The initial hypothesis in this study was that co-administration of compounds acting on different mechanisms involved in controlling food intake would produce larger suppression on food intake than either one of them alone. Furthermore, it was hypothesized that when additive effects of the combination occurred, requiring a smaller dose of individual drugs to decrease food intake, the most common adverse effect of the two drugs, nausea, would be ameliorated. Thus, the acute effects of individual and combined peripheral treatment of Nal and Ex-4 on food intake and CTA were examined. The results revealed that Nal and Ex-4 combination produced greater suppressions of short term food intake than either of them individually. Because the two drugs deviated in their constant relative potency on food intake, there is an inherent difficulty in examining their pharmacological relationship. While RSM can be used where the simple isobolographic method cannot, our assessment of potential synergy is somewhat limited because of the lack of constant relative potency26
. However, our conclusion of additivity is conservative and consistent with the results of the ANOVA. Overall, the anorectic effect of the Nal+Ex-4 combination could be associated with an induced avoidance since there was larger and more rapid suppression of the CS intake in CTA with Nal+Ex-4 than when each compound was administered alone.
Combinational therapies have been applied to treat multiple chronic diseases such as hypertension and diabetes, and many drug combinations to treat obesity are under investigation 5
. One of them is Contrave which combines Nal and bupropion. In this case, Nal the opioid antagonist, was chosen based on its ability to increase the activity of pro-opiomelanocortin neurons in the arcuate nucleus of the hypothalamus to produce anorectic effects7
. In our experiments, we chose Nal based on its effects on the rewarding component of food. Many studies have demonstrated that the opioid system in the striatum contributes to the palatability of foods27,14, 28, 29
. Although Nal alone is associated with minimal weight loss30, 31
, combining it with bupropion has been reported to result in supra-additive effects on food intake and BW reduction7
. In our present research, we combined Nal with the GLP-1 receptor agonist Ex-4. Initially Ex-4 was chosen for its satiety action. However, recent work has also suggested direct effects of GLP-1 on the reward system32, 33
. Thus the combination would affect multiple systems underlying feeding control. The combination produced additive effects on short term intake suppression. Moreover, consecutive once a day Nal+Ex-4 treatment for 4 days revealed a persistent effect of short term intake reduction but no effects on BW. It also appeared that some tolerance occurred at this dose combination over 4-day consecutive treatment. Although not statistically significant, a clear trend of reduced intake suppression was found for 20hr accumulative food intake ().
While the GLP-1 agonist Ex-4 is an FDA approved anti-diabetic drug, its use has produced significant weight loss in human34
. Besides the gut, GLP-1 is expressed in neurons in the nucleus of the solitary tract (NST) and the GLP-1 receptors are expressed there and widely throughout other brain regions35, 36, 37
. These NTS GLP-1 containing neurons largely project to the paraventricular nucleus of the hypothalamus (PVN) and, a significant amount project to other forebrain regions including the ventral tegmental area(VTA) and the nucleus accumbens (NAc)32, 33, 38
. Central GLP-1 pathway activation results not only in food intake reduction but also in food avoidance. For example, injections of GLP-1 in the amygdala can produce malaise as measured by a CTA test39, 40
while intracerebral ventricular GLP-1 antagonists block CTA induced by LiCl 41
. Furthermore, peripheral LiCl injection activates GLP-1 containing neurons in the NTS and those neurons project to the PVN42
. These data suggest that central GLP-1 systems are involved in CTA learning. In this study, Ex-4 and Nal+Ex-4 combination were administered peripherally. Previous studies have suggested that the sites of action of peripherally administered GLP-1 agonists can be both peripheral and central43, 44,45
. Furthermore, it has been demonstrated that Ex-4 can penetrate the brain rapidly46
. Accordingly, our food intake and CTA results with Ex-4 could be induced by both peripheral and central activation of the GLP-1 receptors47
. Although it is unclear whether the food intake and avoidance effects here involve the same populations of GLP-1 neurons or neurons that express GLP-1 receptors, recent studies showing food intake affected by GLP-1 neurons and receptors in the VTA and NAc32, 33
suggest that GLP-1 analogs may also be viewed as agents that acts on not only satiety but also reward mechanisms. Given that the opioid receptors are also expressed in similar brain regions, the interactions between Nal and Ex-4 could potentially occur at multiple central or peripheral sites to control ingestion.
The ability of Ex-4 to induce malaise or visceral illness varies across different animal models. In the rat, there have been mixed results with two commonly used methods to examine the aversive properties of anorexigenic drugs, pica and CTA23
. Pica refers to ingestion of non-nutritive substances such as clay or Kaolin. Rodents show Kaolin hyperphagia in response to toxic treatments such as LiCl injection48, 49
. When using pica behavior to measure the aversive property of the drug in SD rats, none of the tested Ex-4 doses (0.1–10µg/Kg) induced kaolin intake50
. In Wistar and SD rats, peripheral Ex-4 can induce a CTA or decrease locomotor activity at a dose much higher than the threshold dose for reducing food intake 51,50
. However, Hayes and colleagues recently demonstrated that peripheral Ex-4 induces both CTA (0.25–3.0µg/Kg) and pica (3µg/Kg b.i.d.) 52
. Our data are similar to those of Hayes and colleagues. The reasons for the difference among the various experiments are not clear. However, the paradigm we employed with 3 pairings is considered to be especially sensitive.
The aversive effects of Ex-4 have not been as widely reported in experiments with mice and primates. One study demonstrated that the effective peripheral dose to reduce food intake is 50 times lower than the dose to induce CTA in mice53
. In nonhuman primates, a dose dependent reduction in meal size in response to Ex-4 has been reported21, 54
. At doses (0.1 to 3.0µg/Kg) tested, meal numbers were not changed and the monkeys did not show any obvious signs of malaise54
. These data translate better to the responses in humans taking Ex-4 for diabetes treatment. Although nausea is the most commonly reported adverse effect of Exenatide (~38%), the symptom declines or stops over time11, 55
. It has also been suggested that the occurrence of nausea can be greatly reduced if the GLP-1 agonist is taken by gradually increasing the dose. Overall, it is suggested that malaises related to Ex-4 can be improved or excluded overtime in humans.
The results of our study, nevertheless, are in line with the rat data revealing that Ex-4 reduces food intake and produces robust conditioned food avoidance. In Experiment 2, group assignment was reorganized for another CTA with a different CS after the initial conditioned avoidance acquisition. With this method, some rats ended up receiving saline or Nal as the US treatment for the 2nd or 3rd CTA acquisition and some rats repeatedly received the US that contained either Ex-4 alone or combined with Nal. At first glance, this design may complicate the interpretation of the results because the intake of the three different CSs at the first trial differed. However, the fact that there were no group differences in the initial intakes of all three CSs supports the following implications. First, across the doses tested, the aversive effect of Ex-4 was greater than that of Nal. When Nal alone was the US, only the higher dose 3.2mg/Kg reduced CS intake (~22%), but this reduction was not statistically significant. At the lower dose 1mg/Kg, Nal did not reduce intake of either NaCl or citric acid CS during the test trial. However, significant CTA was formed at every doses of Ex-4 tested. At the lowest Ex-4 dose tested (1µg/Kg), rats developed a 50% CS intake reduction by the 1-bottle test trial with either NaCl or citric acid as the CS. Second, in contrast to human data, previous experience with Ex-4 did not ameliorate its aversive effects. That is, no matter whether the rats were naïve or experienced with a US that included Ex-4, they developed avoidance to the CS and the rate of CTA acquisition was not decreased or increased with previous Ex-4 experience. It is possible that acclimating rats to Ex-4 treatments for an extended period of time would ameliorate CTA in the future. However, there was no evidence for this in this study. Finally, in the group that received the combination treatments, the rats acquired avoidance sooner and stronger when the low dose Ex-4 (1µg/Kg) was combined with either high or low dose Nal. Thus, these data suggest that the aversive effect of the combined treatment (Nal+Ex-4) was primarily from Ex-4 and, the combination can produce additive effects on conditioned avoidance.
Data presented here support that the Nal and Ex-4 combinations have additive effects on food intake suppression. This is consistent with the idea of using compounds that act on both the satiety and hedonic aspects of food intake to develop combinational therapy for obesity. However, inconsistent with the assumption that compounds that interact in an additive or synergistic fashion may have the advantage of minimizing dose-dependent adverse effects associated with individual compound, the data indicate that these drug combinations actually produce stronger malaise side effects in rat. Such data enhance the importance of examining not only the desired but also the adverse effects of combinational therapy at early stages of drug development.