Generalization tests using different dosing routes from those of drug discrimination training in rats

Abstract

The purpose of this study was to investigate the discriminative stimulus properties of morphine and codeine using different administration routes to that used at drug discrimination training. Rats were trained to discriminate morphine at 3 mg/kg from saline by the intraperitoneal route in a standard two-lever drug discrimination paradigm. Generalization of morphine by the subcutaneous and the oral routes, and codeine by the intraperitoneal and the oral routes to the discriminative stimulus properties of the morphine training dose were investigated. Morphine at 3 mg/kg by the subcutaneous route generalized to the morphine training dose and 10 of 12 rats showed 80% or more morphine-lever responses. In the administration of morphine by the oral route, morphine at 30 mg/kg generalized to the morphine training dose and all rats showed 80% or more morphine-lever responses within the range of 3 to 30 mg/kg. In the administration of codeine by the intraperitoneal route, codeine at 20 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 3 to 20 mg/kg. In the administration of codeine by the oral route, codeine at 60 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 10 to 60 mg/kg. Thus, the discriminative stimulus properties of morphine and codeine were comparable when using different administration routes to those at discrimination training.

INTRODUCTION

In drug discrimination studies, the same administration route is normally used at drug discrimination training and generalization tests. In drug discrimination training, intraperitoneal (ip) and subcutaneous (sc) routes were used in many cases (Chen et al., 2013; Harris et al., 2014; Eppolito et al., 2014; Qiu et al., 2015). However, there were some cases in which different administration routes were used in the generalization tests due to solubility (Carroll et al., 2006; Heal et al., 2013). In addition, it is recommended to use the intended clinical route of administration in the preclinical studies of new developmental drugs (European Medicines Agency, 2006). However, some pharmaceuticals have multiple clinical routes of administration, such as morphine by oral, rectal, subcutaneous, intravenous, epidural and intrathecal, or fentanyl by percutaneous, intravenous, epidural and intrathecal routes; and it is considered difficult to conduct discrimination training and generalizations tests which encompass such a variety of routes. Therefore, it is thought reasonable to select an administration route by which the animals acquire discrimination drug from the vehicle fastest during discrimination training and use the expected clinical route of administration in the generalization tests. Since drug discrimination studies usually use known dependence-producing drugs, a suitable route of administration for discrimination training can be selected from the literature available. However, when conducting generalization tests using the expected clinical route of administration, there is a high possibility that this route will differ from that used during discrimination training. Therefore, it is imperative that research be conducted into the effects of different routes of administration in generalization tests to those used at discrimination training.

Generalization tests in nicotine (Craft and Howard, 1988) and cocaine (de la Garza and Johanson, 1986) using different administration routes to those during drug discrimination training have been reported. In discrimination training using morphine via the ip route, the results of generalization tests using morphine via the oral (po) route have been reported (Gianutsos and Lal, 1975), but not using codeine via the po route. In this study, we conducted generalization tests using morphine administered by different routes to that used at discrimination training (ip) in rats. Then, in order to test this theory also applies for other drugs, generalization of the opioid codeine, when administered via the oral route, to morphine (ip) was observed.

MATERIALS AND METHODS

Subjects

Sixty male Long-Evans rats (Charles River Laboratories Japan, Inc., Kanagawa, Japan) were purchased at 7 to 8 weeks of age and body weights were maintained at 290 to 330 g (80% free-feeding weight). These rats were individually housed in stainless steel wire mesh cages (29W × 22D × 21H cm) with stainless steel chip trays and wood chip bedding material (Sunflake, Charles River Laboratories Japan, Inc.) and wood blocks for gnawing were placed in the cages during the housing period. Feed was provided at 15 ± 5 g/day (CRF-1: Oriental Yeast Co., Ltd., Tokyo, Japan) after termination of the experiment to maintain body weights at approximately 80% free-feeding weight. Water was available ad libitum for all animals in their home cages. The room temperature and humidity were set at 23 ± 2°C and 55 ± 15%, respectively. The room was illuminated from 7:00 AM to 7:00 PM. All experimental procedures were approved and conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Ina Research Inc., which is fully accredited by AAALAC International.

Apparatus

An operant test chamber (241W × 305D × 292H mm, Med Associates Inc., St. Albans, VT, USA) with levers, lamps and a feed tray on the wall was placed inside a fan-ventilated and sound-attenuated cubicle (670W × 600D × 560H mm) equipped with a room light. A feed dispenser for pellets (45 mg, Bio-Serv, Flemington, NJ, USA) was installed outside the chamber. Experiments and collection of data were controlled by a personal computer system (Med-PC, Med Associates Inc.).

Procedures

Drug discrimination training

After initial shaping of lever pressings under a fixed-ratio 1 (FR1) schedule of food reinforcement, rats were trained to discriminate morphine at 3 mg/kg form saline. Each animal was placed in a chamber 15 min after ip administration of morphine (D) or saline (S). After 15 min, both the right and left levers were set in the chamber and observation for the number of lever pressings was started. On the day of administration of D or S, half of the animals were provided with 1 pellet following lever pressing of the right or left lever, respectively, as the correct response. Pressing of the opposite lever was regarded as the correct response for the remaining half of the animals. After 1 pellet was provided, a 20-sec time-out period was set and the lamp was turned off. Lever pressing during this period was regarded as invalid. Each training session was terminated after the animals acquired 20 pellets or 15 min after the start of the session. The frequency of sessions was limited to once daily for each animal and D and S were administered in a double alternating-sequence schedule (SSDDSSDD etc.). The first FR was set at 1 and then increased every 4 sessions, to 3, then to 5 and finally to 10 in this order. Training to discriminate morphine from saline continued until the following 2 criteria were met for 5 consecutive sessions: 1) the animals acquired 20 pellets/session and, 2) 80% or more of the number of lever pressings up to acquisition of the first pellet on each session and of the total number of lever pressings were correct responses. Once these criteria were met, it was judged that the rat had acquired morphine discrimination from saline. The average number of sessions required to meet the criteria was 52 ± 12 (range: 28 to 74).

Generalization test

The generalization test was started using 16 rats which had acquired morphine discrimination. In the generalization test phase, drug discrimination training was continued to maintain morphine discrimination from saline. The criterion for implementation of the generalization test was to meet the training criteria for at least 3 consecutive sessions, since the rats had already acquired morphine discrimination. Once the training criterion was met, the generalization tests were conducted in ascending order as follows: po morphine, ip codeine, po codeine and sc morphine. (i.e. SSDTDSST…, where T denotes a generalization test). The frequency of generalization tests was limited to once daily for each animal. At 1 hr after po administration or at 30 min after ip and sc administration, the right and left levers were set in the chamber and the count of lever pressings was started. The pretreatment time of morphine and ip codeine were set based on the published literatures (Morgan et al., 1999; Suzuki et al., 1995; Gianutsos and Lal, 1975; Recker and Higgins, 2004) and that of po codeine was set at the same time as po morphine. Following administration with morphine or codeine, the animals were transferred to the chambers 15 min prior to the start of the test. Each session of the generalization test was terminated when the animals reached 10 lever pressings on either lever or 15 min elapsed. The generalization test phase was completed in approximately 120 sessions.

Drugs

Morphine (Morphine hydrochloride hydrate, Takeda Pharmaceutical Industry, Osaka, Japan) and codeine (Codeine phosphate hydrate, Takeda Pharmaceutical Industry) were dissolved in isotonic physiological saline (Otsuka Normal Saline, Otsuka Pharmaceutical Factory Inc., Tokushima, Japan) for ip and sc administration and in water for injection (Otsuka Distilled Water, Otsuka Pharmaceutical Factory Inc.) for po administration. The injection volume was 1 mL/kg for ip and sc administration, and 10 mL/kg for po administration. Training and generalization test doses of ip, sc and po morphine and ip codeine were selected from the published literature (Morgan et al., 1999; Suzuki et al., 1995; Gianutsos and Lal, 1975; Recker and Higgins, 2004). The first dose of po codeine was set at 10 mg/kg, which was the same as the mid dose of ip codeine, to avoid overdosing.

Data analyses

The percentage of morphine-lever responses and the response rates per minute were calculated for each rat. The mean and standard error for groups of 11 to 16 rats were calculated for both parameters at each drug dose tested. Full generalization to the ip morphine training dose was defined as 80% or more of the group mean of morphine-lever responses. The mean group response rates were compared using the Dunnett’s test to investigate behavioral effects (Figs. 1 and 3). The mean group morphine-lever responses and response rates in the dose which showed generalization to the discriminative stimulus effects of morphine by ip administration was compared using the Dunnett’s test (Fig. 2) or Student-t test (Fig. 4) for each drug. In addition, the number of animals that showed generalization to the discriminative stimulus effects of morphine by ip administration were compared using the Chi-square test, since the number of rats used differed among the groups (Figs. 2 and 4).

Fig. 1

Effects of morphine by po administration in rats trained to discriminate 3 mg/kg of morphine from saline (ip). Each point is the mean percentage of morphine-lever responses and response rates with an SEM of 15 or 16 rats. The percentage of morphine-lever responses and response rates were not calculated in cases where a rat made fewer than 10 responses. No statistically significant difference in the response rate compared to W (water for injection).

Fig. 2

Effects of morphine by sc and po administration in rats trained to discriminate 3 mg/kg of morphine from saline (ip). Each column is the mean percentage of morphine-lever responses and response rates with an SEM of 12 or 15 rats. The numbers in parentheses represent the number of rats that showed morphine-lever responses of 80% or more out of the total number of animals that completed 10 lever responses. No statistically significant differences in the morphine-lever responses or the number of rats compared to ip morphine group. *: P < 0.05 vs ip morphine group in response rate.

Fig. 3

Effects of codeine by ip and po administration in rats trained to discriminate 3 mg/kg of morphine from saline (ip). Each point is the mean percentage of morphine-lever responses and response rates with an SEM of 15 rats. No statistically significant differences in response rates compared to saline or W (water for injection).

Fig. 4

Effects of codeine by ip and po administration in rats trained to discriminate 3 mg/kg of morphine from saline (ip). Each column is the mean percentage of morphine-lever responses and response rates with an SEM of 13 or 15 rats. For a more detailed explanation, see Fig. 2. No statistically significant differences in the morphine-lever responses, the number of rats or the response rate compared to ip codeine group.

RESULTS

Morphine by po administration generalized to the discriminative stimulus effects of morphine by ip administration in a dose-dependent manner, and the response rates tended to decrease at 30 mg/kg, although there was no significant difference compared with water for injection (Fig. 1). The number of rats that showed morphine-lever responses of 80% or more was 4 of 16, 9 of 16 and 11 of 15 in morphine at 3, 10 and 30 mg/kg, respectively. Fifteen of 16 rats showed morphine-lever responses of 80% or more at all dose levels of morphine, and only 1 rat did not show such responses. In addition, the dose-response of morphine-lever responses in 5 of 16 rats was not clearly noted; 3 rats that showed morphine-lever responses of 80% or more at 10 mg/kg were noted to decrease in morphine-lever responses at 30 mg/kg and another 2 rats showed morphine-lever responses of 80% or more at 3 and 30 mg/kg (Table 1).

Table 1. Effects of morphine (po) and codeine (ip and po) in each rat trained to discriminate 3 mg/kg of morphine from saline (ip).

Morphine-lever responses (%)
Animal No.	Drug, administration route and dose (mg/kg)
	Morphine (po)				Codeine (ip)				Codeine (po)
	3	10	30		3	10	20		10	30	60
4	0	100	76.9		0	0	100		9.1	0	100
10	66.7	90.9	66.7		0	90.9	66.7		0	16.7	76.9
11	23.1	100	100		100	100	83.3		0	100	90.9
12	9.1	71.4	90.9		9.1	0	9.1		0	0	83.3
20	9.1	0	83.3		9.1	100	E		0	9.1	90.9
26	33.3	100	100		0	0	83.3		16.7	47.4	90.9
33	100	100	E		28.6	83.3	E		0	100	100
34	100	0	90.9		0	100	90.9		0	100	100
36	76.9	100	71.4		9.1	100	100		16.7	55.6	100
37	100	100	100		0	100	100		100	100	100
38	9.1	37.5	90.9		58.8	9.1	83.3		0	90.9	100
50	0	0	58.8		NC	NC	NC		NC	NC	NC
51	100	9.1	100		0	90.9	90.9		23.1	90.9	100
54	0	100	90.9		0	100	100		76.9	100	100
55	0	100	100		83.3	100	100		100	100	55.6
60	76.9	71.4	83.3		58.8	62.5	83.3		0	9.1	100

Response rates (responses/min)
Animal No.	Drug, administration route and dose (mg/kg)
	Morphine (po)				Codeine (ip)				Codeine (po)
	3	10	30		3	10	20		10	30	60
4	54.5	23.1	2.6		40.0	37.5	28.6		44.0	46.2	35.3
10	47.4	33.0	22.0		66.7	36.7	39.1		50.0	90.0	55.7
11	86.7	50.0	66.7		85.7	50.0	5.9		75.0	75.0	34.7
12	55.0	56.0	47.1		66.0	54.5	73.3		75.0	60.0	55.4
20	66.0	50.0	16.0		60.0	75.0	E		54.5	60.0	82.5
26	60.0	42.9	6.3		42.9	37.5	37.9		23.2	81.4	50.8
33	60.0	75.0	E		49.4	55.4	E		46.2	42.9	1.1
34	66.7	85.7	11.4		75.0	60.0	11.4		66.7	85.7	42.9
36	60.0	66.7	31.1		66.0	50.0	30.0		26.7	72.0	50.0
37	54.5	75.0	7.0		54.5	60.0	6.9		66.7	42.9	40.0
38	47.1	73.8	15.0		78.5	55.0	15.3		60.0	60.0	21.4
50	85.7	85.7	51.0		NC	NC	NC		NC	NC	NC
51	75.0	94.3	35.3		75.0	60.0	66.0		86.7	73.3	66.7
54	54.5	35.3	50.8		50.0	18.8	1.7		78.0	54.5	60.0
55	46.2	75.0	42.9		72.0	50.0	85.7		75.0	75.0	34.8
60	78.0	84.0	65.5		85.0	96.0	90.0		60.0	66.0	60.0

E: Data were not calculated because rats made fewer than 10 responses.

NC: Not conducted because morphine (po) did not generalize to the training dose of morphine (ip) in this rat at any dose level.

Morphine at 3 mg/kg by sc administration generalized to the discriminative stimulus effects of morphine by ip administration. The number of rats that showed morphine-lever responses of 80% or more was 10 of 12 animals, with 2 rats not showing such responses. There were no statistically significant differences among the group mean morphine-lever responses and the number of rats that showed morphine-lever responses of 80% or more between morphine at 3 mg/kg by ip and sc administration and morphine at 30 mg/kg by po administration. The response rate of morphine at 30 mg/kg by po administration showed a statistically significant decrease compared to that of morphine at 3 mg/kg by ip administration (Fig. 2).

Codeine by ip and po administration generalized to the discriminative stimulus effects of morphine by ip administration in a dose-dependent manner, and the response rates tended to decrease, although there was no significant difference compared with saline or water for injection (Fig. 3). The number of rats that showed morphine-lever responses of 80% or more was 2 of 15, 10 of 15 and 11 of 13 in codeine at 3, 10 and 20 mg/kg by ip administration, respectively, and 2, 8 and 13 of 15 in codeine at 10, 30 and 60 mg/kg by po administration, respectively. Fourteen of 15 rats by ip and po administration showed morphine-lever responses of 80% or more at all dose levels of codeine, with 1 different rat not showing such responses at each dose level. Similarly to morphine, there were rats which were not noted with dose-dependent morphine-lever responses, but only 1 animal each by ip and po administration (Table 1). There were no statistically significant differences between the group mean morphine-lever responses, the number of rats, or response rates of codeine at 20 mg/kg by ip administration and at 60 mg/kg by po administration (Fig. 4).

DISCUSSION

In rats that had undergone drug discrimination training using ip morphine, sc and po administration of morphine generalized to ip morphine under the conditions of this study. The sc morphine generalized to the training dose of morphine at 3 mg/kg, and the po morphine generalized at 10-fold this dose level (30 mg/kg). In discrimination training, sc morphine at 3 mg/kg is a commonly used route and dose combination, as is ip administration at this dose (Nishida et al., 1989; Easterling and Holtzman, 1998; Li et al., 2013). This means that sc morphine at 3 mg/kg induces sufficient discrimination stimulus effects. Therefore, in this study, sc morphine at the single dose level of 3 mg/kg was used and results showed generalization to ip morphine. In the rats that trained to discriminate ip morphine at 10 mg/kg from saline, morphine at 50 mg/kg by po administration showed generalization to the training dose of ip morphine, but not morphine at 10 or 20 mg/kg by po (Gianutsos and Lal, 1975). It is well known that the training dose influences generalization with other drugs in drug discrimination studies (Sannerud and Ator, 1995; Järbe et al., 1998; Grabus et al., 1999). Therefore, the difference noted in the results for po administration was considered to be a reflection of the difference in generalizing dose levels. Morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) are the major metabolites of morphine in humans and rats (Christrup, 1997; Ogura, 2000; De Gregori et al., 2012). It is reported that these metabolites are similarly produced in iv and sc administration and hepatic microsomes in rats (Kuo et al., 1991; Wu et al., 1997; Van Crugten et al., 1997; Ogura, 2000). In rats, M6G generalized to the discriminative stimulus effects of morphine, but not M3G (Easterling and Holtzman, 1998). This fact suggests that the morphine metabolite M6G is also related to the discriminative stimulus effects of morphine. Therefore, the discriminative stimulus effects of morphine also include those of M6G. Thus, although there are differences in absorption and metabolism rates between the difference routes of administration, there should not be any large differences between the ip, sc and po routes employed in this study as far as discriminative stimulus effects are concerned.

In this study, the response rate in po morphine at 30 mg/kg tended to decrease. Morphine shows sufficient antinociceptive effects in a tail-flick test at 30 mg/kg by po (Okura et al., 2008), at 3 or 10 mg/kg by ip (Li et al., 2013; Unal et al., 2013) and by sc administration (He et al., 2009), suggesting that the plasma concentrations at these dose levels are comparable. However, ip morphine at 3 and 10 mg/kg decreases the response rates to approximately 80 and 50%, respectively, but not at 0.3 and 1 mg/kg (Morgan et al., 1999), and slight sedation is noted in some rats in our laboratory (data not shown). Therefore, it is suggested that morphine induces decreases in the response rates at the pharmacological effective doses in rats.

Codeine at 20 mg/kg by ip and at 60 mg/kg by po administration completely generalized to the discriminative stimulus effects of ip morphine and induced decreases in the response rates in the same manner as that of morphine by sc administration (Recker and Higgins, 2004). Codeine shows antinociceptive effects in a tail immersion test at 16 mg/kg by ip administration (Molina et al., 1983) and in a tail-flick test at 25 mg/kg by sc administration (Chen et al., 2005). Similar to morphine, the doses which codeine generalized to the discriminative stimulus effects of morphine are the same as the pharmacological effective doses. There is no report that investigated antinociceptive effects by po codeine. However, as mentioned above, codeine may also induce behavioral disruption at the pharmacological effective doses in rats.

Codeine by po administration likewise completely generalized to morphine at the training dose. There have been some reports published on generalization tests conducted using different routes of administration to discriminative training. In the rats that trained to discriminate po nicotine from saline, nicotine by po, sc, ip and transdermal (td) administration showed generalization to the training dose of po nicotine. However, nicotine administered by po, sc and ip showed similar dose-effect curves of po nicotine-lever responses but by td, the curve shifted approximately 10-fold rightward, suggesting that an approximately equal cue occurs with nicotine by po, ip, and sc administration, and that the td cue is considerably weaker (Craft and Howard, 1988). As a cause of the weak cue noted by td administration, it is pointed out that percutaneous absorption of nicotine is poor. This means that the blood concentration by td administration is lower than that of other administration routes at the same dose level. In the rhesus monkeys that trained to discriminate intramuscular (im) cocaine from saline, cocaine by im and intravenous administration showed cocaine-appropriate responses in a dose-dependent manner (de la Garza and Johanson, 1986). However, by po administration, responses to cocaine were noted, but not in a dose dependent manner at all dose levels, indicating the effects of uncontrolled pharmacokinetic factors (de la Garza and Johanson, 1986). On the other hand, in the rats that trained to discriminate ip methadone from saline, ip and sc methadone and heroine, sc morphine and sc methadone isomers, (+) methadone and (-) methadone, fully generalized to ip methadone, but not ip morphine or methadone isomers (Vann et al., 2009). In this case, morphine and methadone isomers generalized to ip methadone only via the different administration route, sc, at discrimination training. These results suggest that the different plasma concentrations affected the results of the generalization test. Thus, these facts indicate that plasma concentrations, regardless of administration route, are important in generalization tests. Therefore, it is important that dose selection for generalization tests takes into consideration pharmacokinetic factors, not only when different routes of administration are used, but also when the same routes are used in the generalization tests to those used at discrimination training.

In the generalization test to ip morphine using morphine by po and codeine by ip and po administration, most of the animals showed 80% or more ip morphine-lever responses. However, 1 of 16 animals for morphine by po and 1 of 15 animals each for codeine by ip and po showed responses of less than 80%. In addition, 5 of 16 animals for morphine by po and 1 of 15 animals each for codeine by ip and po did not show clear dose-response. As mentioned above, it is possible that the different plasma concentrations produced by the different routes of administration might have affected the results in these animals, but details are unclear.

In conclusion, the discriminative stimulus properties of morphine and codeine were comparable when using different administration routes to those at discrimination training. Therefore, it is suggested under the conditions of this study, that the use of different administration routes in generalization tests to those at discrimination training does not affect evaluation of the potential for discriminative stimulus properties when appropriate doses are selected.

ACKNOWLEDGMENTS

The authors thank Ms. Penelope Naruta for providing technical assistance.

Conflict of interest

The authors declare that there is no conflict of interest.

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