Abstract
Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor implicated in the regulation of energy homeostasis. Here, we report the biologic effects of a highly optimized BRS-3 agonist, (2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-{[1-(trifluoromethyl)cyclopropyl]methyl}-1H-imidazol-2-yl)propan-2-ol (MK-5046). Single oral doses of MK-5046 inhibited 2-h and overnight food intake and increased fasting metabolic rate in wild-type but not Brs3 knockout mice. Upon dosing for 14 days, MK-5046 at 25 mg · kg−1 · day−1 reduced body weight of diet-induced obese mouse by 9% compared with vehicle-dosed controls. In mice, 50% brain receptor occupancy was achieved at a plasma concentration of 0.34 ± 0.23 μM. With chronic dosing, effects on metabolic rate, rather than food intake, seem to be the predominant mechanism for weight reduction by MK-5046. The compound also effectively reduced body weight in rats and caused modest increases in body temperature, heart rate, and blood pressure. These latter effects on temperature, heart rate, and blood pressure were transient in nature and desensitized with continued dosing. MK-5046 is the first BRS-3 agonist with properties suitable for use in larger mammals. In dogs, MK-5046 treatment produced statistically significant and persistent weight loss, which was initially accompanied by increases in body temperature and heart rate that abated with continued dosing. Our results demonstrate antiobesity efficacy for MK-5046 in rodents and dogs and further support BRS-3 agonism as a new approach to the treatment of obesity.
Introduction
Obesity is a rapidly growing pandemic. It contributes to a number of serious diseases and comorbidities, including diabetes mellitus, hypertension, coronary heart disease, and certain types of cancer (Guh et al., 2009; Pi-Sunyer, 2009). Despite advances in understanding its pathogenesis, current pharmacotherapy for obesity remains limited in both the degree of achievable weight loss and the safety/tolerability of the drugs. Thus, discovery of new targets and therapeutic agents is a focal point for combating this epidemic.
Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor primarily expressed in the brain. Despite its sequence similarity to the other bombesin receptor subfamily members, the neuromedin B and gastrin-releasing peptide receptors, BRS-3 does not exhibit a high affinity for the known endogenous bombesin family peptides, and its natural ligand remains unknown (Jensen et al., 2008). Mice lacking BRS-3 develop obesity and impaired glycemic regulation (Ohki-Hamazaki et al., 1997). Both hyperphagia and reduced energy expenditure contribute to the obesity phenotype in these mice (Ohki-Hamazaki et al., 1997; Ladenheim et al., 2008). We recently demonstrated that pharmacological activation of BRS-3 with Bag-1, a potent and selective agonist, increases fasting metabolic rate (MR) and decreases food intake and body weight in mice (Guan et al., 2010). The anorectic effect of Bag-1 does not require several well known pathways for energy regulation, such as neuropeptide Y, melanocortin receptor 4, endocannabinoid receptor 1, and leptin. Hence, BRS-3 agonists represent a novel approach for the treatment of obesity, either alone or in combination with other agents.
MK-5046 is a novel BRS-3 agonist, with improved BRS-3 potency, specificity, and pharmacokinetic properties that allows in-depth investigation of BRS3 agonism in preclinical species and is also potentially suitable for use in humans. MK-5046 binds to BRS-3 with high affinity (mouse Ki = 1.6 nM; human Ki = 25 nM) and exhibits no appreciable binding activity at the neuromedin B and gastrin-releasing peptide receptors, as well as many other receptors, ion channels, and enzymes (Sebhat et al., 2010). In a cell-based Ca2+ mobilization functional assay, MK-5046 activates human BRS-3 with similar agonist efficacy as the peptide BRS-3 agonist [d-Phe6,β-Ala11,Phe13,Nle14]Bn-(6-14) (Mantey et al., 1997). In this study, we used MK-5046 to explore the mechanisms underlying regulation of energy homeostasis by the BRS-3 pathway under short- and long-term treatment conditions in mice, rats, and dogs. In addition, the effects of MK-5046 on body temperature, heart rate, and blood pressure were investigated.
Materials and Methods
Chemicals.
MK-5046 [(2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-{[1-(trifluoromethyl)cyclopropyl]methyl}-1H-imidazol-2-yl)propan-2-ol] (Sebhat et al., 2010), [3H]Bag-2 (compound 21c in He et al., 2010), and [3H]Bag-3 (compound 3a in Liu et al., 2010) were synthesized at Merck Research Laboratories (Rahway, NJ). Sibutramine and AM251 were purchased from Sigma-Aldrich (St. Louis, MO).
Animals.
Male C57BL/6N mice were purchased from Taconic Farms (Germantown, NY). Male CD or Crl:CD(SD)DIOBR rats were purchased from Charles River Laboratories (Wilmington, MA). Beagle dogs were obtained from Marshall Farms (North Rose, NY) or Merck Research Laboratories (internal colony). Unless otherwise noted, animals were housed in a temperature- (22°C) and humidity-controlled (30–70%) environment with a 12-h light/dark cycle and with food and water available ad libitum. For the diet-induced obese (DIO) model, mice and rats were fed a high-fat diet [S3282 (Bio-Serv, Frenchtown, NJ) or 12492 (Research Diets, Inc., New Brunswick, NJ)] for at least 14 weeks. Brs-3 knockout mice (Brs3−/Y) were kindly provided by Dr. James Battey (National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD) (Ladenheim et al., 2008) and were backcrossed onto the C57BL/6 background for at least six generations. All animal procedures were performed in compliance with the National Institutes of Health guidelines and approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.
BRS-3 Binding and Agonism Assays.
Receptor binding was performed as described previously (Liu et al., 2002) using membranes from Chinese hamster ovary or HEK293 cells overexpressing the indicated receptor. For human BRS-3 neuromedian B receptor and gastrin releasing peptide receptor binding, 30 pM 125I-[d-Tyr6,β-Ala11,Phe13,Nle14]bombesin-(6-14) (PerkinElmer Life and Analytical Sciences, Waltham, MA) was used, whereas 660 pM [3H]Bag-3 was used for rat and mouse BRS-3 binding. The Ki values are calculated from the IC50 values using the equation of Cheng and Prusoff (1973). For functional assays, agonist-induced mobilization of intracellular Ca2+ was measured in HEK293AEQ cells overexpressing BRS-3 by use of an aequorin bioluminescence assay (Sano et al., 2004).
Food Intake Assay.
Male wild-type and Brs3 knockout mice were maintained on a high-fat diet (60% kcal from fat; Research Diets D12492) and a moderately fat diet (32% kcal from fat; Research Diets D12266B), respectively, to match their body weight. At the time of testing, the animals were approximately 30 weeks of age, with average body weights of 49.7 g for wild-type mice and 51.9 g for Brs3 knockout mice. MK-5046 or vehicle (0.25% methylcellulose/10% Tween 80) was administered by oral gavage approximately 30 min before the onset of the dark phase without food available. Food was provided 5 min before the onset of the dark cycle, and consumption was measured 2 and 18 h later.
MR Measurement.
Wild-type and Brs3 knockout mice were individually housed and maintained on a high-fat diet (53% kcal from fat; Bio-Serv S3282) and a moderately high-fat diet (Research Diets D12266B), respectively, to minimize body weight differences. One week before MR study, the Brs3 knockout mice were switched to high-fat diet (S3282) to standardize diets during testing. At the time of study, animals (24–52 weeks of age; average body weight 52.3 g for wild-type mice and 62.8 g for Brs3 knockout mice) had been conditioned to oral dosing and acclimated to the MR apparatus. MR was measured with indirect calorimetry (Oxymax System; Columbus Instruments, Columbus, OH) at 29–30°C (thermoneutrality). Motor activity (total and ambulating) was measured by infrared beam interruption. On the day before dosing (at 1500, with lights out 6:00 PM—6:00 AM), mice were placed into the MR chambers with access to water but not to food. The next morning (9:00 AM, 3 h into light phase), vehicle or MK-5046 was administered by oral gavage, and the response was measured over the next 6 h. The 3-h period from 6:00 AM to 9:00 AM was used to calculate the baseline resting MR. The 1-h period immediately after dosing was excluded because of re-equilibration of the chambers and activity related to dosing. The time interval from 1 to 6 h after dosing was considered the response period. Resting MR was determined for the baseline and response periods [only periods of inactivity (<50 beam breaks per 17 min) were included in the calculation]. The average MR response (kcal per hour per mouse) was expressed as the percentage change from the average baseline resting MR. Final data are presented as the net percentage change relative to vehicle control.
For the chronic MR study, male DIO mice (7 months of age; 53.4 g) were studied under standard housing conditions (22°C, 12-h light/dark cycle). The baseline fasting, resting MR was determined (7:00 AM—2:00 PM), and each mouse was then implanted subcutaneously with an osmotic infusion pump (Alzet Model 2002, 0.5 μl/h; Alzet, Cupertino, CA) releasing 25 mg · kg−1 · day−1 MK-5046 (or vehicle). The evening before MR measurements, mice were placed into MR chambers and fasted, and MR was measured the next day. In a companion study, weight-matched mice with implanted vehicle-containing pumps were calorically restricted to match the body weight loss achieved by treatment with MK-5046 (the required degree of caloric restriction was determined in pilot experiments). The mice were fed once per day, 1 to 2 h before the dark period. On the day before the MR determinations, the mice received their prefasting food followed by fasting of equal duration before the MR measurements for the ad libitum and caloric restriction groups. Metabolic rate measurements were performed on days 6 and 12 in the MK-5046 group and on days 9 and 14 in the caloric restriction group, as described above. Food intake and body weight were recorded daily, with the exception of the day of the MR measurement.
Body Temperature Measurement.
Mice (24–52 weeks of age; average body weight 39 g for wild-type DIO and 49 g for Brs3 knockout mice) were implanted intraperitoneally with radiotelemetry probes (E-mitter; Mini Mitter, Bend, OR) at least 7 days before study and were conditioned to oral dosing. On the day before dosing (at 2:00 PM, with lights out 3:00 PM—3:00 AM), mice were transferred to clean cages, with access to water but not food. The next morning (3 h into light phase, 6:00 AM), vehicle or MK-5046 was administered orally, and the response was measured for the next 6 h. Body temperature (Tb) was recorded at 5-min intervals using Vital View software (Mini Mitter). The 2-h period from 3:30 AM to 5:30 AM was used to calculate baseline Tb. The hour immediately after dosing was excluded because of activity related to dosing. As indicated, the Tb was averaged over the 1- to 2- and 1- to 6-h response periods. After subtraction of baseline Tb, the data are expressed as the change from vehicle for the 1- to 2- (ΔT1–2h) and 1- to 6-h response periods.
Chronic Weight Loss Study.
Male DIO mice were implanted subcutaneously with osmotic infusion pumps (Alzet Model 2002, 0.5 μl/h) containing MK-5046 at doses of 5, 25, or 50 mg · kg−1 · day−1 or vehicle (1:1 DMSO/propylene glycol). Food intake and body weight were recorded daily for 12 days.
Brain Receptor Occupancy.
Male DIO mice and DIO rats (CD strain, 450–770 g; Charles River Laboratories) were dosed with vehicle or MK-5046 either via oral gavage or subcutaneous infusion with an Alzet osmotic pump. At specific times (5–24 h after the last dose), the rodents were euthanized by CO2 asphyxiation, and the brains were quickly removed, frozen in −40°C isopentane, and stored at −80°C. Brain receptor occupancy was determined ex vivo using an autoradiography method with [3H]Bag-2 as the radioligand (Guan et al., 2010). Plasma samples taken with the brain receptor occupancy samples were analyzed for MK-5046 concentration by liquid chromatography-mass spectrometry/mass spectrometry with a limit of quantification of approximately 0.005 μM.
Heart Rate, Tb, Blood Pressure, and Activity Measurement.
Male Obesity-prone rats [Crl:CD(SD)DIOBR; Charles River Laboratories] were raised on a moderately high-fat diet (D12266B) and implanted with telemetry transmitters (TL1M2-C50-PXTor TA11PA-C40; Data Sciences International, St. Paul, MN) in the abdominal aorta, with the transmitter body placed into the peritoneal cavity. After acclimation, animals (8 months of age; 617-g average body weight) were dosed daily at 9:00 AM and 4:00 PM (8 and 1 h before the start of the dark cycle) for 10 days with vehicle (10% Tween 80 in water), MK-5046 (10 mg/kg p.o. b.i.d.), sibutramine (2 mg/kg p.o. each day; dose given at 4:00 PM, vehicle at 9:00 AM), or AM251 (3 mg/kg p.o. each day; dose given at 4:00 PM, vehicle at 9:00 AM). Food intake and body weight were recorded daily. Heart rate, Tb, blood pressure, and locomotor activity were monitored continuously.
Body Weight and Food Intake in Dogs.
Obese (13.5 to 20.2 kg), adult, spayed female Beagle dogs (Merck Research Laboratories) were allowed access for 6 h each day to the amount of food determined previously to maintain pretreatment obese body weight. Water was available continuously. Dogs (six per group) were dosed orally twice daily with vehicle (100% polyethylene glycol 400) or MK-5046 (3 and 10 mg/kg), or once daily with AM251 (2 mg · kg−1) for 28 consecutive days. Food consumption was measured daily, and body weight was measured weekly.
Rectal Temperature and Heart Rate in Dogs.
Male beagle dogs (11.9 kg average body weight, 1 to 3 years old; Marshall Farms), with implanted telemetry transmitters (D70-PCT; Data Sciences International) for heart rate, were dosed with vehicle (10% Tween 80) or MK-5046 (3 mg/kg p.o.) twice daily between 9:00 AM and 10:00 AM (dose 1, light phase 7:00 AM—7:00 PM) and 3:00 PM and 4:00 PM (dose 2) for 4 consecutive days. Heart rate was recorded continuously (30-s averages), and baseline heart rate for each animal was calculated from the 2 days before dosing. Rectal temperatures [Type T Thermocouple thermometer (Barnant Company, Barrington, IL) equipped with an OT-1 Thermocouple sensor (Physitemp Instruments, Clifton, NJ)] were measured daily at 1 h before and 0.5, 2, 4, and 6 h after the first daily dose and 0.5 and 2 h after the second daily dose. Blood samples were obtained at the same time as temperature measurement from the jugular vein.
Statistics.
One-way analysis of variance with repeated measures followed by the Dunnett's post hoc test or two-way analysis of variance with repeated measures followed by Bonferroni's post-test was used for comparing multiple treatments versus the control group. Student's t test (paired or unpaired as appropriate) was used in cases where only two groups were compared. Two-tailed tests were used for all statistical analyses with P < 0.05 as the level of significance.
Results
MK-5046 Inhibits Food Intake and Increases Energy Expenditure.
MK-5046 was tested for its effect on energy intake and expenditure. Oral administration dose-dependently inhibited food intake, both at 2 h (74, 78, and 90% reduction with 3, 10, and 30 mg/kg, respectively; P < 0.01) and overnight (18 and 52% reduction with 10 and 30 mg/kg, respectively; P < 0.05) in DIO mice (Fig. 1A). The anorectic effect was mediated by BRS-3 because it was not observed in Brs3 knockout mice (Fig. 1B).
MK-5046 dose-dependently increased fasting resting MR by −2, 20, 29, 22, and 37% with single doses of 1, 3, 10, 30, and 100 mg/kg, respectively (Fig. 1C). The hypermetabolic effect was not due to increased locomotor activity, which was unchanged (data not shown). To test the specificity of the MK-5046-induced increase in energy expenditure, the effect of MK-5046 (30 mg/kg p.o.) was compared in wild-type DIO and Brs3 knockout mice. MK-5046 treatment increased resting MR in the control but not Brs3 knockout mice (Fig. 1D), demonstrating that the effect requires BRS-3.
Because treatment with the BRS-3 agonist Bag-1 reverses the reduction in mouse Tb that occurs with fasting (Metzger et al., 2010), we also examined the effect of MK-5046. In DIO mice, overnight fasting decreases light-phase Tb to 34.9 ± 0.11°C (n = 25). Subsequent vehicle treatment resulted in a relatively small increase of Tb of approximately 0.6°C. By comparison, treatment of MK-5046 caused a dose-dependent increase in Tb, with a maximal increase of 1.4°C above vehicle and an ED50 of 0.151 ± 0.057 mg/kg (Fig. 1E). The effect of MK-5046 on Tb was compared in wild-type DIO and Brs3 knockout mice. As reported previously (Metzger et al., 2010), the fasting Tb in Brs3 knockout mice (31.9°C) was approximately 3°C lower than that of the wild-type DIO mice. MK-5046 at 30 mg/kg p.o. produced a significant increase in Tb in the wild-type DIO mice but not in Brs3 knockout mice (Fig. 1F). Taken together, these data demonstrate that MK-5046 reduces energy intake and increases energy expenditure via BRS-3.
Chronic MK-5046 Treatment Reduces Body Weight and Food Intake in DIO Mice.
The effect of MK-5046 on body weight was evaluated in DIO mice using continuous subcutaneous infusion to maintain constant drug levels. MK-5046 (5, 25, and 50 mg · kg−1 · day−1 s.c.) significantly reduced body weight over the 14-day period with a maximal reduction of approximately 8 to 9% compared with the vehicle-treated group. It is noteworthy that the maximal weight loss was achieved by day 5 and was maintained thereafter without evidence of tachyphylaxis (Fig. 2A). In addition to the initial decrease in food intake due to surgery seen in all groups, MK-5046 dose-dependently inhibited food intake from days 1 to 4 (Fig. 2B). Plasma levels of MK-5046 reached steady state by day 3 with average concentrations of 0.3, 2.0, and 3.0 μM at doses of 5, 25, and 50 mg · kg−1 · day−1, respectively.
To assess target engagement by BRS-3, we measured BRS-3 receptor occupancy in the hypothalamus by use of an ex vivo autoradiography method (Guan et al., 2010). At the end of the 14-day infusion, brain BRS-3 sites were highly occupied by MK-5046 (63, 92, and 94% for doses of 5, 25, and 50 mg · kg−1 · day−1, respectively). Thus, high levels of receptor occupancy are required for maximal efficacy, and the level of weight loss achieved at 25 to 50 mg · kg−1 · day−1 seems to be the maximal efficacy for MK-5046. By combining data from multiple studies, a correlation between plasma exposure and brain receptor occupancy by MK-5046 was established, allowing estimation of brain BRS-3 engagement from plasma MK-5046 concentrations (Fig. 3). For example, 90% brain BRS-3 receptor occupancy can be achieved at a plasma concentration of 2.1 (mouse) or 1.4 μM (rat) MK-5046. Compared with its potency (Ki) at the receptor, a relatively high plasma concentration of MK-5046 is required for brain receptor occupancy, which is probably a reflection of the high plasma protein binding (99%) by the compound.
BRS-3 Agonist-Induced Relative Increase in MR Does Not Attenuate.
The observation that MK-5046 caused sustained weight loss in this 14-day study while the anorectic effect attenuated after 4 days prompted us to study MR at later treatment times. Small mammals reduce energy expenditure during weight loss due to food scarcity, a regulated, beneficial adaptation (Hill et al., 1985). In DIO mice treated with MK-5046 (25 mg · kg−1 · day−1 s.c. infusion) for 12 days, body weight was reduced by 7.1% on day 5 and 7.9% on day 12 (Fig. 4A). Fasting resting MR was measured on days 6 and 12 and was not significantly changed from baseline or from vehicle-treated control mice (Fig. 4B). In comparison, DIO C57BL/6 mice that were restricted calorically to achieve the same weight loss as caused by MK-5046 had a 7.5 or 8.8% lower MR than ad libitum fed controls at 9 or 14 days, respectively. Corroboration of the predominant effect on MR comes from the observation that, despite their weight loss, the MK-5046-treated mice consumed the same amount of food as vehicle-treated controls (Fig. 4C, days 7–11). Furthermore, to achieve the same weight loss solely by caloric restriction, a 21% reduction in food intake was required (Fig. 4C, days 10–13). Thus, treatment with MK-5046 shows a relative increase in MR in that it suppresses the compensatory reduction in MR that normally occurs with weight loss, and this effect on MR did not attenuate through the course of the study.
Effects of MK-5046 on Heart Rate and Blood Pressure.
Changes in MR and Tb can be accompanied by changes in heart rate and blood pressure. The effects of 10-day treatment with MK-5046 or other anti-obesity drugs, sibutramine (a serotonin-norepinephrine reuptake inhibitor; Heal et al., 1998) and AM251 (a cannabinoid-1 receptor inverse agonist; Thakur et al., 2005), were compared using telemeterized obesity-prone Sprague-Dawley rats. All three compound-treated groups (MK-5046 at 20 mg · kg−1 · day−1 divided into two doses, sibutramine at 2 mg · kg−1 · day−1, and AM251 at 3 mg · kg−1 · day−1) reduced food intake and body weight (Fig. 5, A and B), with weight reductions of 0.4 ± 0.4, 3.3 ± 0.4, 4.1 ± 1.1, and 5.3 ± 0.5% in the vehicle, MK-5046, sibutramine, and AM251 groups, respectively (n = 8 per treatment). On day 11, the plasma concentration of MK-5046 at 6 h after the first daily dose (and 1 h before the second daily dose) was 0.93 ± 0.15 μM, indicating that a high level of drug exposure was maintained for at least 12 to 14 h each day during MK-5046 treatment. Similar to results in mice, in rats MK-5046 caused sustained weight loss, whereas suppression of food intake waned with continued treatment. MK-5046 treatment increased resting and active phase Tb (P < 0.01) and heart rate (nonsignificantly) on the 1st day of treatment, which returned to baseline by day 3 (Fig. 5, C–F). This was accompanied by a transient decrease in night (active) phase activity (Fig. 5, G–H) and a small, transient increase in blood pressure (nonsignificantly) (Fig. 5, I–L). These parameters returned to baseline levels on day 3, with possible reductions in blood pressure, active phase heart rate, and inactive phase Tb thereafter. In contrast, the weight loss evoked by sibutramine was accompanied by a sustained increase in heart rate (P < 0.001 versus vehicle, both day and night), whereas AM251-induced weight loss was associated with an increase in blood pressure (P < 0.05 versus vehicle, night systolic only).
Effect of MK-5046 on Body Weight, Food Intake, Tb, and Heart Rate in Dogs.
To assess BRS-3 pharmacology in larger species, we examined the effect of MK-5046 on body weight and food intake in obese beagle dogs. Oral administration of MK-5046 at 3 and 10 mg/kg b.i.d. for 28 days caused a statistically significant weight loss (approximately 4% reduction at the end of the study), with trough plasma concentrations of 2.7 ± 1.4 and 5.8 ± 1.5 μM, respectively, on day 28. By comparison, AM251 at 2 mg · kg−1 · day−1 reduced body weight by 5.8% (Fig. 6A). MK-5046 treatment also induced a transient suppression of food intake (Fig. 6B).
We next investigated the effect of MK-5046 on Tb and heart rate in another group of dogs. At baseline, the core body temperature was approximately 37.9°C. Initially, oral dosing of MK-5046 at 3 mg/kg b.i.d. increased Tb as much as 0.8°C. The hyperthermic effect subsided with subsequent dosing and approached that of the control groups by day 4 (Fig. 7A). MK-5046 treatment almost doubled the heart rate acutely from an average baseline of 87 bpm during 1st day of dosing. This effect also attenuated with continued dosing and approached vehicle control levels by day 4 (Fig. 7B). Plasma MK-5046 concentrations were similar across 4 days (Fig. 7C), indicating that the diminished Tb and heart rate effects were not a consequence of a decrease in exposure to the drug. Taken together, the effects of MK-5046 on body weight, food intake, Tb, and heart rate in beagle dogs are similar to those observed in rodents.
Discussion
MK-5046 Is an Improved and Effective BRS-3 Agonist.
The discovery of Bag-1, a small molecule BRS-3 agonist, enabled in vivo study of BRS-3 physiology and preclinical pharmacological proof of concept for the treatment of obesity (Guan et al., 2010; He et al., 2010; Liu et al., 2010). Here we report physiologic studies using MK-5046, a highly optimized drug candidate. Compared with Bag-1, MK-5046 maintains high BRS-3 potency (and lack of efficacy in Brs3 knockout mice) while reducing off-target activities and improving pharmacokinetic properties (Sebhat et al., 2010). The improved properties allow high and prolonged brain receptor occupancy so that pharmacodynamic efficacy is achieved with lower oral doses. For example, the minimal effective MK-5046 dose for suppression of 2-h food intake in mice is 3 mg/kg, compared with 50 mg/kg for Bag-1. Likewise, single doses of Bag-1 up to 100 mg/kg failed to inhibit overnight feeding, whereas 10 mg/kg MK-5046 was efficacious.
Chronic BRS-3 Agonism Has a Predominant MR Effect.
The sustained weight loss effect of chronic BRS-3 agonism is mainly achieved by increasing MR, with a much smaller effect on food intake. In Brs3 knockout mice, both diminished MR and increased food intake contribute to the obesity phenotype (Ohki-Hamazaki et al., 1997; Ladenheim et al., 2008). In a pharmacological manner, single doses of BRS-3 agonists act via both reducing food intake and increasing fasting MR. However, with continued treatment, the reduction in food intake attenuates, whereas the MR effect does not. It is notable that a 21% reduction in food intake was required to match the weight loss of mice treated chronically with MK-5046, which reduced their food intake by 4%.
Potential obesity treatments that strongly increase MR include peripheral mechanisms, such as adrenergic (particularly β3-adrenergic) activation (Arch, 2008), chemical uncouplers (such as dinitrophenol) (Tainter et al., 1935), and thyroid hormone (Kaptein et al., 2009); with these mechanisms, there is often a compensatory increase in food intake. In contrast, most centrally acting obesity treatments have beneficial effects on both MR and food intake, with the MR effect generally being modest (Aronne and Thornton-Jones, 2007; Adan et al., 2008). For example, the cannabinoid-1 receptor inverse agonist produced a sustained reduction in food intake through 4 weeks of treatment in dogs while achieving weight loss comparable to that induced by MK-5046 (see below). Thus, the predominant MR effect of chronic BRS-3 agonism suggests a novel niche in the treatment of obesity.
In small mammals, the major mechanism for increasing MR independent of physical activity is sympathetic nervous system stimulation of brown adipose tissue (Cannon and Nedergaard, 2004). It is presumed that BRS-3 agonists activate this pathway because a β-adrenergic blocker reduces the BRS-3 agonist-induced increase in Tb (Metzger et al., 2010). The importance of brown adipose tissue thermogenesis in larger mammals is debated; however, functional brown adipose tissue is present in adult humans (Nedergaard et al., 2007; Virtanen et al., 2009). Thyroid hormone, a major long-term regulator of MR, is unchanged in Brs3 knockout mice (Ohki-Hamazaki et al., 1997), indicating that it may not be as important as sympathetic activity effects.
MK-5046 Is Efficacious in the Dog.
The improved properties of MK-5046 also made feasible the first testing of BRS-3 agonism in a larger, nonrodent species: beagle dogs. Four weeks of treatment with MK-5046 in dogs produced significant weight reduction that reached a plateau. Because larger mammals regulate Tb differently from small mammals (Andrews, 1995; Geiser, 2004), we also measured Tb in the dogs. As in rodents, MK-5046 caused an increase in Tb in dogs that attenuated with continued dosing. It is not known whether this is a primary action of BRS-3 agonists on thermal regulation or a byproduct of the effect on MR. Both are possible because BRS-3 is highly expressed in such areas as the medial preoptic area and diagonal band, paraventricular, dorsomedial, and arcuate hypothalamic nuclei, which are important for Tb control and energy homeostasis (Boulant, 2000; Guan et al., 2010).
MK-5046 Effects on Heart Rate and Blood Pressure.
In the rat, MK-5046 caused increases in heart rate and blood pressure; the former was also observed in treated dogs. Although the sympathetic nervous system regulates heart rate and blood pressure, it also orchestrates coordinated physiologic responses such as “fight-or-flight” and exerts both global and individualized control of homeostatic parameters (Morrison, 2001). As a consequence, the increases in blood pressure and heart rate are not predicted to be solely secondary to MK-5046-elicited MR and Tb changes. Indeed, the rat data with MK-5046 show slightly different kinetics for the blood pressure increase and the Tb and heart rate changes, consistent with individualized regulation of these parameters. BRS-3 mRNA and binding activity are present in brain regions that control heart rate and blood pressure, such as the paraventricular and dorsomedial hypothalamic nuclei, periaqueductal gray, parabrachial nucleus, and nucleus of solitary tract (Loewy and McKellar 1980; Horiuchi et al., 2009; Guan et al., 2010). Thus, a direct action of BRS-3 on central circuitry of heart rate and blood pressure regulation cannot be excluded.
The blood pressure effect of BRS3 agonism was studied briefly in anesthetized and vagotomized dogs, with two compounds increasing (including MK-5046) and one decreasing blood pressure (unpublished observations), making it unclear whether the MK-5046 blood pressure effect was mechanism-based. A significant or prolonged increase in blood pressure or heart rate is unacceptable in a therapeutic agent for obesity. It remains to be tested whether these effects will occur in humans.
In conclusion, MK-5046 is an improved BRS-3 agonist that produces sustained weight loss in mice, rats, and dogs. In a mechanistic manner, a sustained increase in energy expenditure seems to contribute more than does reduction of food intake. MK-5046 also has undesired effects on Tb, heart rate, and blood pressure, which can be monitored and attenuated upon repeated administration. On the basis of the totality of the data, MK-5046 seems suitable for use in proof of concept studies in humans.
Authorship Contributions
Participated in research design: Metzger, Guan, Faidley, Shearman, Kelly, Sebhat, Lin, Lyons, Nargund, Marsh, Strack, and Reitman.
Conducted experiments: Yang, Raustad, Wang, Spann, Kosinski, Yu, Palyha, Kan, Dragovic, and Craw.
Performed data analysis: Metzger, Guan, Faidley, Shearman, Kelly, Sebhat, Lin, Lyons, Nargund, Marsh, Strack, and Reitman.
Wrote or contributed to the writing of the manuscript: Guan, Metzger, and Reitman.
Footnotes
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.174763.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- BRS-3
- bombesin receptor-subtype 3
- MR
- metabolic rate
- MK-5046
- (2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-{[1-(trifluoromethyl)cyclopropyl]methyl}-1H-imidazol-2-yl)propan-2-ol
- Bag-1
- 2-(4-{2-[5-(2,2-dimethylbutyl)-1H-imidazol-2-yl]ethyl}phenyl)pyridine;[3H]Bag-2, 4′-{2-[5-(cyclopentylmethyl)-1H-imidazol-2-yl]-1,2-ditritium-ethyl}biphenyl-2-carboxylic acid
- [3H]Bag-3
- (4′-{2-[5-(cyclohexylmethyl)-1H-imidazol-2-yl](1,2-ditritium)ethyl}-4,5-difluorobiphenyl-2-carboxylic acid
- AM251
- 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide
- DIO
- diet-induced obesity
- DMSO
- dimethyl sulfoxide
- Tb
- core body temperature.
- Received September 2, 2010.
- Accepted October 20, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics