Codeine kinetics as determined by radioimmunoassay Radioimmunoassay (RIA) was used to determine several pharmacokinetic parameters of codeine in man, including the relative bioavailability after oral and intramuscular administration. The study followed a crossover design in 6 healthy, young (/8 to 21 yr), male volunteers. Three subjects received 65 mg codeine phosphate orally in an analgesic mixture which also contained aspirin, phenacetin, and caffeine. At the same time a similar group received an equivalent dose of codeine phosphate in a single intramuscular injection. Two weeks later the study was repeated so that each group received the alternate treatment. Plasma samples were collected at various times after drug administration, and codeine concentrations were determined by a specific RIA procedure. The procedure can detect less than 50 pg of codeine. Following intramuscular administration, peak plasma concentrations (194 to 340 nglm!) were observed between 0.25 to 1 hr; after oral dosing, peak codeine plasma concentrations (102 to 140 nglmt) appeared within 0.75 to I hr. The mean plasma t1;2 and volume of distribution of codeine following intramuscular injection were 3.32 hr and 5. I Llkg, respectively. Oral, relative to intramuscular, bioavailability qf codeine, based on areas under the codeine plasma curves, was 42% to 71% (mean, 53%).
John W. A. Findlay, Ph.D., Robert F. Butz, A.B., and Richard M. Welch, Ph.D. Research Triangle Park, N. C. Department of Medicinal Biochemistry, Wellcome Research Laboratories
Although codeine has long been established as a drug for the relief of moderate pain, little is known about its pharmacokinetics and bioavailability in man. The disposition of codeine in animals after relatively high doses has been extensively studied. 22 Higher concentrations of the drug in urine following hydrolysis of codeine conjugates have facilitated the quantitation of codeine in this fluid,t7. 19 but due to the lack of a sensitive analytic method data Presented in preliminary form at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, New Orleans, La., August, 1976. Received for publication Nov. 13, 1976. Accepted for publication May 27, 1977. Reprint requests to: John W. A. Findlay, Ph.D., Wellcome Research Laboratories, Department of Medicinal Biochemistry, 3030 Cornwallis Road, Research Triangle Park, N. C. 27709.
on plasma or serum concentrations in man after administration of therapeutic doses are limited. Recent approaches to this problem by gas chromatograph y3, 7. 10. 17. 18. 23. and gas chromatography-mass spectrometry 4 appear promising but may be somewhat cumbersome in their application to large numbers of samples. A more recent report of a relatively sensitive thin-layer chromatographic procedure for codeine appears more promising. 8 Radioimmunoassay methods are gaining increasing acceptance as sensitive and specific methods for the quantitation of drugs in biologic fluids. l l We here report the application of a recently developed radioimmunoassay procedureS to a study of the disposition of codeine in man after oral and intramuscular administration.
439
440
Findlay, Butz, and Welch
Clinical Pharmacology and Therapeutics
Table I. Cross-reactivities of various drugs and metabolites with anticodeine-6hemisuccinate-BSA antiserum
Compound
Concentration required to inhibit zero binding* by 50% (ng/ml)
Percentage crossreactivityt
3.4 2.9 4.7 16.5 52.0 72.0 290 1,200 2,500 >10,000 >10,000
100 117 72 21 6.5 (16.n) 5 (7.6+)
Codeine Acety\codeine Ethylmorphine Dih ydrocodeine Morphine Codeine-6-glucuronide Levorphanol Morphine-3-g1ucuronide Norcodeine Nalorphine Meperidine
I
0.3 0.1
"Binding of tracer to antiserum in absence of cold drug. tI.o-CodeineiI.o-A X 100 = percent cross-reactivity for drug A. 16 hr incubation at 0° C. :j:Percentage cross-reactivity for I to 2 hr incubation at 0° C.
Materials and methods
Chemicals. Dichloromethane (glass distilled) was supplied by Burdick and Jackson, Muskegon. Ethanol (absolute) was distilled in glass prior to use. Ethylmorphine hydrochloride (Dionin), morphine, and nalorphine were obtained from Merck & Co. Morphine-3glucuronide and codeine-6-g1ucuronide were supplied by Applied Science Laboratories, Inc. Levorphanol was purchased from Hoffmann-La Roche Inc., and meperidine (Demerol) was obtained from Winthrop Chemical Co. Acetylcodeine was prepared from codeine by acetylation with acetic anhydride in pyridine and norcodeine was synthesized by the method of Von Braun. 21 Subjects. Six normal healthy male subjects, average age, 20 yr (18 to 21), and average weight, 74.1 kg (68.0 to 83.9) participated in the study. None of the subjects had a history of smoking cigarettes (or related materials), and no drugs were taken during the week prior to beginning the study. Food was withheld from subjects for 12 hr preceding administration of drug, and for the ensuing 2-hr period. All caffeine-containing food, drinks, and drugs were withheld for 24 hr preceding drug administration. Crossover protocol. Three subjects (Group A) received 65 mg of codeine phosphate orally in two analgesic tablets which also contained
455 mg aspirin, 325 mg phenacetin, and 65 mg caffeine (Empirin Compound with codeine phosphate No.3). The remaining 3 subjects (Group B) received the same dose of codeine phosphate in saline intramuscularly (deltoid muscle); 2 wk later the groups crossed over, and Group B now received the same dose of codeine phosphate orally in the two tablets while the drug was administered intramuscularly to Group A. On each occasion, blood was collected from each subject prior to drug administration and at 0.25,0.5,0.75, I, 1.5,2,4,6, 8, 12, 24, and 48 hr after dosing. Blood was collected into ethylenediaminetetraacetic acid (EDT A)-containing tubes and plasma was separated and stored at -800 C until assayed for codeine. Assay procedure. The buffer used throughout was 0.05 M in KH 2P04 , 0.9% NaCl, 0.01 M in sodium EDTA, 0.01% ethyl mercurithiosalicylic acid (sodium salt), pH 7.40 (phosphate-buffered saline, PBS), containing the concentrations of bovine serum albumin (BSA) indicated. 3H-Codeine (specific activity, 23 Ci/mmole) was supplied by Amersham/Searle. Charcoal was Schwarz-Mann RIA grade. Radioactivity was quantitated in Riafluor scintillation cocktail (New England Nuclear) on a Packard Model 3320 Tri-Carb liquid scintillation spectrometer. The development of the codeine radioimmunoassay procedure has been
Codeine kinetics
Volume 22 Number 4
described. 5 * The antiserum used in this study was raised to a conjugate of codeine-6hemisuccinate with BSA. The antiserum, which was used at a final dilution of I: 5,000, had similar specificity characteristics to those previously described. 5 Plasma (0.5 ml) was adjusted to pH 14 by addition of 1.0 N NaOH (lOOJLI) and extracted twice with 2.0 ml. of dichloroethane/ethanol (95: 5). The combined organic layers were extracted with 0.05 N H2 S04 (1.0 ml), the acid layer was separated, adjusted to pH 14 with I N NaOH (0.2 ml), and extracted again with the dichloroethane/ethanol mixture (2.0 ml). The organic layers were evaporated to dryness under a stream of dry nitrogen. The residue was dissolved in absolute ethanol (0.5 ml) with vigorous vortex mixing. Solvent was again evaporated as above, the residue was dissolved in absolute ethanol (50 JLI), and the total volume was adjusted to 0.5 ml by addition of 1% BSA-PBS buffer. When necessary, samples were diluted into the range of the assay standard curve with 1% BSA-PBS after extraction. 3H-Codeine diluted in 1% BSA-PBS (250 pg in 200 JLl) was added to either standard codeine (24 pg to 3.12 ng) or unknown buffer extract in 1% BSA-PBS (100 JLI) in 12 x 75 mm polystyrene tubes in an ice water bath. Diluted antiserum (200 JL I in 1% BSA-PBS) was added, the tubes were vortexed, and the contents were incubated at 4° C for 2 hr. Antibody-bound label was separated from free with BSA-coated charcoal and the bound fraction was quantitated in Riafiuor ~ previously described. 5 Background in the assay was measured in tubes in which antiserum was replaced by 1% BSA-PBS, and maximum binding of label in the absence of any codeine standard (Co) was measured by replacing the standard or unknown solution by an aliquot of 1% BSAPBS. Standard curves were expressed as % Co: concentration of standard added on a linear vs log scale. All assay points were run in duplicate. Similar procedures were used in crossreactivity studies, but standard codeine solution (100 JLl) was replaced by aliquots (100 JLI) of solutions of the various compounds in 1% BSA-PBS. Standard curves for these com'Full publication of abstract in Reference 5 is in preparation.
441
100r,----------------------------~
80
60
8 ~
40
20
0.1
lO
10.0
CODEINE CONC. (ng/ml)
Fig. 1. Typical standard curve for codeine radioimmunoassay. Mean± SEM for 11 curves.
pounds were expressed as for codeine, and cross-reactivities were calculated as shown in Table I. Hydrolysis of codeine conjugates in plasma . .B-Glucuronidase 5,000 U, (Miles Research Division; 2,020 Fishman units .B-glucuronidase/mg and 6,200 U aryl sulfatase/ mg), in 0.5 ml of 0.2 M sodium acetate buffer, pH 5.0, was incubated with 0.5 ml of plasma sample for 24 hr at 37°C. After this period, the incubation mixture was basified and extracted with solvent as described above. The plasma extracts were then analyzed by radioimmunoassay as before. Results
Assay. The specificity of this assay procedure for codeine in the presence of its metabolites has been discussed previously. 5 Crossreactivities of various drugs and metabolites with this antiserum are summarized in Table I. The antiserum used in this study recognizes minor changes in substituent on the nitrogen atom very effectively (low cross-reaction with norcodeine, nalorphine, and levorphanol), while changes in substituents on positions 3 and
Clinical Pharmacology and Therapeutics
Findlay, Bulz, and Welch
442
500
~
g400
o w
r=0.999
p
John W. A. Findlay, Ph.D., Robert F. Butz, A.B., and Richard M. Welch, Ph.D. Research Triangle Park, N. C. Department of Medicinal Biochemistry, Wellcome Research Laboratories
Although codeine has long been established as a drug for the relief of moderate pain, little is known about its pharmacokinetics and bioavailability in man. The disposition of codeine in animals after relatively high doses has been extensively studied. 22 Higher concentrations of the drug in urine following hydrolysis of codeine conjugates have facilitated the quantitation of codeine in this fluid,t7. 19 but due to the lack of a sensitive analytic method data Presented in preliminary form at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, New Orleans, La., August, 1976. Received for publication Nov. 13, 1976. Accepted for publication May 27, 1977. Reprint requests to: John W. A. Findlay, Ph.D., Wellcome Research Laboratories, Department of Medicinal Biochemistry, 3030 Cornwallis Road, Research Triangle Park, N. C. 27709.
on plasma or serum concentrations in man after administration of therapeutic doses are limited. Recent approaches to this problem by gas chromatograph y3, 7. 10. 17. 18. 23. and gas chromatography-mass spectrometry 4 appear promising but may be somewhat cumbersome in their application to large numbers of samples. A more recent report of a relatively sensitive thin-layer chromatographic procedure for codeine appears more promising. 8 Radioimmunoassay methods are gaining increasing acceptance as sensitive and specific methods for the quantitation of drugs in biologic fluids. l l We here report the application of a recently developed radioimmunoassay procedureS to a study of the disposition of codeine in man after oral and intramuscular administration.
439
440
Findlay, Butz, and Welch
Clinical Pharmacology and Therapeutics
Table I. Cross-reactivities of various drugs and metabolites with anticodeine-6hemisuccinate-BSA antiserum
Compound
Concentration required to inhibit zero binding* by 50% (ng/ml)
Percentage crossreactivityt
3.4 2.9 4.7 16.5 52.0 72.0 290 1,200 2,500 >10,000 >10,000
100 117 72 21 6.5 (16.n) 5 (7.6+)
Codeine Acety\codeine Ethylmorphine Dih ydrocodeine Morphine Codeine-6-glucuronide Levorphanol Morphine-3-g1ucuronide Norcodeine Nalorphine Meperidine
I
0.3 0.1
"Binding of tracer to antiserum in absence of cold drug. tI.o-CodeineiI.o-A X 100 = percent cross-reactivity for drug A. 16 hr incubation at 0° C. :j:Percentage cross-reactivity for I to 2 hr incubation at 0° C.
Materials and methods
Chemicals. Dichloromethane (glass distilled) was supplied by Burdick and Jackson, Muskegon. Ethanol (absolute) was distilled in glass prior to use. Ethylmorphine hydrochloride (Dionin), morphine, and nalorphine were obtained from Merck & Co. Morphine-3glucuronide and codeine-6-g1ucuronide were supplied by Applied Science Laboratories, Inc. Levorphanol was purchased from Hoffmann-La Roche Inc., and meperidine (Demerol) was obtained from Winthrop Chemical Co. Acetylcodeine was prepared from codeine by acetylation with acetic anhydride in pyridine and norcodeine was synthesized by the method of Von Braun. 21 Subjects. Six normal healthy male subjects, average age, 20 yr (18 to 21), and average weight, 74.1 kg (68.0 to 83.9) participated in the study. None of the subjects had a history of smoking cigarettes (or related materials), and no drugs were taken during the week prior to beginning the study. Food was withheld from subjects for 12 hr preceding administration of drug, and for the ensuing 2-hr period. All caffeine-containing food, drinks, and drugs were withheld for 24 hr preceding drug administration. Crossover protocol. Three subjects (Group A) received 65 mg of codeine phosphate orally in two analgesic tablets which also contained
455 mg aspirin, 325 mg phenacetin, and 65 mg caffeine (Empirin Compound with codeine phosphate No.3). The remaining 3 subjects (Group B) received the same dose of codeine phosphate in saline intramuscularly (deltoid muscle); 2 wk later the groups crossed over, and Group B now received the same dose of codeine phosphate orally in the two tablets while the drug was administered intramuscularly to Group A. On each occasion, blood was collected from each subject prior to drug administration and at 0.25,0.5,0.75, I, 1.5,2,4,6, 8, 12, 24, and 48 hr after dosing. Blood was collected into ethylenediaminetetraacetic acid (EDT A)-containing tubes and plasma was separated and stored at -800 C until assayed for codeine. Assay procedure. The buffer used throughout was 0.05 M in KH 2P04 , 0.9% NaCl, 0.01 M in sodium EDTA, 0.01% ethyl mercurithiosalicylic acid (sodium salt), pH 7.40 (phosphate-buffered saline, PBS), containing the concentrations of bovine serum albumin (BSA) indicated. 3H-Codeine (specific activity, 23 Ci/mmole) was supplied by Amersham/Searle. Charcoal was Schwarz-Mann RIA grade. Radioactivity was quantitated in Riafluor scintillation cocktail (New England Nuclear) on a Packard Model 3320 Tri-Carb liquid scintillation spectrometer. The development of the codeine radioimmunoassay procedure has been
Codeine kinetics
Volume 22 Number 4
described. 5 * The antiserum used in this study was raised to a conjugate of codeine-6hemisuccinate with BSA. The antiserum, which was used at a final dilution of I: 5,000, had similar specificity characteristics to those previously described. 5 Plasma (0.5 ml) was adjusted to pH 14 by addition of 1.0 N NaOH (lOOJLI) and extracted twice with 2.0 ml. of dichloroethane/ethanol (95: 5). The combined organic layers were extracted with 0.05 N H2 S04 (1.0 ml), the acid layer was separated, adjusted to pH 14 with I N NaOH (0.2 ml), and extracted again with the dichloroethane/ethanol mixture (2.0 ml). The organic layers were evaporated to dryness under a stream of dry nitrogen. The residue was dissolved in absolute ethanol (0.5 ml) with vigorous vortex mixing. Solvent was again evaporated as above, the residue was dissolved in absolute ethanol (50 JLI), and the total volume was adjusted to 0.5 ml by addition of 1% BSA-PBS buffer. When necessary, samples were diluted into the range of the assay standard curve with 1% BSA-PBS after extraction. 3H-Codeine diluted in 1% BSA-PBS (250 pg in 200 JLl) was added to either standard codeine (24 pg to 3.12 ng) or unknown buffer extract in 1% BSA-PBS (100 JLI) in 12 x 75 mm polystyrene tubes in an ice water bath. Diluted antiserum (200 JL I in 1% BSA-PBS) was added, the tubes were vortexed, and the contents were incubated at 4° C for 2 hr. Antibody-bound label was separated from free with BSA-coated charcoal and the bound fraction was quantitated in Riafiuor ~ previously described. 5 Background in the assay was measured in tubes in which antiserum was replaced by 1% BSA-PBS, and maximum binding of label in the absence of any codeine standard (Co) was measured by replacing the standard or unknown solution by an aliquot of 1% BSAPBS. Standard curves were expressed as % Co: concentration of standard added on a linear vs log scale. All assay points were run in duplicate. Similar procedures were used in crossreactivity studies, but standard codeine solution (100 JLl) was replaced by aliquots (100 JLI) of solutions of the various compounds in 1% BSA-PBS. Standard curves for these com'Full publication of abstract in Reference 5 is in preparation.
441
100r,----------------------------~
80
60
8 ~
40
20
0.1
lO
10.0
CODEINE CONC. (ng/ml)
Fig. 1. Typical standard curve for codeine radioimmunoassay. Mean± SEM for 11 curves.
pounds were expressed as for codeine, and cross-reactivities were calculated as shown in Table I. Hydrolysis of codeine conjugates in plasma . .B-Glucuronidase 5,000 U, (Miles Research Division; 2,020 Fishman units .B-glucuronidase/mg and 6,200 U aryl sulfatase/ mg), in 0.5 ml of 0.2 M sodium acetate buffer, pH 5.0, was incubated with 0.5 ml of plasma sample for 24 hr at 37°C. After this period, the incubation mixture was basified and extracted with solvent as described above. The plasma extracts were then analyzed by radioimmunoassay as before. Results
Assay. The specificity of this assay procedure for codeine in the presence of its metabolites has been discussed previously. 5 Crossreactivities of various drugs and metabolites with this antiserum are summarized in Table I. The antiserum used in this study recognizes minor changes in substituent on the nitrogen atom very effectively (low cross-reaction with norcodeine, nalorphine, and levorphanol), while changes in substituents on positions 3 and
Clinical Pharmacology and Therapeutics
Findlay, Bulz, and Welch
442
500
~
g400
o w
r=0.999
p
