In a previous study , we demonstrated both the existence of pregnancy-induced analgesia in mice and its enhancement by SCH 32615, an enkephalinase inhibitor. This study indicated that a stressful state, such as pregnancy, was associated with increased activity of endogenous enkephalins and that prevention of their degradation led to demonstrable analgesia. Enkephalins are degraded by aminopeptidase N in addition to enkephalinase . Inhibitors of both enzymes responsible for enkephalin catabolism produce antinociception [3,4]. RB 101 is a newly synthesized inhibitor of both enzymes responsible for enkephalin metabolism [5,6]. Its uniqueness lies in its ability to enter the brain after systemic administration and then, in the presence of brain tissue, to break up into its enzyme-inhibiting components . Perhaps RB 101, by virtue of the fact that it inhibits enkephalin degradation almost completely, induces antinociception in a condition such as pregnancy, which is associated with increased activity of endogenous enkephalins. However, these enzymes have other substrates, and perhaps RB 101 may affect the metabolism of other neurohormones (even though there have been no published reports suggesting this). Further, previous investigations with this drug have suggested that it produces analgesia without many of the unwanted side effects of conventional narcotic analgesics [7,8]. Since the most life-threatening side effect of narcotics is respiratory depression, it was meaningful to investigate the effect of RB 101 on respiration.
We hypothesized that in pregnant mice, RB 101 would elicit antinociception comparable to morphine, but without the unwanted side effect of respiratory depression. To test these hypotheses, we studied hot plate latency (HPL) responses and respiratory rates in pregnant mice before and after the administration of RB 101 or its vehicle or morphine.
Prior approval was obtained from the Animal Care Committee at the Oregon Health Sciences University. The subjects were female Swiss Webster mice (35-65 g, mean 51.1 +/- SD 5.2) with timed pregnancies. Experiments were conducted on Day 17 or Day 18 of pregnancy. Usually, delivery takes place on the 19th gestational day.
Antinociception was measured using the hot plate test. Animals were placed unrestrained on a heated stainless steel surface (31 x 19 cm) maintained at 55 +/- 0.5 degrees C and enclosed on its perimeter by plexiglass. Time to hind paw licking or jumping off the heated surface with both hind limbs was noted as the HPL in seconds. Animals not exhibiting such aversive behavior were removed at 120 s to prevent injury. In testing for antinociception, animals were placed on the hot plate a total of three times: once initially for baseline recording of HPL and then at 30 min and 60 min after treatment. These time points were chosen because they represent the peak activity of subcutaneously administered RB 101 . Animals were randomly assigned to receive a particular treatment in the following groups: Group 1 = RB 101, 150 mg/kg (n = 15); Group 2 = RB 101, 50 mg/kg (n = 15); Group 3 = RB 101 vehicle (ethanol 10%, cremophor 10%, distilled water 80%) (n = 15); Group 4 = morphine 5 mg/kg (n = 14); and Group 5 RB 101 150 mg/kg + naloxone 5 mg/kg (n = 10).
In addition, a separate group of pregnant animals (n = 13) was placed on the hot plate for a total of three times in the same time sequence as the other groups but was not given any injection. This group served as a control for the effect of the hot plate on HPL. Also, animals in Groups 1 and 3 were given RB 101 150 mg/kg or RB 101 vehicle, respectively, on the second day after delivery. All drugs were of similar volume (1 mL) and were given subcutaneously after baseline recording of HPL. The person recording HPL was blinded to the treatment given in every instance.
In separate experiments, respiratory rate was measured as an index of respiratory depression. This was done by placing the mouse in a plastic restrainer with its snout exposed. The end of the sampling tube of an Ohmeda 6000 multigas monitor (BOC Group, Louisville, CO) was placed in close proximity to the nose so that exhaled gases could be sampled. This resulted in a waveform produced by the end-tidal carbon dioxide, which was then recorded. Respiratory rate could subsequently be counted from the hard copy (Figure 1). Oxygen at the rate of 1 L/min was blown across the exposed snout of the mouse as a precaution against hypoxia. Baseline recording of the respiratory rate was made by averaging the rate over a 5-min period. After treatment, recordings were made at 30 min and again at 60 min. Respiratory depression was compared between RB 101 150 mg/kg (n = 16) and morphine 5 mg/kg (n = 16). A control group of animals (n = 15) was administered the RB 101 vehicle. The number of subjects in each group was calculated from previously published work  so that power (1-beta) > 0.9.
Data are expressed as mean +/- SEM. To allow comparison of analgesia among treatment groups, HPL was converted to percentage of maximal possible effect (%MPE), where %MPE = (test latency - control latency/cutoff time [120 s] - control latency) x 100. For comparison of the effects on respiration, postdrug respiratory rates were expressed as a percentage, thus postdrug rate/predrug rate x 100. The results were subjected to analysis of variance with post hoc analysis with the Student-Newman-Keuls test. A P value of <0.05 was considered significant.
Pregnant mice given RB 101 150 mg/kg displayed analgesia that was similar to the analgesia that developed in pregnant mice given morphine 5 mg/kg. The analgesic effects of RB 101 150 mg/kg and morphine 5 mg/kg were significantly different from that of RB 101 50 mg/kg, RB 101 vehicle, and RB 101 150 mg/kg coadministered with naloxone 5 mg/kg at both 30 min and 60 min after treatment (Figure 2). The baseline (control latency) for animals given the combination of naloxone with RB 101 was derived from the mean HPL of animals (n = 15) given only naloxone 5 mg/kg (data not shown). The mean %MPE of animals given RB 101 150 mg/kg on the second day after delivery was 1.1 +/- 2.3 at 30 min and 1.0 +/- 4.2 at 60 min after treatment. This was not significantly different from the mean %MPE of similar postpartum animals given only RB 101 vehicle, 1.7 +/- 3.0 at 30 min (P = 0.9) and -1.5 +/- 3.2 at 60 min (P = 0.6), respectively. The mean HPL of pregnant animals not given any treatment was 10.4 +/- 0.6 when first placed on the hot plate, 12.7 +/- 1.2 at 30 min, and 10.5 +/- 0.9 at 60 min. There was no significant difference among these values.
Respiratory rate was recorded at 30 min and 60 min after drug administration. Morphine 5 mg/kg significantly decreased respiratory rate when compared with RB 101 150 mg/kg and RB 101 vehicle at 30 min posttreatment (Figure 3). At this time point, both morphine 5 mg/kg and RB 101 150 mg/kg produced antinociception of a similar magnitude (Figure 2).
Our results support an earlier report that RB 101 produces naloxone-reversible analgesia comparable to morphine, at one dose of each drug . However, in contrast to that study, we examined pregnant animals. We have previously established that endogenous enkephalins play a part in pregnancy-induced analgesia in mice . Endogenous opiate activity in the brain is increased after artificial stressful stimuli, and this increase parallels an increase in analgesia . Our study provides further evidence that drugs such as RB 101 work by enhancing the already increased endogenous opiate activity in natural stressful states, such as pregnancy.
An earlier study in a similar animal model using SCH 32615, an enkephalinase inhibitor, revealed that there was a ceiling effect in terms of dose response . We thought that RB 101, by virtue of the fact that it almost completely inhibited enkephalin degradation, would not have this limitation. However, we were limited by the lack of solubility of RB 101, and this has been noted before in another study . Perhaps a different formulation for the vehicle may address this problem. Alternatively, a larger volume in which to dissolve the drug may be the answer. We were limited by the fact that more than 1 mL administered subcutaneously in our very small subjects resulted in very large lumps from which fluid could be seen leaking out. This lack of solubility has prompted the synthesis of a newer and more soluble dual inhibitor, RB1000, which is active when given orally (Dr. B.P. Roques, personal communication, 1996).
Our results show that at an equianalgesic dose, morphine produced significantly more depression of respiratory rate than RB 101. Further, studies using other inhibitors of enkephalin metabolism have reported a lack of effect on respiration .1 This suggests that there may be very little endogenous enkephalin release in the vicinity of neurons that control respiratory function. Also, RB 101 is free of other opiate side effects, such as dependence and tolerance [7,8]. This could reflect higher efficacy of endogenous enkephalins at micro receptors when compared with exogenous opioids .
(1) Boudinot E, Denavit-Saubie M, Fournie-Zaluski MC, et al. Respiratory consequences in cats and rats of inhibition of enkephalin-degrading enzymes by kelatorphan [abstract]. J Physiol (Lond) 1988;406:169P.
Measurement of respiratory rate has been validated as a means of measuring opiate-receptor-mediated depression of respiration [9,14,15]. In our study, even though morphine was statistically different than RB 101 150 mg/kg in its effect on respiratory rate at 30 min posttreatment, there was no difference between its effect and that of RB 101 150 mg/kg or RB 101 vehicle at 60 min. Pregnancy is associated with increased serum levels of progesterone. The excitatory effect of progesterone on central neural structures that control respiration may help to explain this aspect of the effect of morphine in our study .
Further studies could be directed toward possible synergism between RB 101 and conventional opioids. Indeed, the preferential binding of [leu] and [met]enkephalin to delta receptors  has led to the speculation that they may be associated with less side effects than those of micro receptor agonists such as morphine . Side effects are dose-related and certainly such a combination could lead to reduction of the dose of both drugs if they interact synergistically. However, it is important to remember that the interaction of endogenous opioids with pain transmission may not be as simple as or analogous to that of acetylcholine at the myoneural junction .
In conclusion, we demonstrated that RB 101, a prodrug inhibitor of the main enzymes responsible for enkephalin degradation produces antinociception in pregnant mice, comparable to morphine. This antinociceptive effect of RB 101 was antagonized by naloxone, thus suggesting its opioid basis. Compared to morphine, an equinociceptive dose of RB 101 did not significantly depress respiratory rate.
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