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Acetylcodeine, an impurity of illicitly manufactured heroin, elicits convulsions, antinociception, and locomotor stimulation in mice
Drug and Alcohol Dependence 65 (2001) 37 – 43 www.elsevier.com/locate/drugalcdep
Acetylcodeine, an impurity of illicitly manufactured heroin, elicits convulsions, antinociception, and locomotor stimulation in mice Carol L. O’Neal a,1, Alphonse Poklis a, Aron H. Lichtman b,* a
b
Department of Pathology, Virginia Commonwealth Uni6ersity, Richmond, VA 23298 -0613, USA Department of Pharmacology and Toxicology, Virginia Commonwealth Uni6ersity, P.O. Box 980613, Richmond, VA 23298 -0613, USA Received 1 June 2000; received in revised form 5 March 2001; accepted 5 March 2001
Abstract Acetylcodeine is one of the major impurities present in illicitly manufactured heroin (diacetylmorphine). Data on its pharmacology and toxicology are limited and its ability to alter the toxic effects of diacetylmorphine is not known. The first objective of the present study was to compare the acute pharmacological and toxicological effects of acetylcodeine to those of codeine and diacetylmorphine in mice by assessing nociception in the tail-flick test, locomotor stimulation, and convulsive behavior. The second goal of this study was to determine whether acetylcodeine would alter the convulsant effects of diacetylmorphine. The antinociceptive potencies of acetylcodeine and codeine were similar, as reflected by their ED50 (95% confidence limits) values of 35 (29–44) and 51 (40–65) mmol/kg, respectively. Acetylcodeine was somewhat less potent than codeine in stimulating locomotor behavior, with ED50 values of 28 (22– 37) and 12 (6 – 24) mmol/kg, respectively. Diacetylmorphine was considerably more potent than the other two drugs, producing antinociception and locomotor stimulation at ED50 values of 2.4 (1.4–4.1) and 0.65 (0.36–1.2) mmol/kg, respectively. On the other hand, the convulsant effects of acetylcodeine (ED50 =138 (121–157) mmol/kg) and diacetylmorphine (ED50 = 115 (81– 163) mmol/kg) were similar in potency and both were more potent than codeine (ED50 =231 (188–283) mmol/kg). Finally, a subthreshold dose of acetylcodeine (72 mmol/kg) decreased the convulsant ED50 dose of diacetylmorphine to 40 (32– 49). These findings suggest that the convulsant effects of acetylcodeine are more potent than predicted by its effects on locomotor activity and antinociception. The observation that acetylcodeine potentiated the convulsant effects of diacetylmorphine suggests a mechanism for some of the heroin-related deaths reported in human addicts. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acetylcodeine; Heroin; Diacetylmorphine; Codeine; Convulsant; Antinociception; Locomotor activity
1. Introduction Heroin availability and use, as well as serious heroinrelated consequences, are increasing after a decline in the US in the mid-1980s. According to the Drug Abuse Warning Network (DAWN) report, heroin toxicity accounted for about one-third of the medical examiners’ cases in 1990 (National Institute on Drug Abuse, 1990). Heroin-related toxicity may be due to the direct effects of the drug and its metabolites, infections or lifestyle * Corresponding author. Tel.: + 1-804-8288480; fax: +1-8048282117. E-mail address: [email protected] (A.H. Lichtman). 1 Present address: Division of Forensic Sciences, Fairfax, VA, USA.
influences related to drug abuse, and direct effects of impurities or dilutents (Karch, 1992). The ability of these impurities to enhance the pharmacological activity of heroin has not been an area of recent study (Soine, 1986; Cooper, 1989). Comprehensive pharmacological and toxicological data are available only for a few of the compounds present in street heroin. Acetylcodeine has been identified as one of the major impurities of origin in heroin, the pure form of which is diacetylmorphine. The amount of acetylcodeine found in illicit heroin was 1–15% (wt./wt.) or 1–80% relative to the amount of diacetylmorphine (Nakamura and Ukita, 1962; Lim and Chow, 1978; Van Vendeloo et al., 1980; Baker and Gough, 1981; O’Neil et al., 1984; Saady, 1989; O’Neil and Pitts, 1992). Data on the pharmacological and toxicological effects of acetyl-
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codeine are limited. In 1938, Small reviewed the results of animal studies comparing the activities of alkaloids found in heroin (Small et al., 1938). Acetylcodeine was approximately two-fold more toxic, with a three-fold greater convulsant activity than diacetylmorphine in mice. The 24 h LD50 of acetylcodeine and codeine were equivalent, and both were approximately one-tenth the potency of that of morphine in mice (Buckett et al., 1964). Because acetylcodeine is a common constituent of street heroin and can be present at high concentrations, this by-product may contribute to the pharmacological and toxicological effects of heroin. The purpose of the present study was to further characterize the pharmacology and toxicology of acetylcodeine in mice. Its effects on antinociception as assessed in the tail-flick test, locomotor activity, and convulsive behavior were compared to those of diacetylmorphine and codeine. Finally, in order to assess the impact of acetylcodeine on the toxic effects of heroin, the dose– response relationship of diacetylmorphine in producing convulsions was evaluated in the presence of a subthreshold dose of acetylcodeine.
evaluated in the tail-flick test had a pre-injection latency outside of this range and was not used in the study. Each subject (n= 6/group) was given a baseline tail-flick test, given an injection of saline, acetylcodeine (11, 22, 44 or 88 mmol/kg), codeine (12, 25, 49 or 98 mmol/kg) or diacetylmorphine (0.24, 0.71, 2.4 or 7.1 mmol/kg), and evaluated in the tail-flick test again 30 min later.
2.4. Locomotor acti6ity The number of interruptions of a light beam in a 16 photocells/cage activity system (Med Associates, East Fairfield, VT) was used to assess locomotor activity. Individual mice were placed in a 25× 20× 33.5 cm clear plastic cage with a wire mesh top for a 10-min adaptation period. Each subject was then removed, given its respective injection of drug or saline, and returned to the cage where activity counts were taken in 10-min periods for a total of 30 min. Subjects (n=12/ group) received saline, acetylcodeine (11, 22, 44 or 88 mmol/kg), codeine (12, 25, 49, or 98 mmol/kg) or diacetylmorphine (0.2, 0.7, 2.4, 7.1 or 24 mmol/kg).
2.5. Con6ulsant acti6ity 2. Materials and methods
2.1. Subjects Male ICR mice, weighing 18– 20 g on delivery, were purchased from Harlan (Dublin, VA). Water and food (Rodent Laboratory Chow, Ralston-Purina Co., St. Louis, MO) were available ad libitum. The mice were housed in the animal care quarters maintained at 229 2°C on a 12 h light/dark cycle and were moved to the test room 24 h prior to testing for adaptation. Each drug or saline was administered subcutaneously (s.c.) at a dose volume of 0.1 ml/10 g of body weight.
2.2. Drugs Diacetylmorphine hydrochloride (heroin) and codeine phosphate were obtained from the National Institute on Drug Abuse (Bethesda, MD). Acetylcodeine hydrochloride was synthesized from codeine phosphate. No impurities in the acetylcodeine were detected by GC–MS analysis.
2.3. Antinociception The tail-flick response to radiant heat was used to assess antinociception (D’Amour and Smith, 1941). The mice were hand-held during assessment and the intensity of the heat stimulus was adjusted to yield control latencies of 2– 4 s with an automatic 10-s cutoff to minimize tissue damage. One mouse out of the 90
The acute toxicity was evaluated by assessing the convulsant response to each drug administered at high doses. Subjects (n= 6) received either saline, codeine (98, 197, 296, or 394 mmol/kg), acetylcodeine (72, 108, 143 215, and 287 mmol/kg) or diacetylmorphine (24, 71, 106, 142, and 236 mmol/kg). The observation period began immediately following the injection and continued for 60 min. About 20 min postinjection, the observer lifted each mouse gently by the tail, observed for the appearance of tonic or clonic convulsions, then gently turned the subject 180° and observed again for convulsions. This 20-min time point was based on observations during pilot experiments. The scores were given as follows (Evans and Balster, 1993): 0=no effect during the entire observation; 1=tonic convulsion when mouse was lifted and turned; 2=tonic convulsion when mouse was only lifted or tonic–clonic convulsion when mouse was lifted and turned; 3=tonic–clonic convulsion when mouse was merely lifted; 4=tonic–clonic convulsion observed either before mouse was lifted or after it was released. An additional experiment was performed to evaluate whether a subthreshold convulsant dose of acetylcodeine could potentiate the convulsant effects of diacetylmorphine. Subjects received either saline or diacetylmorphine (24, 71, 106, or 142 mmol/kg) alone or in combination with acetylcodeine (72 mmol/kg). All
C.L. O’Neal et al. / Drug and Alcohol Dependence 65 (2001) 37–43
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other conditions of the experiment were identical to the above protocol.
2.6. Statistical analyses Tail-flick response times were expressed as percent maximum possible effect (MPE) determined by the equation: 100× [(test response−baseline response)/ (cutoff− baseline response)]. The 100% MPE indicated that the subject did not flick its tail within the 10-s cutoff time, and 0% MPE indicated that the test response did not differ from the baseline response. Locomotor activity data are expressed as percentage of the control group mean counts for each respective drug. Antinociceptive and locomotor activity data were analyzed by ANOVA and Dunnett’s test was used for post-hoc analysis when appropriate. The convulsive data were analyzed by the Kruskal–Wallis test. All differences were considered significant at the P B0.05 level. Naive mice were used for each test. The ED50 values in producing antinociception, locomotor stimulation, and convulsive activity (CD50) were calculated by least squares linear regression analysis. In order to determine the potency of each drug in stimulating locomotor activity the following formula was used: 100×[(activity counts after drug administration− mean activity counts for the saline group)/ (Emax −mean activity counts for the saline group)]. The Emax for locomotor activity stimulation was 2.5× the mean activity counts for the saline group in each experiment. For the convulsive data, scores of 2 and more were defined as convulsive because the data had a bimodal distribution and all the saline-treated mice exhibited scores of zero or one. The potency of acetylcodeine was compared to the other two drugs for each measure by determining the potency ratios (Colquhoun, 1971). The therapeutic indexes of each drug (Eaton and Klaassen, 1996) were calculated by dividing the ED50 values obtained from the antinociception and locomotor stimulation studies into the CD50 value.
Fig. 1. The antinociceptive effects of acetylcodeine ( ), codeine (), and diacetylmorphine () in the tail-flick test. Each subject was assessed in the tail-flick test 30 min after a s.c. injection of saline or drug. The results are presented as means 9S.E. of % MPE, n=6 mice/group.
3.2. Locomotor acti6ity stimulation Fig. 2 depicts the dose-related increases in locomotor activity for acetylcodeine, codeine, and diacetylmorphine. Each drug significantly increased locomotor activity compared to the respective saline-treated control group (PB 0.05). The ED50 (95% C.L.) values of acetylcodeine, codeine, and diacetylmorphine were 28 (22– 37), 12 (6–24), and 0.65 (0.36–1.2) mmol/kg, respectively. Diacetylmorphine was 28 (18–43) fold more potent than acetylcodeine, while acetylcodeine and codeine did not statistically differ in potency.
3. Results
3.1. Antinocicepti6e acti6ity The dose – response relationship of each drug in producing antinociception is shown in Fig. 1. Each drug produced a significant antinociceptive effect (PB 0.05). The ED50 (95% C.L.) values for acetylcodeine, codeine, and diacetylmorphine were 35 (29– 44), 51 (40– 66), and 2.4 (1.4–4.1) mmol/kg, respectively. Diacetylmorphine was 17 (12–24) fold more potent than acetylcodeine (potency ratio with 95% C.L.), and acetylcodeine and codeine did not significantly differ in potency.
Fig. 2. The locomotor effects of acetylcodeine ( ), codeine (), diacetylmorphine (). The results are presented as the percent of the vehicle control activity levels 9 S.E. for the total activity counts during the 30 min observation period, n =12 mice/group.
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Table 1 Convulsant behavior in mice following s.c. injections of acetylcodeine (n=6 mice/group)
Table 3 Convulsant behavior in mice following s.c. injections of diacetylmorphine (n =6 mice/group)
Score
Score
0 1 2 3 4
Saline controls
1 5 0 0 0
Acetylcodeine dose (mmol/kg) 72
108
143
215
287
1 5 0 0 0
0 6 0 0 0
0 2 3 0 1
0 0 1 0 5a
0 0 0 0 2a
0 1 2 3 4
Saline controls
Diacetylmorphine (mmol/kg)
3 3 0 0 0
24
71
106
142
236
3 3 0 0 0
2 4 0 0 0
2 2 0 0 2
1 0 0 0 5
1 0 0 0 5
One mouse given 215 mmol/kg and one mouse given 287 mmol/kg died within 15–25 min after the injection. a
3.3. Acute toxicity studies Results of the acute toxicity studies for acetylcodeine, codeine, and diacetylmorphine are listed in Tables 1–3, respectively. All three drugs produced a significant effect on convulsive behavior (P B 0.05, Kruskal– Wallis test). The CD50 values for acetycodeine, codeine, and diacetylmorphine were 138 (122– 157), 229 (189– 278), and 115 (81–163) mmol/kg, respectively. Diacetylmorphine was two-fold more potent than acetylcodeine, while once again acetylcodeine and codeine did not statistically differ in potency. The effects of acetylcodeine are depicted in Table 1. Spontaneous tonic– clonic convulsions were observed within 5– 10 min postinjection in both mice treated with 287 mmol/kg culminating in the lethality of one of these subjects at 25 min. Spontaneous tonic– clonic convulsions were observed in five of the six mice administered 215 mmol/kg acetylcodeine within 5– 11 min postinjection. One of the mice in this dosage group died within 25 min following tonic– clonic convulsions. As shown in Table 2, none of the mice administered codeine exhibited spontaneous tonic– clonic convulsions (i.e. a score of 4) at any time during the 1-h observation period even at approximately 400 mmol/kg. In contrast, a high dose of diacetylmorphine (236 mmol/kg) produced spontaneous tonic– clonic convulsions marked by
Table 2 Convulsant behavior in mice following s.c. injections of codeine (n=6 or 7 mice/group)
tremors, wet dog shakes, and jerks that began 10–15 min postinjection in five of the six mice assessed (Table 3). Spontaneous convulsions were also observed at 20 min in five of the six mice administered 142 mmol/kg diacetylmorphine. Excitatory responses such as stereotypy, circling, hyperactivity, hyper-responsiveness to stimuli and straub tail were also observed in these mice. Shown in Table 4 is the dose–response relationship of diacetylmorphine in the presence of 72 mmol/kg acetylcodeine. Spontaneous tonic–clonic convulsions were observed in all mice treated with 142, 106 and 71 mmol/kg diacetylmorphine (PB 0.05, Kruskal– Wallis test). One of the six mice in the 24 mmol/kg dosage group also exhibited spontaneous tonic–clonic convulsions. The convulsive effects of diacetylmorphine were potentiated three (2–4) fold by a subthreshold dose of acetylcodeine, decreasing its CD50 from 115 (see Table 3) to 40 (32–49) mmol/kg. The therapeutic index values of acetylcodeine, codeine and diacetylmorphine are listed in Table 5. Acetylcodeine had the lowest therapeutic indexes for both measures, though codeine‘s therapeutic index for antinociception was similar to that of acetylcodeine. In contrast, the therapeutic indexes of diacetylmorphine were considerably greater than that of acetylcodeine as well as codeine.
Table 4 Effect of a subthreshold dose of acetylcodeine (72 mmol/kg) on convulsant behavior elicited by diacetylmorphine (n = 6 mice/group) Score
Score
0 1 2 3 4
Saline controls
3 3 0 0 0
Saline controls
Codeine (mmol/kg) 98
197
296
394
2 4 0 0 0
1 4 1 0 0
0 2 4 0 0
0 0 3 4 0
0 1 2 3 4
2 4 0 0 0
Diacetylmorphine (mmol/kg) + acetylcodeine (72 mmol/kg) Saline
24
71
106
142
1 5 0 0 0
5 0 0 0 1
0 0 0 0 6
0 0 0 0 6
0 0 0 0 6
C.L. O’Neal et al. / Drug and Alcohol Dependence 65 (2001) 37–43 Table 5 The therapeutic index values (CD50/ED50) of acetylcodeine, codeine and diacetylmorphine for producing convulsant effects relative to antinociception and locomotor stimulation Drug
Antinociception
Acetylcodeine Codeine Diacetylmorphine
3.9 4.5 48
a
a
Locomotor stimulation 4.9 19 177
As determined in the tail-flick test.
4. Discussion The results of the tail-flick test in this study are consistent with the data reported in previous studies characterizing acetylcodeine. Small et al. (1938) reported that 27 mmol/kg of either acetylcodeine or codeine produced antinociception in cats. Furthermore, Buckett et al. (1964) found that the antinociceptive potencies of codeine and acetylcodeine in rats were each approximately one tenth of that of morphine. In the present study, the antinociceptive potencies of acetylcodeine and codeine were nearly identical and were at least 12-fold less than that of diacetylmorphine. The stimulatory effects of opioids in mice includes exploring, grooming, salivation, hyper-motility, escape behavior, aggression, hyper-responsiveness to stimuli, wet dog shakes and straub tail. Once again, diacetylmorphine was considerably more potent than either acetylcodeine or codeine in stimulating locomotor activity. No significant difference was observed between acetylcodeine and codeine in stimulating locomotor activity. This is in agreement with a previous study in which acetylcodeine and codeine elicited similar excitatory effects in cats, with ED50 values of 21 and 27 mmol/kg, respectively (Small et al., 1938). Despite the fact that the pharmacology of acetylcodeine was first investigated more than 60 years ago, relatively little has been published on its acute toxicity. In mice, the lethal and convulsant effects of acetylcodeine were considerably more potent than those effects produced by other opiates (Small et al., 1938). The LD50 value of acetylcodeine (i.e. 290 mmol/kg) was less than half the LD50 values of both codeine (i.e. 806 mmol/kg) and diacetylmorphine (i.e. 709 mmol/kg). Similarly, the convulsant dose of acetylcodeine (i.e. 176 mmol/kg) was approximately three times less than the convulsant doses of codeine (i.e. 538 mmol/kg) and diacetylmorphine (i.e. 531 mmol/kg) in mice (Small et al., 1938). Conversely, Buckett, et al. (1964) reported no difference between the LD50 values of acetylcodeine and codeine, 410 and 401 mmol/kg, respectively. However, the cause of death was not determined and the occurrence of convulsive behavior was not assessed. It should be noted that all three drugs elicited tonic– clonic convulsions at the highest doses evaluated,
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though there were some differences in the magnitude of the convulsant effects. Whereas the maximum convulsive effect of both acetylcodeine and diacetylmorphine was a 4 (i.e. tonic–clonic convulsions either before or after being lifted), the maximum effect of codeine was a 3 (i.e. tonic– clonic convulsions while being lifted). Moreover, whereas acetylcodeine and diacetylmorphine produced tonic–clonic convulsions at similar potencies, the convulsions following acetylcodeine administration appeared more violent and persisted longer than diacetylmorphine-induced convulsions. Two mice given acetylcodeine died after exhibiting tonic–clonic convulsions. The CD50 value of acetylcodeine was consistent with the convulsant dose reported by Small et al. (1938), but the CD50 values for diacetylmorphine and codeine observed in the present study were much lower than the convulsant doses reported in the previous study. These disparities may reflect either methodological or strain differences. In particular, our procedure of holding each mouse by its tail and turning it resulted in additional stimulation, whereas the prior studies merely observed the mice for an unspecified amount of time. Although acetylcodeine was considerably less potent than diacetylmorphine in the tail-flick and the locomotor activity experiments, the two compounds had similar CD50 values. Consequently, the therapeutic index of acetylcodeine was substantially lower than that of diacetylmorphine. In the drug combination experiment, 72 mmol/kg of acetylcodeine, a dose that failed to elicit spontaneous convulsions when administered alone, potentiated the convulsive effects of diacetylmorphine. However, this finding does not address whether this drug interaction is simply additive or synergistic. The acute toxicity of acetylcodeine can be eaplained by several possibilities. High doses of morphine and related opioids have been shown to produce tonic– clonic convulsions in animals involving several receptor systems (Jaffe and Martin, 1993). There is evidence of the involvement of m opioid receptor systems in the production of convulsions as the proconvulsant effects of morphine are blocked by naloxone (Gilbert and Martin, 1975; Umans and Inturrisi, 1982; Frenk, 1983; Lauretti et al., 1994). Conversely, other investigators reported that naloxone was ineffective in antagonizing the convulsant effects of morphine (Snyder et al., 1980; Kolesnikov et al., 1997). Delta agonists have also been shown to produce tonic–clonic convulsions that were antagonized by naltrindole, a selective, competitive delta opioid receptor antagonist (Frenk, 1983; Dykstra et al., 1993; Pakarinen et al., 1995). Opiate-induced convulsions may be elicited indirectly by antagonizing inhibitory neurotransmitters such as GABA or enhancing excitatory neurotransmission with NMDA (Frenk, 1983; Lauretti et al., 1994; Kolesnikov et al., 1997). In addition, opioids may not elicit convulsions through a single mechanism, but may interact through a combina-
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tion of receptor systems. As an alkaloid that is similar to morphine, acetylcodeine could induce convulsions by activating a combination of the above receptor systems. However, acetylcodeine is also structurally related to thebaine which is believed to mediate its proconvulsant effect at the level of the spinal cord (Gilbert and Martin, 1975; Tortella et al., 1984). The metabolite norcodeine is not believed to be a factor in the toxicity of acetylcodeine because it was found to be less toxic than codeine, unlike normeperidine and normorphine (Miller and Anderson, 1954). A factor that was not examined in the present study was the impact of repeated drug administration. Whereas this study evaluated acute effects of each drug, chronic heroin abusers often administer the drug several times a day, with daily doses exceeding 200 mg. Tolerance to some of the effects of opioids is well documented, but the evidence that tolerance develops to the convulsant activity of opioids is mixed. Potentiation of the convulsant effects has been observed (Frenk, 1983). The chronic abuse of heroin can expose the abuser to increasing amounts of acetylcodeine, therefore the low therapeutic index of acetylcodeine and its propensity to lower the seizure threshold of diacetylmorphine is of importance. Consequently, chronic studies would further elucidate the role of acetylcodeine in the toxicity of heroin. In conclusion, the results of the present study demonstrate that in mice acetylcodeine is considerably more toxic in eliciting convulsive behavior than what is predicted by its effects on locomotor and nociceptive behavior. The observation that a subthreshold dose of acetylcodeine potentiated the convulsant effects of diacetylmorphine suggests that the presence of acetylcodeine in street heroin may pose an increased risk of toxicity in opioid users. Acknowledgements The authors thank Dr William Glassco for synthesizing acetylcodeine HCl and Dr Forrest L. Smith for valuable statistical advice. This research was supported by NIDA grant DA 02396. References Baker, P.B., Gough, T.A., 1981. The separation and quantitation of the narcotic components of illicit heroin using reversed-phase high performance liquid chromatography. J. Chromatogr. Sci. 19, 483 – 489. Buckett, W.R., Farquharson, M.E., Haining, C.G., 1964. The analgesic properties of some 14-substituted derivatives of codeine and codeinone. J. Pharm. Pharmacol. 16, 174 –182. Colquhoun, D., 1971. Lectures on Biostatistics: an Introduction to Statistics with Applications in Biology and Medicine. Clarendon Press, Oxford.
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Acetylcodeine, an impurity of illicitly manufactured heroin, elicits convulsions, antinociception, and locomotor stimulation in mice Carol L. O’Neal a,1, Alphonse Poklis a, Aron H. Lichtman b,* a
b
Department of Pathology, Virginia Commonwealth Uni6ersity, Richmond, VA 23298 -0613, USA Department of Pharmacology and Toxicology, Virginia Commonwealth Uni6ersity, P.O. Box 980613, Richmond, VA 23298 -0613, USA Received 1 June 2000; received in revised form 5 March 2001; accepted 5 March 2001
Abstract Acetylcodeine is one of the major impurities present in illicitly manufactured heroin (diacetylmorphine). Data on its pharmacology and toxicology are limited and its ability to alter the toxic effects of diacetylmorphine is not known. The first objective of the present study was to compare the acute pharmacological and toxicological effects of acetylcodeine to those of codeine and diacetylmorphine in mice by assessing nociception in the tail-flick test, locomotor stimulation, and convulsive behavior. The second goal of this study was to determine whether acetylcodeine would alter the convulsant effects of diacetylmorphine. The antinociceptive potencies of acetylcodeine and codeine were similar, as reflected by their ED50 (95% confidence limits) values of 35 (29–44) and 51 (40–65) mmol/kg, respectively. Acetylcodeine was somewhat less potent than codeine in stimulating locomotor behavior, with ED50 values of 28 (22– 37) and 12 (6 – 24) mmol/kg, respectively. Diacetylmorphine was considerably more potent than the other two drugs, producing antinociception and locomotor stimulation at ED50 values of 2.4 (1.4–4.1) and 0.65 (0.36–1.2) mmol/kg, respectively. On the other hand, the convulsant effects of acetylcodeine (ED50 =138 (121–157) mmol/kg) and diacetylmorphine (ED50 = 115 (81– 163) mmol/kg) were similar in potency and both were more potent than codeine (ED50 =231 (188–283) mmol/kg). Finally, a subthreshold dose of acetylcodeine (72 mmol/kg) decreased the convulsant ED50 dose of diacetylmorphine to 40 (32– 49). These findings suggest that the convulsant effects of acetylcodeine are more potent than predicted by its effects on locomotor activity and antinociception. The observation that acetylcodeine potentiated the convulsant effects of diacetylmorphine suggests a mechanism for some of the heroin-related deaths reported in human addicts. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acetylcodeine; Heroin; Diacetylmorphine; Codeine; Convulsant; Antinociception; Locomotor activity
1. Introduction Heroin availability and use, as well as serious heroinrelated consequences, are increasing after a decline in the US in the mid-1980s. According to the Drug Abuse Warning Network (DAWN) report, heroin toxicity accounted for about one-third of the medical examiners’ cases in 1990 (National Institute on Drug Abuse, 1990). Heroin-related toxicity may be due to the direct effects of the drug and its metabolites, infections or lifestyle * Corresponding author. Tel.: + 1-804-8288480; fax: +1-8048282117. E-mail address: [email protected] (A.H. Lichtman). 1 Present address: Division of Forensic Sciences, Fairfax, VA, USA.
influences related to drug abuse, and direct effects of impurities or dilutents (Karch, 1992). The ability of these impurities to enhance the pharmacological activity of heroin has not been an area of recent study (Soine, 1986; Cooper, 1989). Comprehensive pharmacological and toxicological data are available only for a few of the compounds present in street heroin. Acetylcodeine has been identified as one of the major impurities of origin in heroin, the pure form of which is diacetylmorphine. The amount of acetylcodeine found in illicit heroin was 1–15% (wt./wt.) or 1–80% relative to the amount of diacetylmorphine (Nakamura and Ukita, 1962; Lim and Chow, 1978; Van Vendeloo et al., 1980; Baker and Gough, 1981; O’Neil et al., 1984; Saady, 1989; O’Neil and Pitts, 1992). Data on the pharmacological and toxicological effects of acetyl-
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codeine are limited. In 1938, Small reviewed the results of animal studies comparing the activities of alkaloids found in heroin (Small et al., 1938). Acetylcodeine was approximately two-fold more toxic, with a three-fold greater convulsant activity than diacetylmorphine in mice. The 24 h LD50 of acetylcodeine and codeine were equivalent, and both were approximately one-tenth the potency of that of morphine in mice (Buckett et al., 1964). Because acetylcodeine is a common constituent of street heroin and can be present at high concentrations, this by-product may contribute to the pharmacological and toxicological effects of heroin. The purpose of the present study was to further characterize the pharmacology and toxicology of acetylcodeine in mice. Its effects on antinociception as assessed in the tail-flick test, locomotor activity, and convulsive behavior were compared to those of diacetylmorphine and codeine. Finally, in order to assess the impact of acetylcodeine on the toxic effects of heroin, the dose– response relationship of diacetylmorphine in producing convulsions was evaluated in the presence of a subthreshold dose of acetylcodeine.
evaluated in the tail-flick test had a pre-injection latency outside of this range and was not used in the study. Each subject (n= 6/group) was given a baseline tail-flick test, given an injection of saline, acetylcodeine (11, 22, 44 or 88 mmol/kg), codeine (12, 25, 49 or 98 mmol/kg) or diacetylmorphine (0.24, 0.71, 2.4 or 7.1 mmol/kg), and evaluated in the tail-flick test again 30 min later.
2.4. Locomotor acti6ity The number of interruptions of a light beam in a 16 photocells/cage activity system (Med Associates, East Fairfield, VT) was used to assess locomotor activity. Individual mice were placed in a 25× 20× 33.5 cm clear plastic cage with a wire mesh top for a 10-min adaptation period. Each subject was then removed, given its respective injection of drug or saline, and returned to the cage where activity counts were taken in 10-min periods for a total of 30 min. Subjects (n=12/ group) received saline, acetylcodeine (11, 22, 44 or 88 mmol/kg), codeine (12, 25, 49, or 98 mmol/kg) or diacetylmorphine (0.2, 0.7, 2.4, 7.1 or 24 mmol/kg).
2.5. Con6ulsant acti6ity 2. Materials and methods
2.1. Subjects Male ICR mice, weighing 18– 20 g on delivery, were purchased from Harlan (Dublin, VA). Water and food (Rodent Laboratory Chow, Ralston-Purina Co., St. Louis, MO) were available ad libitum. The mice were housed in the animal care quarters maintained at 229 2°C on a 12 h light/dark cycle and were moved to the test room 24 h prior to testing for adaptation. Each drug or saline was administered subcutaneously (s.c.) at a dose volume of 0.1 ml/10 g of body weight.
2.2. Drugs Diacetylmorphine hydrochloride (heroin) and codeine phosphate were obtained from the National Institute on Drug Abuse (Bethesda, MD). Acetylcodeine hydrochloride was synthesized from codeine phosphate. No impurities in the acetylcodeine were detected by GC–MS analysis.
2.3. Antinociception The tail-flick response to radiant heat was used to assess antinociception (D’Amour and Smith, 1941). The mice were hand-held during assessment and the intensity of the heat stimulus was adjusted to yield control latencies of 2– 4 s with an automatic 10-s cutoff to minimize tissue damage. One mouse out of the 90
The acute toxicity was evaluated by assessing the convulsant response to each drug administered at high doses. Subjects (n= 6) received either saline, codeine (98, 197, 296, or 394 mmol/kg), acetylcodeine (72, 108, 143 215, and 287 mmol/kg) or diacetylmorphine (24, 71, 106, 142, and 236 mmol/kg). The observation period began immediately following the injection and continued for 60 min. About 20 min postinjection, the observer lifted each mouse gently by the tail, observed for the appearance of tonic or clonic convulsions, then gently turned the subject 180° and observed again for convulsions. This 20-min time point was based on observations during pilot experiments. The scores were given as follows (Evans and Balster, 1993): 0=no effect during the entire observation; 1=tonic convulsion when mouse was lifted and turned; 2=tonic convulsion when mouse was only lifted or tonic–clonic convulsion when mouse was lifted and turned; 3=tonic–clonic convulsion when mouse was merely lifted; 4=tonic–clonic convulsion observed either before mouse was lifted or after it was released. An additional experiment was performed to evaluate whether a subthreshold convulsant dose of acetylcodeine could potentiate the convulsant effects of diacetylmorphine. Subjects received either saline or diacetylmorphine (24, 71, 106, or 142 mmol/kg) alone or in combination with acetylcodeine (72 mmol/kg). All
C.L. O’Neal et al. / Drug and Alcohol Dependence 65 (2001) 37–43
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other conditions of the experiment were identical to the above protocol.
2.6. Statistical analyses Tail-flick response times were expressed as percent maximum possible effect (MPE) determined by the equation: 100× [(test response−baseline response)/ (cutoff− baseline response)]. The 100% MPE indicated that the subject did not flick its tail within the 10-s cutoff time, and 0% MPE indicated that the test response did not differ from the baseline response. Locomotor activity data are expressed as percentage of the control group mean counts for each respective drug. Antinociceptive and locomotor activity data were analyzed by ANOVA and Dunnett’s test was used for post-hoc analysis when appropriate. The convulsive data were analyzed by the Kruskal–Wallis test. All differences were considered significant at the P B0.05 level. Naive mice were used for each test. The ED50 values in producing antinociception, locomotor stimulation, and convulsive activity (CD50) were calculated by least squares linear regression analysis. In order to determine the potency of each drug in stimulating locomotor activity the following formula was used: 100×[(activity counts after drug administration− mean activity counts for the saline group)/ (Emax −mean activity counts for the saline group)]. The Emax for locomotor activity stimulation was 2.5× the mean activity counts for the saline group in each experiment. For the convulsive data, scores of 2 and more were defined as convulsive because the data had a bimodal distribution and all the saline-treated mice exhibited scores of zero or one. The potency of acetylcodeine was compared to the other two drugs for each measure by determining the potency ratios (Colquhoun, 1971). The therapeutic indexes of each drug (Eaton and Klaassen, 1996) were calculated by dividing the ED50 values obtained from the antinociception and locomotor stimulation studies into the CD50 value.
Fig. 1. The antinociceptive effects of acetylcodeine ( ), codeine (), and diacetylmorphine () in the tail-flick test. Each subject was assessed in the tail-flick test 30 min after a s.c. injection of saline or drug. The results are presented as means 9S.E. of % MPE, n=6 mice/group.
3.2. Locomotor acti6ity stimulation Fig. 2 depicts the dose-related increases in locomotor activity for acetylcodeine, codeine, and diacetylmorphine. Each drug significantly increased locomotor activity compared to the respective saline-treated control group (PB 0.05). The ED50 (95% C.L.) values of acetylcodeine, codeine, and diacetylmorphine were 28 (22– 37), 12 (6–24), and 0.65 (0.36–1.2) mmol/kg, respectively. Diacetylmorphine was 28 (18–43) fold more potent than acetylcodeine, while acetylcodeine and codeine did not statistically differ in potency.
3. Results
3.1. Antinocicepti6e acti6ity The dose – response relationship of each drug in producing antinociception is shown in Fig. 1. Each drug produced a significant antinociceptive effect (PB 0.05). The ED50 (95% C.L.) values for acetylcodeine, codeine, and diacetylmorphine were 35 (29– 44), 51 (40– 66), and 2.4 (1.4–4.1) mmol/kg, respectively. Diacetylmorphine was 17 (12–24) fold more potent than acetylcodeine (potency ratio with 95% C.L.), and acetylcodeine and codeine did not significantly differ in potency.
Fig. 2. The locomotor effects of acetylcodeine ( ), codeine (), diacetylmorphine (). The results are presented as the percent of the vehicle control activity levels 9 S.E. for the total activity counts during the 30 min observation period, n =12 mice/group.
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Table 1 Convulsant behavior in mice following s.c. injections of acetylcodeine (n=6 mice/group)
Table 3 Convulsant behavior in mice following s.c. injections of diacetylmorphine (n =6 mice/group)
Score
Score
0 1 2 3 4
Saline controls
1 5 0 0 0
Acetylcodeine dose (mmol/kg) 72
108
143
215
287
1 5 0 0 0
0 6 0 0 0
0 2 3 0 1
0 0 1 0 5a
0 0 0 0 2a
0 1 2 3 4
Saline controls
Diacetylmorphine (mmol/kg)
3 3 0 0 0
24
71
106
142
236
3 3 0 0 0
2 4 0 0 0
2 2 0 0 2
1 0 0 0 5
1 0 0 0 5
One mouse given 215 mmol/kg and one mouse given 287 mmol/kg died within 15–25 min after the injection. a
3.3. Acute toxicity studies Results of the acute toxicity studies for acetylcodeine, codeine, and diacetylmorphine are listed in Tables 1–3, respectively. All three drugs produced a significant effect on convulsive behavior (P B 0.05, Kruskal– Wallis test). The CD50 values for acetycodeine, codeine, and diacetylmorphine were 138 (122– 157), 229 (189– 278), and 115 (81–163) mmol/kg, respectively. Diacetylmorphine was two-fold more potent than acetylcodeine, while once again acetylcodeine and codeine did not statistically differ in potency. The effects of acetylcodeine are depicted in Table 1. Spontaneous tonic– clonic convulsions were observed within 5– 10 min postinjection in both mice treated with 287 mmol/kg culminating in the lethality of one of these subjects at 25 min. Spontaneous tonic– clonic convulsions were observed in five of the six mice administered 215 mmol/kg acetylcodeine within 5– 11 min postinjection. One of the mice in this dosage group died within 25 min following tonic– clonic convulsions. As shown in Table 2, none of the mice administered codeine exhibited spontaneous tonic– clonic convulsions (i.e. a score of 4) at any time during the 1-h observation period even at approximately 400 mmol/kg. In contrast, a high dose of diacetylmorphine (236 mmol/kg) produced spontaneous tonic– clonic convulsions marked by
Table 2 Convulsant behavior in mice following s.c. injections of codeine (n=6 or 7 mice/group)
tremors, wet dog shakes, and jerks that began 10–15 min postinjection in five of the six mice assessed (Table 3). Spontaneous convulsions were also observed at 20 min in five of the six mice administered 142 mmol/kg diacetylmorphine. Excitatory responses such as stereotypy, circling, hyperactivity, hyper-responsiveness to stimuli and straub tail were also observed in these mice. Shown in Table 4 is the dose–response relationship of diacetylmorphine in the presence of 72 mmol/kg acetylcodeine. Spontaneous tonic–clonic convulsions were observed in all mice treated with 142, 106 and 71 mmol/kg diacetylmorphine (PB 0.05, Kruskal– Wallis test). One of the six mice in the 24 mmol/kg dosage group also exhibited spontaneous tonic–clonic convulsions. The convulsive effects of diacetylmorphine were potentiated three (2–4) fold by a subthreshold dose of acetylcodeine, decreasing its CD50 from 115 (see Table 3) to 40 (32–49) mmol/kg. The therapeutic index values of acetylcodeine, codeine and diacetylmorphine are listed in Table 5. Acetylcodeine had the lowest therapeutic indexes for both measures, though codeine‘s therapeutic index for antinociception was similar to that of acetylcodeine. In contrast, the therapeutic indexes of diacetylmorphine were considerably greater than that of acetylcodeine as well as codeine.
Table 4 Effect of a subthreshold dose of acetylcodeine (72 mmol/kg) on convulsant behavior elicited by diacetylmorphine (n = 6 mice/group) Score
Score
0 1 2 3 4
Saline controls
3 3 0 0 0
Saline controls
Codeine (mmol/kg) 98
197
296
394
2 4 0 0 0
1 4 1 0 0
0 2 4 0 0
0 0 3 4 0
0 1 2 3 4
2 4 0 0 0
Diacetylmorphine (mmol/kg) + acetylcodeine (72 mmol/kg) Saline
24
71
106
142
1 5 0 0 0
5 0 0 0 1
0 0 0 0 6
0 0 0 0 6
0 0 0 0 6
C.L. O’Neal et al. / Drug and Alcohol Dependence 65 (2001) 37–43 Table 5 The therapeutic index values (CD50/ED50) of acetylcodeine, codeine and diacetylmorphine for producing convulsant effects relative to antinociception and locomotor stimulation Drug
Antinociception
Acetylcodeine Codeine Diacetylmorphine
3.9 4.5 48
a
a
Locomotor stimulation 4.9 19 177
As determined in the tail-flick test.
4. Discussion The results of the tail-flick test in this study are consistent with the data reported in previous studies characterizing acetylcodeine. Small et al. (1938) reported that 27 mmol/kg of either acetylcodeine or codeine produced antinociception in cats. Furthermore, Buckett et al. (1964) found that the antinociceptive potencies of codeine and acetylcodeine in rats were each approximately one tenth of that of morphine. In the present study, the antinociceptive potencies of acetylcodeine and codeine were nearly identical and were at least 12-fold less than that of diacetylmorphine. The stimulatory effects of opioids in mice includes exploring, grooming, salivation, hyper-motility, escape behavior, aggression, hyper-responsiveness to stimuli, wet dog shakes and straub tail. Once again, diacetylmorphine was considerably more potent than either acetylcodeine or codeine in stimulating locomotor activity. No significant difference was observed between acetylcodeine and codeine in stimulating locomotor activity. This is in agreement with a previous study in which acetylcodeine and codeine elicited similar excitatory effects in cats, with ED50 values of 21 and 27 mmol/kg, respectively (Small et al., 1938). Despite the fact that the pharmacology of acetylcodeine was first investigated more than 60 years ago, relatively little has been published on its acute toxicity. In mice, the lethal and convulsant effects of acetylcodeine were considerably more potent than those effects produced by other opiates (Small et al., 1938). The LD50 value of acetylcodeine (i.e. 290 mmol/kg) was less than half the LD50 values of both codeine (i.e. 806 mmol/kg) and diacetylmorphine (i.e. 709 mmol/kg). Similarly, the convulsant dose of acetylcodeine (i.e. 176 mmol/kg) was approximately three times less than the convulsant doses of codeine (i.e. 538 mmol/kg) and diacetylmorphine (i.e. 531 mmol/kg) in mice (Small et al., 1938). Conversely, Buckett, et al. (1964) reported no difference between the LD50 values of acetylcodeine and codeine, 410 and 401 mmol/kg, respectively. However, the cause of death was not determined and the occurrence of convulsive behavior was not assessed. It should be noted that all three drugs elicited tonic– clonic convulsions at the highest doses evaluated,
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though there were some differences in the magnitude of the convulsant effects. Whereas the maximum convulsive effect of both acetylcodeine and diacetylmorphine was a 4 (i.e. tonic–clonic convulsions either before or after being lifted), the maximum effect of codeine was a 3 (i.e. tonic– clonic convulsions while being lifted). Moreover, whereas acetylcodeine and diacetylmorphine produced tonic–clonic convulsions at similar potencies, the convulsions following acetylcodeine administration appeared more violent and persisted longer than diacetylmorphine-induced convulsions. Two mice given acetylcodeine died after exhibiting tonic–clonic convulsions. The CD50 value of acetylcodeine was consistent with the convulsant dose reported by Small et al. (1938), but the CD50 values for diacetylmorphine and codeine observed in the present study were much lower than the convulsant doses reported in the previous study. These disparities may reflect either methodological or strain differences. In particular, our procedure of holding each mouse by its tail and turning it resulted in additional stimulation, whereas the prior studies merely observed the mice for an unspecified amount of time. Although acetylcodeine was considerably less potent than diacetylmorphine in the tail-flick and the locomotor activity experiments, the two compounds had similar CD50 values. Consequently, the therapeutic index of acetylcodeine was substantially lower than that of diacetylmorphine. In the drug combination experiment, 72 mmol/kg of acetylcodeine, a dose that failed to elicit spontaneous convulsions when administered alone, potentiated the convulsive effects of diacetylmorphine. However, this finding does not address whether this drug interaction is simply additive or synergistic. The acute toxicity of acetylcodeine can be eaplained by several possibilities. High doses of morphine and related opioids have been shown to produce tonic– clonic convulsions in animals involving several receptor systems (Jaffe and Martin, 1993). There is evidence of the involvement of m opioid receptor systems in the production of convulsions as the proconvulsant effects of morphine are blocked by naloxone (Gilbert and Martin, 1975; Umans and Inturrisi, 1982; Frenk, 1983; Lauretti et al., 1994). Conversely, other investigators reported that naloxone was ineffective in antagonizing the convulsant effects of morphine (Snyder et al., 1980; Kolesnikov et al., 1997). Delta agonists have also been shown to produce tonic–clonic convulsions that were antagonized by naltrindole, a selective, competitive delta opioid receptor antagonist (Frenk, 1983; Dykstra et al., 1993; Pakarinen et al., 1995). Opiate-induced convulsions may be elicited indirectly by antagonizing inhibitory neurotransmitters such as GABA or enhancing excitatory neurotransmission with NMDA (Frenk, 1983; Lauretti et al., 1994; Kolesnikov et al., 1997). In addition, opioids may not elicit convulsions through a single mechanism, but may interact through a combina-
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tion of receptor systems. As an alkaloid that is similar to morphine, acetylcodeine could induce convulsions by activating a combination of the above receptor systems. However, acetylcodeine is also structurally related to thebaine which is believed to mediate its proconvulsant effect at the level of the spinal cord (Gilbert and Martin, 1975; Tortella et al., 1984). The metabolite norcodeine is not believed to be a factor in the toxicity of acetylcodeine because it was found to be less toxic than codeine, unlike normeperidine and normorphine (Miller and Anderson, 1954). A factor that was not examined in the present study was the impact of repeated drug administration. Whereas this study evaluated acute effects of each drug, chronic heroin abusers often administer the drug several times a day, with daily doses exceeding 200 mg. Tolerance to some of the effects of opioids is well documented, but the evidence that tolerance develops to the convulsant activity of opioids is mixed. Potentiation of the convulsant effects has been observed (Frenk, 1983). The chronic abuse of heroin can expose the abuser to increasing amounts of acetylcodeine, therefore the low therapeutic index of acetylcodeine and its propensity to lower the seizure threshold of diacetylmorphine is of importance. Consequently, chronic studies would further elucidate the role of acetylcodeine in the toxicity of heroin. In conclusion, the results of the present study demonstrate that in mice acetylcodeine is considerably more toxic in eliciting convulsive behavior than what is predicted by its effects on locomotor and nociceptive behavior. The observation that a subthreshold dose of acetylcodeine potentiated the convulsant effects of diacetylmorphine suggests that the presence of acetylcodeine in street heroin may pose an increased risk of toxicity in opioid users. Acknowledgements The authors thank Dr William Glassco for synthesizing acetylcodeine HCl and Dr Forrest L. Smith for valuable statistical advice. This research was supported by NIDA grant DA 02396. References Baker, P.B., Gough, T.A., 1981. The separation and quantitation of the narcotic components of illicit heroin using reversed-phase high performance liquid chromatography. J. Chromatogr. Sci. 19, 483 – 489. Buckett, W.R., Farquharson, M.E., Haining, C.G., 1964. The analgesic properties of some 14-substituted derivatives of codeine and codeinone. J. Pharm. Pharmacol. 16, 174 –182. Colquhoun, D., 1971. Lectures on Biostatistics: an Introduction to Statistics with Applications in Biology and Medicine. Clarendon Press, Oxford.
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