscular syndrome associated with administration of clofibrate. New Engl. J. Med. 279, 856-860. LI~vINsoN H. Z. & LEVI~SON A. R. (1973) Action of the lipid antagonist et hyl-p-chlorophenoxy-isobutyrate (ECPIB) on insect growth and reproduction. J. Insect Physiol. 19, 1727-1734. M ~ ' n N B. K. (1965) Potential effect of the plasma proteins on drug distribution. Nature, Lond. 207, 274-276.
1977. Vol, 58C, pp. 173 to 175. Pergamon
Press. Printed in Great Britain
STUDY OF CLOFIBRIC ACID DISTRIBUTION, METABOLISM AND MODEL, IN CRAB P A C H Y G R A P S U S M A R M O R A T U S (DECAPODA, BRACHYURA) JACQUES LAUTIER 1, JEAN-LOuIS CHANAL 2, JACQUES GUIBERT 2 and JEAN-G. LAGARRIGUE 1 1Laboratoire de Physiologie des Invert~brts, U.S.T.L., Place E. BataiUon, 34060 Montpellier Cedex, France and 2Laboratoire de Physique, Facult6 de Pharmacie, Avenue C. Flahault, 34000 Montpellier, France
(Received 9 February 1977) Abstract--I, The DL50 of clofibric acid which is injected intravenously in crab Pachygrapsus marmoratus is 173.5 mg/kg. 2. Hypolipidemic drug distribution is distinct accdding to the organ. The fixation power of stomach and hepatopancreas is high; on the other hand, crab muscle fixes less clofibric acid than a mammal's; ovary radioactivity is higher than the testicular one. 3. The difference between males and females seems to consist in higher urinary excretion of labelled compound in males. 4. Metabolisation which begins after 8 hr increases slowly and is more important in males. 5. Pharmacokinetic model indicates that clofibric acid is distinctly distributed according to organs and sex.
INTRODUCTION
In consideration of their therapeutic importance, there are numerous hypolipidemic drugs. They belong to various chemical groups and their efficiency in pharmacology in mammals has been proved but their action is not yet known. In Invertebrates, these lipid depressants have been little studied. As far as we know, only Levinson & Levinson (1973) studied ethyl-p-chlorophenoxyisobutyrate (ECPIB) in Dermestes maculatus; these authors established that this hypolipidemic drug retarded the growth of these insects and suppressed their fecundity. In our study on lipid metabolism in the crab Pachy9rapsus marmoratus, we made a pharmacokinetic study of clofibric acid. MATERIALS AND METHODS
Pachygrapsus marmoratus were collected from the Mediterranean shore at Frontignan-Plage and acclimatised to aquarium sea water (salinity 37 + lYoo;temperature: 20°C) for at least 1 week before use. 1. Determination of urinary excretion and clofibric acid distribution in hemolymph and different organs The pharmacokinetic study is founded on utilisation of labelled clofibric acid (carboxyl t4C) with a specific activity of 20 mCi/mmole. The drug is administered by intravenous injection in proportion of 50 mg/kg dissolved in 10Yo ethanol at the rate of 10 mg/ml. This injection is slowly made in a thoracic metamer through the articular membrane of a crab leg; these animals are in C4 intermolt stage. Immediately after the injection, the crab is kept in 200 ml of sea water during a time which corresponds to hemolymph coagulation at the injection place. So, we can fix by difference the radioactivity which has really been injected in the animal. The crabs which are individually put into another series of boxes containing 200 ml of sea water, are then killed at different times. For each animal, the radioactivity in hemolymph and organs previously weighed and dissolved in toluene (hepatopancreas, stomach, gonads and muscle) is determined with a Packard Tricarb liquid 173
scintillation counter model 3320. In a parallel way, the urinary radioactivity is measured with an Instagel scintillation solution.
2. Investigation of urinary metabolite Urine, which is directly taken out of the urinary pore at times 6, 10, 24, 48 hr after labelled clofibric acid injection, is put on plates of silica gel (Merck, Kieselgel 60 F 254). Three solvent systems are used: solvent I--chloroform, acetic acid (95/5) (v/v); solvent II--toluene, acetone (50/50) (v/v); solvent III--benzene, dioxan, acetic acid (90/25/4)
(v/v/v).
The thin-layer plates are put into contact with Kodirex sensitized plates allowing localisation of labelled spots after development. RESULTS AND DISCUSSION
1. Determination of DL5o Toxicity is studied in C,, male and female crabs whose weight is between 4.5 and 5.5 g. Clofibric acid injection at increasing quantities in a thoracic metamer allows a calculation of DLso according to the Behrens and Karber formula. The value which is found (173.5 mg/kg) is obviously lower than the one determined in the mouse. 2. Determination of urinary excretion and clofibric acid distribution in hemolymph and different organs Results (Table I) are expressed for hemolymph as a percentage of radioactivity per organ in proportion to initial dose and for urines in cumulative percentages. The radioactivity decrease in hemolymph is slow up to 8 hr, significant between 8 and 24 hr and lower between 24 and 48 hr. In males, the rates are always higher. In the organs, the radioactivity maximum is reached faster in females (5 hr) than in males (8 or 24 hr) and hepatopancreas has highest rates. Distribution in other organs is smaller with differences between the sexes in the gonads where ovary radioac-
JACQUES LAUTIER
174
et
al.
Table 1. Distribution of clofibric acid in the crab Pachyorapsus marmoratus in proportion with initial dose
Urine Hemolymph Hepatopancreas Stomach Testicles or ovaries
1 or 2 hr
5 hr
8 hr
24 hr
2.91 4- 1.57" 6.32 4- 3.25t 39.72 4- 10.23 34.71 4- 3.36 5.59 4- 0.36 4.18 4- 0.39 1.92 4- 0.02 1.91 4- 0.05 0.30 4- 0.10 1.60 4- 0.58
6.63 + 3.14 14.94 4- 8.10 34.54 4- 10.89 27.57 4- 12.46 5.22 4- 0.32 4.48 4- 0.63 2.30 4- 0.27 2.10 4- 0.05 0.32 4- 0.02 1.48 _ 0.14
23.15 4- 5.50 15.32 4- 4.11 23.63 4- 7.58 23.39 4- 6.37 6.17 4- 0.44 3.86 4- 0.56 1.84 4- 0.17 1.44 4- 0.13 0.38 4- 0.07 1.00 4- 0.09
41.61 4- 6.75 34.70 4- 7.72 11.44 + 2.41 6.66 4- 4.25 7.29 _+ 0.22 3.60 4- 0.42 2.99 4- 0.06 1.55 4- 0.25 0.32 4- 0.02 1.63 4- 0.68
48 hr
53.49 37.92 5.21 4.17 6.94 7.47 2.88 4.97 0.31 6.93
4- 6.96 4- 8.24 4- 2.64 4- 2.08 4- 0.79 4- 3.68 4- 0.26 4- 2.73 _+ 0.05 _+ 3.26
* Males. t Females. tivity of l g of the organ (Table 2) indicates the fixation power of the drug and its metabolites. In hepatopancreas, elofibric acid accumulation is pronounced from the first hour after injection. In females, this mechanism is much slower. After 8 hr, this proportion increases considerably in both sexes, thus proving in hepatopancreas an important accumulation of doffbric acid and its metabolites. The stomach and gonads show the same radioactivity, small at first and increasing later on. However, the fixation power of the stomach is higher than that of the hepatopancreas. The ovary fixes more clofibric acid than the testicle. It is practically impossible to determine exactly the muscular mass of crab. This proportion is lower than one during the first 8 hr. So the clofibric acid injected is not fixed at this time; this takes place after the 24th hr but the fixation power is relatively slow contrary to what happens in mammals (Langer & Levy, 1968). The urinary elimination of clofibric acid and its metabolites is different in both sexes. After 8 hr, it is higher in males than in females. This difference appears at the 48th hour.
Table 2. Proportion study: radioactivity of 1 g of organ relative to radioactivity of one millilitre hemolymph in the crab Pachyorapsus marmoratus 1 or
Hepatopancreas/ Hemolymph Stomach/ Hemolymph Testicles or ovaries/ Hemolymph Muscle/ Hemolymph
2hr
5 hr
8 hr 24hr
8 hr
1.15" 0.53t 0.89 0.70 0.59 0.59 0.70 0.66
1.78 1.50 2.76 1.70 0.92 0.74 0.65 0.89
2.91 0.92 2.74 1.08 1.24 1.12 0.78 0.73
28.14 29.17 32.13 38.57 12.90 17.08 3.51 7.26
6.22 8.24 6.29 9.36 2.93 1.68 2.76 3.03
3. Investigation of urinary metabolites Study of chromatograms shows an absence of metabolism, 6 hr after clofibric acid injection. There is only one spot corresponding to the unaltered drug J R : = 0.52 (solvent I) 0.25 (solvent II) 0.30 (solvent
liD]. After 8 hr, metabolism begins. At that time, chromatograms show two spots: that of the unaltered drug and that of a metabolite [R I = 0.09 (solvent I) 0.05 (solvent II) 0.07 (solvent III)]; with these solvents, there is only one urinary metabolite of clofibric acid in crab. By chromatographic spots elution it is possible to estimate the percentage of clofibric acid urinary metabolite (Table 3). Metabolism increases slowly. It is more important in male than in female; this might explain the total urinary elimination in males 48 hr after injection. 4. Pharmacokinetic model determination This determination is based on hemolymph data; urinary data, which are less clear for precise results, will serve as a control for the proposed model according to the hemolymph graph. A dialysis experiment, the protocol of which has been described by Dubreuil (1974) and Blanchard (1975), shows that clofibric acid binding to hemolymph proteins is about 19%. Martin 0965) showed that this protein binding did not disturb the drug distribution between different tissues and direct elimination or metabolism. It is possible to develop a pharmacokinetic model including two compartments distinguished by their interchange kinetics: a central
k~2
* Males. 1" Females. k21
p21
Table 3. Percentage of clofibric acid urinary metabolite in the crab Pachygrapsus marmoratus 6 hr
10 hr
24 hr
48 hr
0% 0%
12.2% 6.2%
26.2% 17.9%
87.9% 31.6%
Rel
Males Females
Fig. 1. Model with two clofibric acid compartments in the crab Pachygrapsus marmoratus.
175
Study of clofibric acid distribution compartment 1 or C constituted by hemolymph and a peripheral compartment 2 or P by hepatopancreas, stomach and gonads. In referring to the model, the clofibric acid concentration in the central compartment follows the relation:
dC/dt
=
-kl2C
-
k~lC + k2xP.
The integration of this equation by Laplace conversion gives the general relation: C = Ae - ~ + Be -a~ in which A, B, ~ and fl are complicated constants including distribution and elimination stages of drug from the central compartment. F r o m graphic and mathematic analysis, we may evaluate the different parameters ct, fl, A, B and the interchange constants in females and males. Females: C = 1.09e-°'°2t + 1.58 e -°'°6t
k12 = 0.01 h r - 1 ; k21 = 0.036hr-1; k~ = 0.033 h r - 1 Males: C = 1.43e -°'°16t + 1.15e -°'°~t kl2 = 0.008 h r - 1 ;
k21 = 0.034 h r - 1., kel = 0.040 h r - 1. These calculated values were put on an analogue computer, a Telemecanique Alpam S100 calculator connected with a XY Bryans 26000 recorder.
In females, the simulated graphs and experimental points agree for the peripheral compartment. In males, correspondence is also reached but only up to 8 hr; later, the central compartment and urinary elimination graphs came below experimental points in consequence of a higher clofibric acid metabolism. The pharmacokinetic model of clofibric acid in Pachygrapsus marmoratus agrees well that this drug is distributed differently according to organs and sex. The fixation in the peripheral compartment from 8 hr after hypolipidemic drug injection corresponds to the metabolite and unaltered drug accumulation.
REFERENCES
BLANCHARDM. S. (1975) Etude du module pharmacocin~tique d'un sulfamide fi propri~t~s diur~tiques. Th~se Doctorat de Pharmacie, Universit~ de Montpellier, 476, 1-135. DUBREUIL J. (1974) Etude pharmacocin~tique de la bro-. mocresotinamide et d'analogues structuraux. Th~se Doctorat de Pharmacie, Universit6 de Montpellier, 482, 1-174. LANGERT. & LEVY R. I. (1968) Acute muscular syndrome associated with administration of clofibrate. New Engl. J. Med. 279, 856-860. LI~vINsoN H. Z. & LEVI~SON A. R. (1973) Action of the lipid antagonist et hyl-p-chlorophenoxy-isobutyrate (ECPIB) on insect growth and reproduction. J. Insect Physiol. 19, 1727-1734. M ~ ' n N B. K. (1965) Potential effect of the plasma proteins on drug distribution. Nature, Lond. 207, 274-276.