A Dose-Ranging Study of the Pharmacokinetics of Codeine Phosphate following Intravenous Administration to Rats

JAYMIN SHAH’*’ AND WILLIAM D. MASON* Received July 24, 1989, from the *University of Missouri-Kansas City, Schools of Pharmacy and Medicine, 241 1 Holmes Street, Kansas City, MO 64708. Accepted for ublication April 25, 1990. 3401 Hillview Avenue, A3-165, Palo Alto, C!A 94304.

*Present address: Syntex Research, Department of Drug Metabolism,

Abstract 0 The linearity of the pharmacokinetics of codeine was exam- ined in male Sprague-Dawley rats given iv bolus doses of 1, 1.5, 3, and 4 mglkg of codeine phosphate. Codeine and morphine were determined in serial blood samples utilizing HPLC with electrochemical detection. Codeine exhibits characteristics consistent with a two-compartment pharrnacokinetic model. The kinetics of codeine are linear in the iv dose range 1 4 mg/kg. The ratio AUC,,,,,,,,:AUC,delne increases dispro- portionately with increasing doses of codeine.

Codeine has been used extensively as an analgesic and antitussive for about one and a half centuries. The suggestion that codeine m a y exert its moderate analgesic effect by biotransformation t o morphine was first proposed in 1948.1 This hypothesis is fur ther supported by the low affinity of codeine for the opiate receptors when compared wi th that of morphine.2 The biologic disposition of codeine in various experimental animals after relatively high doses has been extensively studied.3 Studies done in male rats have shown that about half the dose of codeine undergoes O-demethyl- ation.4 The morphine formed by metabolic conversion of codeine has been found i n plasma, urine, bile, and feces.”7 Morphine has also been determined t o be present in the brain of rats following large doses of codeine.8

There are n o animal d a t a in the l i terature for the concen- t ra t ions of morphine following administration of relatively low doses of codeine; this is mainly due t o lack of analytic techniques of sufficient sensitivity and specificity. Early studies relied on a colorimetric assay which is not very sensitive.9 Recent reports of the application of gas chroma- tographylo and gas chromatography-mass spectrometry11 appear promising b u t m a y be somewhat cumbersome in their application for analysis of a large number of samples. A more recent assay for codeine using radioimmunoassay12 lacks specificity between the drug a n d i t s major metabolite. We have recently reported an HPLC method13 for direct estima- tion of codeine and morphine in rat whole blood.

The a i m of the present investigation w a s therefore to conduct an iv dose-ranging s tudy of codeine in rats to establish the pharmacokinetic parameters of codeine and morphine and also to investigate whether or not the phar- macokinetics of codeine are dose dependent.

Experimental Section Animal Experiments-Male Sprague-Dawley rats, weighing 250-

300 g, were used throughout the study. The surgical and serial blood sampling procedures reported by Upton14 and Waynforth’s were adapted with minor modifications. The method involved exterioriza- tion of the right jugular vein by means of a cannula (silicon tubing, 0 . 0 2 id . ) which travelled subcutaneously from the vein and emerged at the back of the neck. The cannula was used to inject drug intravenously and also to withdraw serial blood samples for the pharmacokinetic studies. The technique is well suited to a wide range

of experimental situations requiring multiple samples. The animals were allowed to recover from surgery for 72 h, during which time they had free access to food and water. Three animals were used in each dose group. Each group received one of the four doses of codeine phosphate (1.0, 1.5, 3.0, or 4.0 mgkg) in solution via the indwelling cannula on the fourth day after surgery. The cannula was flushed with normal saline after drug administration. Blood samples (120 4) were withdrawn at 0, 1,3,5,9,12,15,20,30,40,50,60,90,120,150, 180, and 240 min after drug administration. Sampling withdrawal was immediately followed by fresh blood replacement from a donor animal in order to maintain the hematocrit levels. Aliquots (100 pL) for whole blood analysis were withdrawn from the collection tube into a 15-mL conical centrifuge tube containing a drop of heparin (5 units/mL). Tubes were capped and mixed immediately by vortexing, and they were stored at -30 “C.

The specimens were analyzed using a previously reported13 HPLC assay procedure for simultaneous determination of codeine and its metabolites in rat whole blood using electrochemical detection. Sample volume was increased beyond the 2-h collection to increase the sensitivity of the assay. The sensitivity of the assay was 2 ng/mL for both compounds.

Sample Analysis-Whole blood samples containing the internal standard (dihydromorphinone) were extracted at pH 8.9 into a 298 1-butano1:methyl tertiary butyl ether organic solvent system and back extracted into 25 mM phosphate buffer. One hundred-microliter samples were injected onto the column. Calibration standards were prepared in a similar fashion and injected along with the samples. Analysis was done using a 5-pm phenyl column with detection by electrochemical oxidation a t + 1.2 V versus Ag/AgCl.

Pharmacokinetic Analysis-Initial parameter estimates for com- partmental model pharmacokinetic analysis were obtained by curve stripping the whole blood concentration versus time curve for codeine. The concentration versus time data were then fitted by nonlinear regression analysis16 to two- and three-compartment pharmacoki- netic models. During the fitting procedure, each concentration was weighted by the reciprocal of its square. Goodness-of-fit was evalu- ated using statistical methods [Akaike’s Information Criteria ( A I W and the F-test181. Included was also the analysis of residuals for randomness and normality and visual examination of the nonlinear regression plots. In this approach, the number of exponential terms is selected on the basis of the residual sums of squares and the number of parameters according to the “principle of parsimony.” Total body clearance (CL) was computed by dividing the dose by AUC,. Volume of distribution (Vd,) was calculated by dividing CL by the terminal elimination rate constant (PI . Mean residence time and Vd,, for codeine were calculated using statistical moments.19

Maximum blood concentration (Cb,max) and time to maximum concentration (tmax) for morphine were determined from visual inspection. The areas under the whole blood concentration versus time curve (AUC) for parent drug and morphine were calculated following trapezoidal method up to the last determined whole blood concentration and the ratios were compared.

Statistical evaluations were performed using one-way analysis of variance (ANOVA) to compare mean values of codeine among different dose groups.

Results A typical semilogarithmic plot of codeine and morphine

blood concentrations versus time following a 3-mg/kg iv dose

0022-354919 1/0300-0229$01.00/0 0 7991, American Pharmaceutical Association

Journal of Pharmaceutical Sciences / 229 Vol. 80, No. 3, March 7991

of codeine phosphate is shown in Figure 1. Pharmacokinetic parameters of codeine and morphine related to iv dosing are summarized in Table I.

An open two-compartment pharmacokinetic model was found to be adequate to describe the whole blood concentra- tion of codeine in the rat. All animals, except three, exhibited a biphasic decline in the concentration-time profile. The area under the concentration-time curve ranged from 9.1 to 35.2 pg * min/mL and was linear with dose (Figure 2). The corre- lation coefficient was 0.999. The CL was 6.6 5 1.8,7.2 * 1.8, 6.6 2 1.1, and 6.1 t 0.8 Ukgh for 1-, 1 .5 , 3-, and 4-mglkg doses of codeine phosphate, respectively, and was not signif- icantly different among doses (p r 0.05). The average value of p was 0.021 2 0.006 min-'. The mean Vd, was 5.49 * 1.11 L * kg-' and appeared to be dose independent. The mean residence time and Vd,, for codeine averaged 36.3 2 7.3 min and 3.96 t 0.80 L kg-', respectively.

Codeine is rapidly demethylated to morphine in the rat following iv dosing, as indicated by the appearance of mor- phine in the blood in the first few samples following dosing. The maximum blood concentration of morphine ranges from 5.8 to 76.8 ng/mL, and the mean t,,, for maximum morphine concentration is 3.2 min. The ratio AUC,, hine:AUCedeine increases from 0.009 to 0.115 with increasing2oses ofcodeine.

Discussion The pharmacokinetics of codeine is best described by a

two-compartment open system in the rat. The codeine is administered directly into the central compartment compris- ing the rapidly perfused tissues. Following administration, codeine is distributed between the central and peripheral compartments, and is assumed to be metabolized and elimi- nated from the central compartment. The average CL of 6.6 L * h-' * kg-' is about twice the liver blood A o w , ~ ~ indicating a significant amount of extrahepatic elimination of codeine. The mean Vd value 5.5 L * kg-' suggests extensive distri- bution of the &ug into the extravascular region. The consist- ency of clearance at increasing dose levels of codeine admin- istered to rats shows dose proportionality with the area under the curve.

Peak blood metabolite concentrations, -0.9 to 4.8% of the peak codeine blood concentrations, were found in the samples collected a few minutes after iv codeine administration. This indicates a capacity for rapid metabolic conversion of the parent drug that is limited by tissue uptake. Thereafter, metabolite concentrations decreased more slowly than co- deine concentrations.

A few minutes after iv codeine dosing, the codeine concen- trations are relatively high compared with morphine concen- trations, but 1-2 h after the dose, the ratio of morphine to

1E4

1 0 25 50 75 100 125 150 175 200

TIME (MINUTES)

Figure 1-Typical plot of experimental blood codeine (0) and morphine (A) concentrations following a 3.0-mgkg iv dose. The solid line indicates the nonlinear least squares regression fitted curve.

codeine rises sharply. This change may be due to distribution of morphine and codeine with a slower release of morphine from some tissues. Animal studies have shown the presence of morphine in serum for long periods after administration,21 suggesting extensive tissue uptake or enterohepatic recircu- lation22 of the drug. Examination of the metabo1ite:parent drug AUC ratio (Figure 2) shows disproportionate changes in concentration of metabolite following the higher dose of codeine. The data indicate increase in morphine concentra- tion following higher doses of iv codeine. Morphine is sequen- tially metabolized to morphine glucuroniden and also under- goes enterohepatic recirculation due to bacterial hydrolysis in the gUt.22 Increased morphine concentration following higher doses of iv codeine may be due to maturation of morphine metabolism. An alternate hypothesis is that codeine de- creases gastrointestinal tract motility, thereby increasing the transit time of morphine glucuronide in the intestinal tract. This allows longer exposure to bacterial glucuronide hydrol- ysis which favors enterohepatic recycling. Cascade design experiments in rats have shown that -1&30% of morphine excreted in the bile is reabsorbed from the intestinal tract, and the recycling continues until the dose is completely eliminated.22.24

In summary, it can be stated that codeine obeys linear kinetics at iv doses between 1 and 4 mg/kg. Codeine is rapidly demethylated to morphine in the body. There is a dispropor- tionate increase in the area of morphine and the ratio AUCmorphine:AUCcodeine with increasing doses of codeine.

Table CPharmacokinetic Parameters Obtalned from Whole Blood Codeine and Morphine a8 a Functlon ol Dose

Dose, mg/kg" 1 1.5 3 4

Codeine

Parameter

p, min ~ ' 0.024 2 0.01 1 0.019 -+ 0.004 0.022 t 0.001 0.018 t 0.002 CL, Uh/kg 6.6 t 1.8 7.2 t 0.8 6.6 t 1.1 6.1 2 0.8

Vd,, Ukg 4.9 -t 1.3 6.3 t 1.1 4.9 t 0.9 5.8 2 0.9 M ~ T , min 36.1 -+ 11.5 38.2 2 4.6 33.4 2 7.0 37.8 rt 8.5 Vd** Ukg 3.7 f 0.6 4.6 2 0.4 3.6 t 0.1 3.9 2 1.4

G,maxt WmL 5.8 '' 1.2 25.6 2 2.1 56.1 t 15.1 76.8 t 29.6 Lax, min 5.7 2 8.1 2.3 t 1.2 3.0 t 2.0 4.0 t 4.4 AUC,,,,,, pg. min/mL 0.079 t 0.024 0.363 t 0.083 2.39 t 0.35 3.85 2 1.61

AUC,,, pg . min/mL 9.5 2 2.4 12.5 t 1.3 27.8 t 4.7 39.7 -+ 5.6

Morphine

AUCm:AUC, 0.009 2 o.Oo0 0.031 t 0.007 0.09 t 0.01 0.12 2 0.02

a Mean -+ SD of three animals in each dose group.

230 / Journal of Pharmaceutical Sciences Vol. 80, No. 3, March 1997

5 00E4

3 4 DOE4 z \ n I 6 300E4 z w

W 2 0 0 E 4 tl 0 0 0 3 4 I DOE4

v

z

0 00 0 1 2 3 4

DOSE (MG/KC)

Flgure 2-Plot of total AUC of codeine as a function of dose (mglkg). The inset shows a plot of the ratio AUC,,,ab,lle:AUCd,.g as a function of iv dose (rng/kg) of codeine.

References and Notes 1. Sanfilippo, G. Boll. Soc. Ztal. Biol. Sper. 1948,24, 723-726. 2. Pert, C. B.; Snyder, S. H. Proc. Natl. Acad. Sci. U S A . 1973,70,

3. Way, E. L.; Adler, T. K. Bull. WHO 1962,26, 51-66. 4. Yeh, S.Y.; Woods, L. A, J. Pharmacol Exp. Ther. 1969, 166,

5. Johannesson, T.; Schou, J. Acta Pharmacol. Toxicol. 1963, 20

2243-2247.

86-95.

165-173.

6. Johannesson, T.; Woods, L. A. Acta Pharmacol. Toxicol. 196421,

7. Yoshimura, H.; Mori, M.; Oguri, K.; Tsukamoto, H. Biochem. Pharmacol. 1970,19,2353-2360.

8. Dahlstrom, B.; Paalzow, L. Opiates and Endogenous Oprord Peptides; Kosterlitz, H. W., Ed.; North Holland Publishing: Amsterdam, New York, Oxford, 1976, pp. 395398.

9. Woods, L. A.; Muehlenbeck, H. E.; Mellett, L. B. J . Pharmacol.

381-396.

1956,116,117-125. 10. Brunson, M. K.; Nash, J. F. Clin Chem. 1975,21, 1956-1960. 11. Ebbighausen, W. 0. R.; Mowat, J. H.; Vestergaard, P.; Kline,

N. S. Adv. Biochem. Psychopharmacol. 1973, 7 , 135-146. 12. Findlay, J. W. A.; Butz, R. F.; Welch, R. M. Fed. Proc. 1976,35,

531. 13. Shah, J.; Mason, W. D. Anal. Lett. 1987, 9, 1493-1501. 14. Upton, R. A. J. Pharm. Sci. 1975,64, 112-114. 15. Waynforth, H. In Experimental and Surgical Techniques in the

16. PCNONLIN, Statistical Consultants, Inc. (SCI), Lexington, KY. 17. Yamaoka, K.; Nakagawa, T.; Uno, T. J. Pharmacokinet. Bio-

18. Boxenbaum, H. G.; Riegelman, S.; Elashoff, R. M. J. Pharmaco-

19. Gibaldi, M.; Perrier, D. Pharmacokinetics, 2nd ed.; Marcel Dek-

20. Bischoff, K.; Dedrick, R.; Zaharko, D.; Longstrength, J. J . Pharm.

21. Spector, S . J. Pharmacol. Exp. Ther. 1971,178, 253-258. 22. Walsh, C.; Levine, R. J. Pharmacol. Exp. Ther. 1975, 195,

23. Iwamoto, K.; Klassen C. J. Pharmacol. Ezp. Ther. 1977, 200,

24. Shah, J.; Mason, W., unpublished results.

Rat; Academic: London, 1980.

pharm. 1978,6, 165-175.

Kinet. Biophurm. 1974,2, 123-148.

ker: New York, 1982; pp 409-417.

Sci. 1971, 60, 1128-1133.

303-310.

236-244.

Journal of Pharmaceutical Sciences I 231 Vol. 80, No. 3, March 1991