Drug Metabolism Reviews
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Correlation of Multiple Biological Techniques Henry J. Laurencot To cite this article: Henry J. Laurencot (1990) Correlation of Multiple Biological Techniques, Drug Metabolism Reviews, 22:6-8, 789-802, DOI: 10.3109/03602539008991469 To link to this article: http://dx.doi.org/10.3109/03602539008991469
Published online: 22 Sep 2008.
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Date: 11 November 2015, At: 11:13
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DRUG METABOLISM REVIEWS, 22(6-8), 789-802 (1990)
CORRELATION OF MULTIPLE BIOLOGICAL TECHNIQUES* HENRY J. LAURENCOT Research Investigator Animal Science Research Hoffmann-LaRoche Inc. 340 Kingsland Street Nutley, New Jersey 07110
I.
INTRODUCTION ..........................................................
789
11.
BIOACTIVITY ..............................................................
790
111.
BIOAVAILABILITY .......................................................
792
References. ..................................................................
.802
I. INTRODUCTION The use of biological techniques has been helpful in evaluating the human food safety of edible animal tissues which contain residues derived from animal drugs or feed additives. Drug residues are usually determined *This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 7 89 Copyright 0 1991 by Marcel Lkkker, Inc
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790
LAURENCOT
by administering ''C-radiolabeled drug to the target species for several days and assaying the edible tissues for radioactivity over an extended withdrawal period. The assays give total concentration of residues based on the specific radioactivity of the carbon-labeled drug. The radioactive residues may represent the parent drug compound, metabolites, conjugates, degradation products, or endogenous compounds. In today's talk I am going to present our results on the bioavailability studies and biological activity assays of the residues of the polyether antibiotic lasalocid, which are found in animal tissues.
11. BIOACTIVITY As shown in Fig. I the tissue samples for these studies are derived from the target animal feeding study with I4C-labeled drug. The bioavailability studies give us information on the absorption and solubilities of the residues. The thin-layer chromatography (TLCtbioautography assays show which residues are biologically active. A comparison can be made between the absorption of total radioactivity of whole tissues to that of the soluble fraction or insoluble bound residue fraction of the same tissue and to the absorption of the parent drug compound. The treatment compound in the target animal feeding study was lasalocid (Fig. 2). It was enriched in I4C isotope in the 13, 17, and 21 positions. I-I4C-Butyrate was used as the precursor in the fermentation. Lasalocid is used as a coccidiostat in chickens and for improved growth feed efficiency in cattle. Lasalocid is a noncarcinogen. The compound was 94% chemically pure lasalocid sodium, 4% methyloleate, and 2% water. The radiochemical purity of lasalocid sodium was 100%. The specific radioactivity was 3270 d p d p g as the sodium salt. The tissue assay method for lasalocid is a TLCbioautographical method for chicken skidfat tissue as reported by MacDonald et al. in 1978 [ I ] . The sample was homogenized and extracted with a mixture of benzene and chloroform, defatted on a silica column, and developed on a silica gel thin layer plate. The plate is overlayed with seeded agar and incubated overnight at 37°C. The zones of inhibition are visualized with a 0.1% TTC (2,3,5 triphenyl tetrazolium chloride) + 0.2% glucose solution. The plate is photographed and the area of the zones are measured and quantitated (Fig. 3). The TLC-bioautographic method for lasalocid gives positive response for biological activity. The radioisotopic scans of the plates can correlate the
BIOAVAILABILITY AND BIOACTIVITY
79 I
TOTAL TLSSUE-14C RESIDUE
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I
I
BIOLOGICAL SENSORS
I
BIOAVAILABILITY BIOAUTOGRAPHY
J COMPARISON OF ABSORPTION AND SOLUBILITIES
BIOLOGICAL ACTIVITY
.
.c WHOLE TISSUES
FRACTIONATED TISSUES
FRACTION
PARENT COMPOUND
RESIDUES
FIG. 1. Origin of tissue samples.
labeled parent compound or any of the possible metabolites with biological activity. This correlation has been made with tissue, feces, urine, and feed extracts. The biological activity response has been limited to intact lasalocid. The quantitation of this method has been validated with HPLCfluorescence detection as reported by Weiss and MacDonald in 1985 (21. Sixteen percent (16%) of the total bovine liver residue is intact lasalocid and biologically active at zero time withdrawal (Table 1). Additional examples of the biological evaluation of lasalocid are given in the presentation by Dr. Weiss (see page 829 of the present issue of Drug Mefabolism Reviews).
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792
LAURENCOT
Molecular Weight: 612.8
Empirical Formula: C34H5308Na
0
Denotes carbon 13.17 and 21 enriched in14C isotopewhen sodium (l-14C) butyrate had been added as a precursor in the fermentation.
FIG. 2. L a ~ a l o c i d - ~treatment ~C compound. 111. BIOAVAILABILITY
The many advantages in using the rat model in bioavailability studies of labeled residues were first presented in detail by Dr. Hugo Gallo-Torres in 1977 [3]. Figure 4 is a schematic representation of the animal model used in our studies and taken from his earlier work. An update on the use of these procedures by Dr. Gallo-Torres is given in this issue (see page 707). The rat is one of the most commonly used species in biological and toxicological research. We used this model for the determination of the absorption of labeled residues from whole and fractionated tissues of chicken and steer.
TABLE 1 TLC Bioautography for Lasalocid Primary Screen for Activity: Correlates TLC-radioisotope scans with microbiological activity on overlay Used with tissue, fecal, urine, and feed extracts Activity limited to intact lasalocid Quantation validated with HPLC-fluorescence 16% of total liver radioactivity is lasalocid and biologically active at zero time withdrawal
BIOAVA ILABI LITY AND BIOACTIVITY
1
793
Homogenize and Extract
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Aqueous Wash Silica Column Column Wash
-
- MeOH Elute
-
Benzene and MeCl
Column Eluate Concentrate and Apply to Plate
1
Thin-Layer Chromatography Develop Plates Overlay Plate with Seeded Agar Bacillus TA NRRL B-3167 24-hr Incubation at 37OC. Visualize Response with TTC-Glucose Interpret/ Quantitate
FIG. 3. Tissue assay flow diagram. The use of proper controls is very important for assay and interpretation of results. Table 2 presents the data for a control experiment which determines the absorption of the parent compound ''C-lasalocid in the rat model when fed grain rat chow. This is what I call a type-I study. Two hundred and thirty gram male rats were treated via the stomach cannula at a dose of 1 mg/kg with ''C-lasalocid sodium. The specific radioactivity in this experiment was 3500 d p d p g , and the total treatment radioactivity was 790,000 dpdrat. The radioactivity of this dose is relatively high when compared to edible tissue residues. The tissue distribution and reproducibility between rats are given for the nonabsorbed parent compound and its possible metabolites as an average of 37% found in the feces and g.i. tract contents. The amount of radioactivity found as absorbed in the bile, carcass, g.i. tract tissue, liver, and urine was 61%. The average recovery was 98%.
LAURENCOT
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794
Bile Salt Solution
FIG. 4. Schematic representation of the animal model used in the present studies. A cannula, placed in the upper part of the common bile duct. is used to collect bile, uncontaminated with pancreatic juice. The pancreatic secretions are left undisturbed. Steady-state conditions are achieved by continuous intragastric infusion of solutions of bile salts. The gastric catheter is also used for quantitative delivery of test compounds into the gastrointestinal tract. In this type-I control experiment, the percent of the radioactivity absorbed from the parent compound was not dependent on the sex of the rat or the route of administration, either by stomach cannula or mixed in the rat chow and fed orally. The average percent of treatment recovery was 98%, and the animal survival rate was 95% in the control studies.
66.28 16 96.5077
60.8669 98.3642 51.3706 100.3690 64.3434 99.9876
58.6237 98.8227
63.7149 96.1342
58.6706 0.1279" 0.0742 0.9412 1.0530 48.3151 0.2185" 0.1133 1.4621 1.2616 63.2165 0.072ga 0.0341 0.7300 0.2900
0.0534 1.1797 1.2580
0.0665'
56.0661
65.4197 0.1298' 0.0434 0.5201 0.1686
60.3354 0.1520" 0.1266 0.8140 2.2869
37.4974 48.9984
Average
35.6442
40.1990
30.226 1
-
37.0527 0.4447
32.4193
32.2067 0.2126
Rat 5 47.6475 1.3509
Rat 4 35.3608 0.2834
Rat 3 39.9622 0.2368
Rat 2
30.0862 0.1399
"Below Minimal Detectable % Treatment.
Subtotal Total Recovery
Subtotal Absorbed Bile Carcass GI Tract Tissue Liver Urine
Nonabsorbed Feces GI Tract Contents
Rat 1
% Recovery
Percent Treatment Radioactivity Recovered in Urine, Feces, Bile, and Tissues of 5 Male Rats Treated Orally via Stomach Cannula at a Dose of 1 mgkg Lasalocid-''C Sodium During the 48-Hour Withdrawal Period
TABLE 2
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796
LAURENCOT
Table 3 presents the data for the distribution of radioactivity in liver tissues derived from Ia~alocid-~~C-fed chickens treated at 0.0075% in the feed for 21 days. The tissue was homogenized in a solution of absolute ethanol-3A:chloroform:1NHCI; 155: I ; v/v/v, and exhaustively extracted with 95% ethanol-3A. The ppm fresh weight, ppm, and % radioactivity found in the ethanol-soluble and in the ethanol-insoluble fractions are given for 4 days of the withdrawal period. At zero day (0-hr) withdrawal 64% of the radioactivity was found in the ethanol-soluble fraction and 36% in the ethanol-insoluble fraction. At days 1-4 withdrawal, the reverse is true. Here the majority of the radioactivity is found in the ethanolinsoluble fraction. This is an indication of a change in the nature of the radioactive residues found in the livers with time. Table 4 compares the concentration and bioavailability of radioactive liver residues derived from Ia~alocid-~~C fed to chickens which were treated at 0.0075% and 0.0125% in the feed for 21 days. The concentration of radioactivity found in the liver of birds treated with la~alocid-'~C at 0.0075% in the feed was 4.05 ppm at 0-hr withdrawal, and 14.3% of this radioactivity was bioavailable in the rat model. In the birds treated at 0.0125% in the feed, the livers contained 11.93 ppm at 0-hr withdrawal, and 18.9% of this radioactivity was bioavailable. The concentrations found in the livers at 0-hr withdrawal for the two treatments not only show the expected dose response to liver concentration, but also an increase in the percent radioactivity that is bioavailable in the rat with increased dose. This would indicate a higher percentage of radioactivity in the soluble fraction at 0-hr withdrawal. The data presented in Tables 3 and 4 show a rapid decrease in the concentration of radioactivity in the whole liver, in the percent of radioactivity that is bioavailable to the rat,
TABLE 3 Distribution of Radioactivity in Liver Tissues Derived from Lasalocid14C-Fed Chicken Livers at 0.0075% in the Feed for 21 Days Ethanol-soluble fraction
Ethanol-insoluble fraction
Withdrawal day
PPm fresh weight
ppm
%
PPm
%
0 I 2 3 4
3.84 I .75 1.29 1.36 0.95
2.46 0.55 0.43 0.36 0.25
63.9 31.4 33.3 26.5 26.3
I .39 I .20 0.86 1 .OO 0.70
36. I 68.6 66.7 73.5 73.7
797
BIOAVAILABILITY AND BIOACTIVITY
TABLE 4
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Comparison of the Concentration and Bioavailability of Radioactive Liver Residues Derived from Lasal~id-'~C Fed to Chickens Treated at 0.0075% and 0.0125% in the Feed for 21 Days Withdrawal time (days)
PPm fresh weight
% Not
bioavailable
% Bioavailable
PPm fresh wt. bioavailable
1. 0.0075% in Feed (5.30 mglkglday), 37 days pretreatment
0
4.05
85.68
14.32
0.58
11. 0.0125% in Feed (9.69 mglkglday), 34 days pretreatment
0 1 2 3 4
I I .93 2.63 1.72 1.59 1.37
5
1.15
81.10 93.43 97.57 97.86 97.48 98.28
18.90 6.57 2.43 2.14 2.52 1.72
2.25 0.17 0.04 0.03 0.03 0.02
and the percent of radioactivity that is found in the ethanol-soluble fraction during the first 24 h of withdrawal. During days 2-5 of withdrawal, both treatments show a low, similar concentration of radioactivity in liver and a much slower elimination of radioactivity with time. Just over 2% of the total radioactivity in the livers are bioavailable in the rat model during this withdrawal period where approx. 30% of the total radioactivity is found in the ethanol-soluble fraction and 70% in the ethanol-insoluble fraction. Figure 5 shows the concentration of 14C-lasalocidand its possible metabolites found in the liver of chickens treated for 21 days with medicated feed containing ''C-lasalocid at a concentration of 125 ppm in the feed and during the 5-day withdrawal period. The total liver radioactivity, as ppm, is plotted for 0, I , 2, 3, 4, and 5 days withdrawal. There is a distinct biphasic withdrawal curve. In the first phase, 0-24 h, the half-life of elimination of the total radioactivity (t,,J is I I h. In the second phase the t1,2 is 6 days. The calculated ppm for that fraction of the first phase which is rapidly eliminated is given in hours and ppm by subtraction of the second phase. This rapidly eliminated fraction has a half-life of elimination of radioactivity (tIl2) of 6.3 h and has a correlation to solubility and bioavailability of the residues. Figure 6 shows again the biphasic curve for the total concentration of radioactivity in the 0-5-day withdrawal chicken livers and the curve and data for the bioavailability of the total liver residues. The data for the solubility and the bioavailability shows the amount absorbed to be associated
798
LAURENCOT
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\
t,,=ll
hr
I
0.5
‘
0
1% t 1n=6.3tr
1 1
1
I
I
1
2
3
4
WITHDRAWAL TIME
(DAYS)
FIG. 5. The concentration of la~alocid-’~C and its possible metabolites found in the liver of chickens during the 5-day withdrawal period. with the soluble fraction. Nineteen percent (19%)of the total radioactivity of the zero-day withdrawal livers was absorbed. Just over 2% of the 25-day withdrawal liver radioactivity was absorbed. Table 5 presents the data used for the determination of the half-life of elimination of the radioactivity from the chicken livers during the 5-day withdrawal period. The data for the determination of the half-life of elimination of the radioactivity absorbed from these livers when fed to rats are also given. The concentrations used to determine the half-life of elimination of radioactivity for the first phase of elimination, day 0 and day 1 of withdrawal, and the second phase of elimination, days 2-5, were the assayed values for both the chicken liver and rat bioavailability data. The fast phase of elimination is that fraction of the first phase remaining after subtraction of the second phase contribution to total concentration. The concentrations of the second phase at 0- and I-day withdrawal are calculated by linear regression analysis from the 2-5-day assay data. The assay data, linear regression analysis of the second phase, and the calculated fast phase fractions of the first phase of elimination are plotted in Fig. 7 for the chicken liver and rat bioavailability data.
5
BIOAVAILABILITY AND BIOACTIVITY
799
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A
0 t Y
0.01
' 0
I
I
I
I
I
1
2
3
4
5
WIll-DRAWAL TIME
(DAYS)
FIG. 6. The concentration of la~alocid-'~C and its possible metabolites found in the liver of chickens and its concentration found bioavailable in the rat during the 5-day withdrawal period. The radioactivity of the chicken livers had a half-life of elimination in the first phase of 1 1 h, in the second phase 6 days, and in the fast phase 6.3 h. The radioactivity of the chicken livers bioavailable in the rat model had a half-life of elimination in the first phase of 6.5 h, in the second phase 4 days, and in the fast phase 5.8 h. The rates of elimination of radioactivity from the fast phase of elimination in the chicken livers (6.3 h), and the first phase (6.5 h), and fast phase (5.8 h) of the radioactivity of the chicken liver that were bioavailable in the rat model are almost identical. The data shows that almost all of the radioactivity absorbed by the rat when fed these chicken livers is derived from the soluble fraction of the chicken livers. The radioactivity of this fraction is rapidly eliminated in the chicken livers and greatly reduced in bioavailability to the rat during the first 24 h of withdrawal. The half-life of elimination of the fast phase of bioavailable radioactivity is 5.8 h. Table 6 gives a comparison of the concentration and bioavailability of radioactive whole liver tissue residues derived from ''C-lasalocid fed to steers which were treated at 0.5, 1.0, and 2.0 mg/kg/day for 14 days. The treatment was administered twice a day by capsule. The withdrawal times are from the last capsule treatment. When the steers were treated at a dose of 0.5 mg/kg/day, the liver contained radioactivity equivalent to 4.5 ppm lasalocid or its possible metabolites. At a dose of 1.0 mg/kg/day, the liver
800
LAURENCOT
TABLE 5 '*C Residues in Chicken Liver: Total and Bioavailable ppm During a 5-Day Withdrawal Period
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ppm Liver-I4C residues Total ppm
Withdrawal time (days) 0 1 2 3 4 5 tl/Z(h)
t,,,(days)
Bioavailable ppm
Linear regression analysis phases
-
Linear regression analysis phases
Assayed
1st
2nd
Fast
Assayed
1st
2nd
Fast
11.93 2.63 1.72 1.59 I .37
11.93 2.63
9.80 0.69
0.06 0.05 0.04 0.04 0.03 0.02
2.20 0.12
-
2.25 0.17 0.04 0.03 0.03 0.02
2.25 0.17
1.15
2.13 1.94 1.75 1.55 1.36 1.17
-
11.0
-
6.3
6.5
6.0
-
-
-
-
-
-
-
-
-
4.0
contained 5.9 pprn and at a dose of 2.0 mg/kg/day, the concentration of radioactivity was 10.2 pprn showing a dose response. The percent of radioactivity of the steer liver absorbed by the rat was 21.6 (0.5 mg/kg/day), 23.2 ( I .O mg/kg/day), and 27.1 (2.0 mg/kg/day). This increase in the percent of radioactivity bioavailable in the rat model with increase in dose to
TABLE 6 Comparison of Concentration and Bioavailability of Radioactive Steer Liver Residues in the Bile Cannulated Rat" Liver Withdrawal time Dose (mg/kg)
Days
+H
Steer
PPm fresh weight
% Not bio-
% Bio-
available
available
ppm fresh wt/bioavailable
78.44 76.83 72.88
21.56 23.17 27.12
0.965 1.364 2.765
~~
0.5 I .o 2.0
0 : 17 0 : 19 0 : 20
II 18 16
4.476 5.886 10.196
asteen were treated with lasal~cid-'~C at 0.5, 1.0, and 2.0 mg/kg/day for 14 days.
BIOAVAILABILITY AND BIOACTIVITY
80 1
-
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-c
FRESHWT.
.,y;.,, -\:.,,
0.1
,= =6.5
/t
.\?. A*
:
TOTAL
hr
-L
..*. FAST PHASE
t ,m=5.8
__ __ __ -~
Cl---------..r~-
SECOND PHASE
hr
810-, AVAILABLE_ _ -
I I -.-
+FRESHWT. SECOND PHASE FASTPHASE
-
. -.-.
0.01
'
0
112
=4 days
~.-
I
I
I
I
I
1
2
3
4
5
WITHDRAWAL TIME
(DAYS)
FIG. 7. Comparison of depletion rates of total chicken liver radioactivity and its bioavailable radioactivity when fed to rats during the 5-day withdrawal period. the steers was also seen with chickens. This increase is associated with the soluble fraction of the liver. Table 7 gives the data for the bioavailability of the fractionated steer liver radioactivity derived from steers treated at 1 mg/kg/day with ''C-lasalocid for 14 days and sacrificed 19 h after the last capsule treatment. The ethanolinsoluble fraction contained 18% of the total radioactivity. Five percent (5%) of this fraction was absorbed or 0.9% of the total radioactivity. The ethanol-soluble fraction contained 82% of the total radioactivity. Forty percent (40%) of this fraction was absorbed or 33% of the total radioactivity. In the data presented for the chicken and steer liver residues derived from ''C-lasalocid, approximately 75% of the total radioactivity is not absorbed, and 25% is absorbed at 0-h withdrawal. This is much less than the absorption of the parent compound. There is a very rapid elimination of the absorbable fraction of the total radioactivity in the first 24 h of withdrawal to a level of approximately 6%. The absorption of radioactivity after 24 h withdrawal was approximately 2% of total residues for the 5-day withdrawal period. The low absorption of total residues and the usual withdrawal times for slaughter, as followed in industrial practice, should allow for appropriate
LAURENCOT
802
TABLE 7 Bioavailability of the Radioactivity of the Ethanol-Soluble and the Ethanol-Insoluble Fractions of Steer Liver'
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Percent
Individual tissue fractions Ethanol-insoluble Ethanol-soluble Tissue fraction contribution as percent of 1 g tissue Ethanol-insoluble Ethanol-soluble Total contribution percent
Absorbed
Nonabsorbed
Total
4.90 40.20
95.10 59.80
100.00 100.00
0.89 32.89
17.28 48.94
18.17 81.82
33.18
66.22
100.00
"The steer was treated at 1.0 mg/kg/day for 14 days and sacrificed 19 h after the last capsule treatment. adjustments to be made to the minimal levels of residues remaining in the tissues of animals intended for human consumption.
REFERENCES [ I ] A. MacDonald, G. Chen, P Duke, A . Popick, R. Saperstein. M. Kaykaty, C. Crowley, H. Hutchinson, and J. Westheimer, in Densiromerry in Thin-Layer Chromatography (J. C. Touchstone and J. Sherma, eds.), Chapter 9, Wiley-Interscience, New York, 1978, pp. 20 1-222. [2] G. Weiss and A. MacDonald, J. Assoc. O f . Anal. Chem., 68, 971 ( 1985). [3] H. E. Gallo-Torres, J. Toxicol. Envir. Healrh, 2 , 827 (1977).
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Correlation of Multiple Biological Techniques Henry J. Laurencot To cite this article: Henry J. Laurencot (1990) Correlation of Multiple Biological Techniques, Drug Metabolism Reviews, 22:6-8, 789-802, DOI: 10.3109/03602539008991469 To link to this article: http://dx.doi.org/10.3109/03602539008991469
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [University of California, San Diego]
Date: 11 November 2015, At: 11:13
Downloaded by [University of California, San Diego] at 11:13 11 November 2015
DRUG METABOLISM REVIEWS, 22(6-8), 789-802 (1990)
CORRELATION OF MULTIPLE BIOLOGICAL TECHNIQUES* HENRY J. LAURENCOT Research Investigator Animal Science Research Hoffmann-LaRoche Inc. 340 Kingsland Street Nutley, New Jersey 07110
I.
INTRODUCTION ..........................................................
789
11.
BIOACTIVITY ..............................................................
790
111.
BIOAVAILABILITY .......................................................
792
References. ..................................................................
.802
I. INTRODUCTION The use of biological techniques has been helpful in evaluating the human food safety of edible animal tissues which contain residues derived from animal drugs or feed additives. Drug residues are usually determined *This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 7 89 Copyright 0 1991 by Marcel Lkkker, Inc
Downloaded by [University of California, San Diego] at 11:13 11 November 2015
790
LAURENCOT
by administering ''C-radiolabeled drug to the target species for several days and assaying the edible tissues for radioactivity over an extended withdrawal period. The assays give total concentration of residues based on the specific radioactivity of the carbon-labeled drug. The radioactive residues may represent the parent drug compound, metabolites, conjugates, degradation products, or endogenous compounds. In today's talk I am going to present our results on the bioavailability studies and biological activity assays of the residues of the polyether antibiotic lasalocid, which are found in animal tissues.
11. BIOACTIVITY As shown in Fig. I the tissue samples for these studies are derived from the target animal feeding study with I4C-labeled drug. The bioavailability studies give us information on the absorption and solubilities of the residues. The thin-layer chromatography (TLCtbioautography assays show which residues are biologically active. A comparison can be made between the absorption of total radioactivity of whole tissues to that of the soluble fraction or insoluble bound residue fraction of the same tissue and to the absorption of the parent drug compound. The treatment compound in the target animal feeding study was lasalocid (Fig. 2). It was enriched in I4C isotope in the 13, 17, and 21 positions. I-I4C-Butyrate was used as the precursor in the fermentation. Lasalocid is used as a coccidiostat in chickens and for improved growth feed efficiency in cattle. Lasalocid is a noncarcinogen. The compound was 94% chemically pure lasalocid sodium, 4% methyloleate, and 2% water. The radiochemical purity of lasalocid sodium was 100%. The specific radioactivity was 3270 d p d p g as the sodium salt. The tissue assay method for lasalocid is a TLCbioautographical method for chicken skidfat tissue as reported by MacDonald et al. in 1978 [ I ] . The sample was homogenized and extracted with a mixture of benzene and chloroform, defatted on a silica column, and developed on a silica gel thin layer plate. The plate is overlayed with seeded agar and incubated overnight at 37°C. The zones of inhibition are visualized with a 0.1% TTC (2,3,5 triphenyl tetrazolium chloride) + 0.2% glucose solution. The plate is photographed and the area of the zones are measured and quantitated (Fig. 3). The TLC-bioautographic method for lasalocid gives positive response for biological activity. The radioisotopic scans of the plates can correlate the
BIOAVAILABILITY AND BIOACTIVITY
79 I
TOTAL TLSSUE-14C RESIDUE
Downloaded by [University of California, San Diego] at 11:13 11 November 2015
I
I
BIOLOGICAL SENSORS
I
BIOAVAILABILITY BIOAUTOGRAPHY
J COMPARISON OF ABSORPTION AND SOLUBILITIES
BIOLOGICAL ACTIVITY
.
.c WHOLE TISSUES
FRACTIONATED TISSUES
FRACTION
PARENT COMPOUND
RESIDUES
FIG. 1. Origin of tissue samples.
labeled parent compound or any of the possible metabolites with biological activity. This correlation has been made with tissue, feces, urine, and feed extracts. The biological activity response has been limited to intact lasalocid. The quantitation of this method has been validated with HPLCfluorescence detection as reported by Weiss and MacDonald in 1985 (21. Sixteen percent (16%) of the total bovine liver residue is intact lasalocid and biologically active at zero time withdrawal (Table 1). Additional examples of the biological evaluation of lasalocid are given in the presentation by Dr. Weiss (see page 829 of the present issue of Drug Mefabolism Reviews).
Downloaded by [University of California, San Diego] at 11:13 11 November 2015
792
LAURENCOT
Molecular Weight: 612.8
Empirical Formula: C34H5308Na
0
Denotes carbon 13.17 and 21 enriched in14C isotopewhen sodium (l-14C) butyrate had been added as a precursor in the fermentation.
FIG. 2. L a ~ a l o c i d - ~treatment ~C compound. 111. BIOAVAILABILITY
The many advantages in using the rat model in bioavailability studies of labeled residues were first presented in detail by Dr. Hugo Gallo-Torres in 1977 [3]. Figure 4 is a schematic representation of the animal model used in our studies and taken from his earlier work. An update on the use of these procedures by Dr. Gallo-Torres is given in this issue (see page 707). The rat is one of the most commonly used species in biological and toxicological research. We used this model for the determination of the absorption of labeled residues from whole and fractionated tissues of chicken and steer.
TABLE 1 TLC Bioautography for Lasalocid Primary Screen for Activity: Correlates TLC-radioisotope scans with microbiological activity on overlay Used with tissue, fecal, urine, and feed extracts Activity limited to intact lasalocid Quantation validated with HPLC-fluorescence 16% of total liver radioactivity is lasalocid and biologically active at zero time withdrawal
BIOAVA ILABI LITY AND BIOACTIVITY
1
793
Homogenize and Extract
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Aqueous Wash Silica Column Column Wash
-
- MeOH Elute
-
Benzene and MeCl
Column Eluate Concentrate and Apply to Plate
1
Thin-Layer Chromatography Develop Plates Overlay Plate with Seeded Agar Bacillus TA NRRL B-3167 24-hr Incubation at 37OC. Visualize Response with TTC-Glucose Interpret/ Quantitate
FIG. 3. Tissue assay flow diagram. The use of proper controls is very important for assay and interpretation of results. Table 2 presents the data for a control experiment which determines the absorption of the parent compound ''C-lasalocid in the rat model when fed grain rat chow. This is what I call a type-I study. Two hundred and thirty gram male rats were treated via the stomach cannula at a dose of 1 mg/kg with ''C-lasalocid sodium. The specific radioactivity in this experiment was 3500 d p d p g , and the total treatment radioactivity was 790,000 dpdrat. The radioactivity of this dose is relatively high when compared to edible tissue residues. The tissue distribution and reproducibility between rats are given for the nonabsorbed parent compound and its possible metabolites as an average of 37% found in the feces and g.i. tract contents. The amount of radioactivity found as absorbed in the bile, carcass, g.i. tract tissue, liver, and urine was 61%. The average recovery was 98%.
LAURENCOT
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794
Bile Salt Solution
FIG. 4. Schematic representation of the animal model used in the present studies. A cannula, placed in the upper part of the common bile duct. is used to collect bile, uncontaminated with pancreatic juice. The pancreatic secretions are left undisturbed. Steady-state conditions are achieved by continuous intragastric infusion of solutions of bile salts. The gastric catheter is also used for quantitative delivery of test compounds into the gastrointestinal tract. In this type-I control experiment, the percent of the radioactivity absorbed from the parent compound was not dependent on the sex of the rat or the route of administration, either by stomach cannula or mixed in the rat chow and fed orally. The average percent of treatment recovery was 98%, and the animal survival rate was 95% in the control studies.
66.28 16 96.5077
60.8669 98.3642 51.3706 100.3690 64.3434 99.9876
58.6237 98.8227
63.7149 96.1342
58.6706 0.1279" 0.0742 0.9412 1.0530 48.3151 0.2185" 0.1133 1.4621 1.2616 63.2165 0.072ga 0.0341 0.7300 0.2900
0.0534 1.1797 1.2580
0.0665'
56.0661
65.4197 0.1298' 0.0434 0.5201 0.1686
60.3354 0.1520" 0.1266 0.8140 2.2869
37.4974 48.9984
Average
35.6442
40.1990
30.226 1
-
37.0527 0.4447
32.4193
32.2067 0.2126
Rat 5 47.6475 1.3509
Rat 4 35.3608 0.2834
Rat 3 39.9622 0.2368
Rat 2
30.0862 0.1399
"Below Minimal Detectable % Treatment.
Subtotal Total Recovery
Subtotal Absorbed Bile Carcass GI Tract Tissue Liver Urine
Nonabsorbed Feces GI Tract Contents
Rat 1
% Recovery
Percent Treatment Radioactivity Recovered in Urine, Feces, Bile, and Tissues of 5 Male Rats Treated Orally via Stomach Cannula at a Dose of 1 mgkg Lasalocid-''C Sodium During the 48-Hour Withdrawal Period
TABLE 2
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796
LAURENCOT
Table 3 presents the data for the distribution of radioactivity in liver tissues derived from Ia~alocid-~~C-fed chickens treated at 0.0075% in the feed for 21 days. The tissue was homogenized in a solution of absolute ethanol-3A:chloroform:1NHCI; 155: I ; v/v/v, and exhaustively extracted with 95% ethanol-3A. The ppm fresh weight, ppm, and % radioactivity found in the ethanol-soluble and in the ethanol-insoluble fractions are given for 4 days of the withdrawal period. At zero day (0-hr) withdrawal 64% of the radioactivity was found in the ethanol-soluble fraction and 36% in the ethanol-insoluble fraction. At days 1-4 withdrawal, the reverse is true. Here the majority of the radioactivity is found in the ethanolinsoluble fraction. This is an indication of a change in the nature of the radioactive residues found in the livers with time. Table 4 compares the concentration and bioavailability of radioactive liver residues derived from Ia~alocid-~~C fed to chickens which were treated at 0.0075% and 0.0125% in the feed for 21 days. The concentration of radioactivity found in the liver of birds treated with la~alocid-'~C at 0.0075% in the feed was 4.05 ppm at 0-hr withdrawal, and 14.3% of this radioactivity was bioavailable in the rat model. In the birds treated at 0.0125% in the feed, the livers contained 11.93 ppm at 0-hr withdrawal, and 18.9% of this radioactivity was bioavailable. The concentrations found in the livers at 0-hr withdrawal for the two treatments not only show the expected dose response to liver concentration, but also an increase in the percent radioactivity that is bioavailable in the rat with increased dose. This would indicate a higher percentage of radioactivity in the soluble fraction at 0-hr withdrawal. The data presented in Tables 3 and 4 show a rapid decrease in the concentration of radioactivity in the whole liver, in the percent of radioactivity that is bioavailable to the rat,
TABLE 3 Distribution of Radioactivity in Liver Tissues Derived from Lasalocid14C-Fed Chicken Livers at 0.0075% in the Feed for 21 Days Ethanol-soluble fraction
Ethanol-insoluble fraction
Withdrawal day
PPm fresh weight
ppm
%
PPm
%
0 I 2 3 4
3.84 I .75 1.29 1.36 0.95
2.46 0.55 0.43 0.36 0.25
63.9 31.4 33.3 26.5 26.3
I .39 I .20 0.86 1 .OO 0.70
36. I 68.6 66.7 73.5 73.7
797
BIOAVAILABILITY AND BIOACTIVITY
TABLE 4
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Comparison of the Concentration and Bioavailability of Radioactive Liver Residues Derived from Lasal~id-'~C Fed to Chickens Treated at 0.0075% and 0.0125% in the Feed for 21 Days Withdrawal time (days)
PPm fresh weight
% Not
bioavailable
% Bioavailable
PPm fresh wt. bioavailable
1. 0.0075% in Feed (5.30 mglkglday), 37 days pretreatment
0
4.05
85.68
14.32
0.58
11. 0.0125% in Feed (9.69 mglkglday), 34 days pretreatment
0 1 2 3 4
I I .93 2.63 1.72 1.59 1.37
5
1.15
81.10 93.43 97.57 97.86 97.48 98.28
18.90 6.57 2.43 2.14 2.52 1.72
2.25 0.17 0.04 0.03 0.03 0.02
and the percent of radioactivity that is found in the ethanol-soluble fraction during the first 24 h of withdrawal. During days 2-5 of withdrawal, both treatments show a low, similar concentration of radioactivity in liver and a much slower elimination of radioactivity with time. Just over 2% of the total radioactivity in the livers are bioavailable in the rat model during this withdrawal period where approx. 30% of the total radioactivity is found in the ethanol-soluble fraction and 70% in the ethanol-insoluble fraction. Figure 5 shows the concentration of 14C-lasalocidand its possible metabolites found in the liver of chickens treated for 21 days with medicated feed containing ''C-lasalocid at a concentration of 125 ppm in the feed and during the 5-day withdrawal period. The total liver radioactivity, as ppm, is plotted for 0, I , 2, 3, 4, and 5 days withdrawal. There is a distinct biphasic withdrawal curve. In the first phase, 0-24 h, the half-life of elimination of the total radioactivity (t,,J is I I h. In the second phase the t1,2 is 6 days. The calculated ppm for that fraction of the first phase which is rapidly eliminated is given in hours and ppm by subtraction of the second phase. This rapidly eliminated fraction has a half-life of elimination of radioactivity (tIl2) of 6.3 h and has a correlation to solubility and bioavailability of the residues. Figure 6 shows again the biphasic curve for the total concentration of radioactivity in the 0-5-day withdrawal chicken livers and the curve and data for the bioavailability of the total liver residues. The data for the solubility and the bioavailability shows the amount absorbed to be associated
798
LAURENCOT
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\
t,,=ll
hr
I
0.5
‘
0
1% t 1n=6.3tr
1 1
1
I
I
1
2
3
4
WITHDRAWAL TIME
(DAYS)
FIG. 5. The concentration of la~alocid-’~C and its possible metabolites found in the liver of chickens during the 5-day withdrawal period. with the soluble fraction. Nineteen percent (19%)of the total radioactivity of the zero-day withdrawal livers was absorbed. Just over 2% of the 25-day withdrawal liver radioactivity was absorbed. Table 5 presents the data used for the determination of the half-life of elimination of the radioactivity from the chicken livers during the 5-day withdrawal period. The data for the determination of the half-life of elimination of the radioactivity absorbed from these livers when fed to rats are also given. The concentrations used to determine the half-life of elimination of radioactivity for the first phase of elimination, day 0 and day 1 of withdrawal, and the second phase of elimination, days 2-5, were the assayed values for both the chicken liver and rat bioavailability data. The fast phase of elimination is that fraction of the first phase remaining after subtraction of the second phase contribution to total concentration. The concentrations of the second phase at 0- and I-day withdrawal are calculated by linear regression analysis from the 2-5-day assay data. The assay data, linear regression analysis of the second phase, and the calculated fast phase fractions of the first phase of elimination are plotted in Fig. 7 for the chicken liver and rat bioavailability data.
5
BIOAVAILABILITY AND BIOACTIVITY
799
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A
0 t Y
0.01
' 0
I
I
I
I
I
1
2
3
4
5
WIll-DRAWAL TIME
(DAYS)
FIG. 6. The concentration of la~alocid-'~C and its possible metabolites found in the liver of chickens and its concentration found bioavailable in the rat during the 5-day withdrawal period. The radioactivity of the chicken livers had a half-life of elimination in the first phase of 1 1 h, in the second phase 6 days, and in the fast phase 6.3 h. The radioactivity of the chicken livers bioavailable in the rat model had a half-life of elimination in the first phase of 6.5 h, in the second phase 4 days, and in the fast phase 5.8 h. The rates of elimination of radioactivity from the fast phase of elimination in the chicken livers (6.3 h), and the first phase (6.5 h), and fast phase (5.8 h) of the radioactivity of the chicken liver that were bioavailable in the rat model are almost identical. The data shows that almost all of the radioactivity absorbed by the rat when fed these chicken livers is derived from the soluble fraction of the chicken livers. The radioactivity of this fraction is rapidly eliminated in the chicken livers and greatly reduced in bioavailability to the rat during the first 24 h of withdrawal. The half-life of elimination of the fast phase of bioavailable radioactivity is 5.8 h. Table 6 gives a comparison of the concentration and bioavailability of radioactive whole liver tissue residues derived from ''C-lasalocid fed to steers which were treated at 0.5, 1.0, and 2.0 mg/kg/day for 14 days. The treatment was administered twice a day by capsule. The withdrawal times are from the last capsule treatment. When the steers were treated at a dose of 0.5 mg/kg/day, the liver contained radioactivity equivalent to 4.5 ppm lasalocid or its possible metabolites. At a dose of 1.0 mg/kg/day, the liver
800
LAURENCOT
TABLE 5 '*C Residues in Chicken Liver: Total and Bioavailable ppm During a 5-Day Withdrawal Period
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ppm Liver-I4C residues Total ppm
Withdrawal time (days) 0 1 2 3 4 5 tl/Z(h)
t,,,(days)
Bioavailable ppm
Linear regression analysis phases
-
Linear regression analysis phases
Assayed
1st
2nd
Fast
Assayed
1st
2nd
Fast
11.93 2.63 1.72 1.59 I .37
11.93 2.63
9.80 0.69
0.06 0.05 0.04 0.04 0.03 0.02
2.20 0.12
-
2.25 0.17 0.04 0.03 0.03 0.02
2.25 0.17
1.15
2.13 1.94 1.75 1.55 1.36 1.17
-
11.0
-
6.3
6.5
6.0
-
-
-
-
-
-
-
-
-
4.0
contained 5.9 pprn and at a dose of 2.0 mg/kg/day, the concentration of radioactivity was 10.2 pprn showing a dose response. The percent of radioactivity of the steer liver absorbed by the rat was 21.6 (0.5 mg/kg/day), 23.2 ( I .O mg/kg/day), and 27.1 (2.0 mg/kg/day). This increase in the percent of radioactivity bioavailable in the rat model with increase in dose to
TABLE 6 Comparison of Concentration and Bioavailability of Radioactive Steer Liver Residues in the Bile Cannulated Rat" Liver Withdrawal time Dose (mg/kg)
Days
+H
Steer
PPm fresh weight
% Not bio-
% Bio-
available
available
ppm fresh wt/bioavailable
78.44 76.83 72.88
21.56 23.17 27.12
0.965 1.364 2.765
~~
0.5 I .o 2.0
0 : 17 0 : 19 0 : 20
II 18 16
4.476 5.886 10.196
asteen were treated with lasal~cid-'~C at 0.5, 1.0, and 2.0 mg/kg/day for 14 days.
BIOAVAILABILITY AND BIOACTIVITY
80 1
-
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-c
FRESHWT.
.,y;.,, -\:.,,
0.1
,= =6.5
/t
.\?. A*
:
TOTAL
hr
-L
..*. FAST PHASE
t ,m=5.8
__ __ __ -~
Cl---------..r~-
SECOND PHASE
hr
810-, AVAILABLE_ _ -
I I -.-
+FRESHWT. SECOND PHASE FASTPHASE
-
. -.-.
0.01
'
0
112
=4 days
~.-
I
I
I
I
I
1
2
3
4
5
WITHDRAWAL TIME
(DAYS)
FIG. 7. Comparison of depletion rates of total chicken liver radioactivity and its bioavailable radioactivity when fed to rats during the 5-day withdrawal period. the steers was also seen with chickens. This increase is associated with the soluble fraction of the liver. Table 7 gives the data for the bioavailability of the fractionated steer liver radioactivity derived from steers treated at 1 mg/kg/day with ''C-lasalocid for 14 days and sacrificed 19 h after the last capsule treatment. The ethanolinsoluble fraction contained 18% of the total radioactivity. Five percent (5%) of this fraction was absorbed or 0.9% of the total radioactivity. The ethanol-soluble fraction contained 82% of the total radioactivity. Forty percent (40%) of this fraction was absorbed or 33% of the total radioactivity. In the data presented for the chicken and steer liver residues derived from ''C-lasalocid, approximately 75% of the total radioactivity is not absorbed, and 25% is absorbed at 0-h withdrawal. This is much less than the absorption of the parent compound. There is a very rapid elimination of the absorbable fraction of the total radioactivity in the first 24 h of withdrawal to a level of approximately 6%. The absorption of radioactivity after 24 h withdrawal was approximately 2% of total residues for the 5-day withdrawal period. The low absorption of total residues and the usual withdrawal times for slaughter, as followed in industrial practice, should allow for appropriate
LAURENCOT
802
TABLE 7 Bioavailability of the Radioactivity of the Ethanol-Soluble and the Ethanol-Insoluble Fractions of Steer Liver'
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Percent
Individual tissue fractions Ethanol-insoluble Ethanol-soluble Tissue fraction contribution as percent of 1 g tissue Ethanol-insoluble Ethanol-soluble Total contribution percent
Absorbed
Nonabsorbed
Total
4.90 40.20
95.10 59.80
100.00 100.00
0.89 32.89
17.28 48.94
18.17 81.82
33.18
66.22
100.00
"The steer was treated at 1.0 mg/kg/day for 14 days and sacrificed 19 h after the last capsule treatment. adjustments to be made to the minimal levels of residues remaining in the tissues of animals intended for human consumption.
REFERENCES [ I ] A. MacDonald, G. Chen, P Duke, A . Popick, R. Saperstein. M. Kaykaty, C. Crowley, H. Hutchinson, and J. Westheimer, in Densiromerry in Thin-Layer Chromatography (J. C. Touchstone and J. Sherma, eds.), Chapter 9, Wiley-Interscience, New York, 1978, pp. 20 1-222. [2] G. Weiss and A. MacDonald, J. Assoc. O f . Anal. Chem., 68, 971 ( 1985). [3] H. E. Gallo-Torres, J. Toxicol. Envir. Healrh, 2 , 827 (1977).
