The Regulation of Feeding and Metabolic Rate and the Prevention of Murine Cancer Cachexia with a Small-Molecule Melanocortin-4 Receptor Anta
http://www.100md.com
内分泌学杂志 2005年第6期
Neurocrine Biosciences (S.M., A.C.F., C.C., S.R.J.H., B.A.F., B.T.B.), San Diego, California 92130; and Department of Pediatrics (G.B.B., A.H., D.L.M.), Center for the Study of Weight Regulation (D.L.M.), Oregon Health & Sciences University, Portland, Oregon 97239
Address all correspondence and requests for reprints to: Daniel L. Marks M.D., Ph.D., Department of Pediatrics, Mailcode CDRCP, 707 Southwest Gaines Road, Portland, Oregon 97239. E-mail: marksd@ohsu.edu.
Abstract
Cachexia is metabolic disorder characterized by anorexia, an increased metabolic rate, and loss of lean body mass. It is a relatively common disorder, and is a pathological feature of diseases such as cancer, HIV infection, and renal failure. Recent studies have demonstrated that cachexia brought about by a variety of illnesses can be attenuated or reversed by blocking activation of the melanocortin 4 subtype receptor (MC4-R) within the central nervous system. Although the potential use of central MC4-R antagonists for the treatment of cachexia was supported by these studies, utility was limited by the need to deliver these agents intracerebroventricularly. In the current study, we present a series of experiments demonstrating that peripheral administration of a small molecule MC4-R antagonist can effectively stimulate daytime (satiated) food intake as well as decrease basal metabolic rate in normal animals. Furthermore, this compound attenuated cachexia and preserved lean body mass in a murine cancer model. These data clearly demonstrate the potential of small molecule MC4-R antagonists in the treatment of cachexia and underscore the importance of melanocortin signaling in the development of this metabolic disorder.
Introduction
INVOLUNTARY WEIGHT LOSS is a feature of many acute and chronic diseases. Unlike simple starvation, weight loss during illness is not associated with a significant degree of protective metabolic or behavioral responses, and it leads to enhanced loss of lean body mass. This type of weight loss, known as cachexia, is found in patients with a variety of diseases including cancer, renal failure, heart failure, and chronic infections (1). The severity of cachexia in these illnesses is often the primary determining factor in both quality of life and in eventual mortality (1, 2). Unfortunately, despite our increased understanding of this process, there is currently no safe and effective treatment for this condition. Extensive trials with antiinflammatory agents have shown modest benefit, and significant gastrointestinal and renal side effects are common (3). Perhaps the most promising agents in early trials were the progestational agents (e.g. megesterol acetate), but more recent trials have shown that the modest weight gain produced by these agents is primarily due to gains in body fat and water retention, with little beneficial effects on lean body tissues (4). GH has also received attention as a potential anticachexia agent, but its usefulness is limited by cost, difficulty of delivery, worsened insulin resistance, potential to promote the growth of tumors in individuals with cancer, and GH resistance in most forms of cachexia (5, 6). Thus, there is a critical need for the development of new agents with efficacy in treating cachexia brought about by a variety of diseases.
There is a growing body of evidence to indicate that cachexia in disorders as different as HIV disease and congestive heart failure may have a common mechanism that involves the action of elevated levels of circulating cytokines (7, 8, 9, 10, 11). This is also true in chronic renal failure, where circulating concentrations of cytokines such as IL-1, IL-6, and TNF- are increased in patients with uremia and correlate with the degree of cachexia in these individuals (12, 13). Collectively, the existing animal and human data strongly suggest that increases in the level of circulating inflammatory cytokines can produce all of the major features of cachexia, including anorexia, increased metabolic rate, and loss of lean body mass. The association between elevated circulating cytokines and cachexia has led to the proposal of numerous hypotheses regarding the mechanism underlying this process. At this point, most authors reason that one important mechanism involves the action of cytokines released during inflammation and malignancy on the central nervous system. Ultimately, these cytokines are thought to alter the release and function of a number of key neurotransmitters, thereby altering appetite, metabolic rate, and nutrient partitioning (1, 14, 15, 16, 17).
The hypothalamic arcuate nucleus is known to be critical for the control of body weight and is responsive to numerous circulating compounds, including cytokines (18, 19). Proopiomelanocortin (POMC) is a propeptide precursor that is produced in neurons found in the hypothalamic arcuate nucleus (20). POMC neurons are thought to provide an important tonic inhibition of food intake and energy storage, primarily via production and release of -MSH from the POMC precursor. -MSH binds to central melanocortin receptors (particulary the type 4 melanocortin receptor, MC4-R) where it acts to inhibit food intake (21). Recently, our own lab and others (22, 23, 24, 25) have demonstrated that cachexia in acute and chronic disease models in rodents can be reversed by genetic or pharmacologic blockade of central melanocortin signaling via the MC4-R. Thus, antagonists of central MC4-R are plausible candidates for novel therapeutics for cachexia.
We describe here the characteristics of NBI-12i as a potent, selective, and bioavailable small molecule MC4-R antagonist. We have examined the effects of peripheral administration of NBI-12i on food intake in normal mice, receptor knockout mice, and for its ability to attenuate cachexia in a murine cancer model.
Materials and Methods
In vitro characterization
Cell culture.
Human embryonic kidney (HEK) 293 cells expressing human MC1, MC3, MC4. and MC5 receptors, and mouse MC3 and MC4 receptors, were obtained as previously described (27). All of these cell lines were cultured in DMEM, supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 0.2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively), and 200 μg/ml G418. Cells were grown in a humidified atmosphere of 7% CO2 at 37 C.
Preparation of cell membranes.
P2 membrane fractions of cells were prepared using a high-pressure nitrogen cell and differential centrifugation as previously described (28) The protein concentration in the membrane pellet was determined using the Coomassie method (Pierce, Rockford, IL), using BSA as the standard. Membranes were stored at –80 C before use.
Radioligand binding assays.
Binding assays were set up in low-binding 96-well plates (no. 3605; Corning, Palo Alto, CA). The assay buffer was 25 mM HEPES, 1.5 mM CaCl2, 1 mM MgSO4, 100 mM NaCl (pH 7). The following were added sequentially to the plates: 75 μl [125I]NDP-MSH [specific activity 2200 Ci/mmol, PerkinElmer Life Sciences (Boston, MA)], 75 μl unlabeled ligand, and 50 μl membrane suspension. The final concentration of [125I]NDP-MSH used was 200 pM. The assay was incubated for 90 min at room temperature, then bound and free radioligand were separated by rapid filtration as previously described (28). Glass fiber grade C filters were pretreated for 20 min with 0.1% polyethylenimine. Radioactivity on filters (Auger electrons) was counted using a Topcount NXT (Packard, Meriden, CT) at 35% efficiency, and the total amount of radioligand added to the assay was measured using a Packard Cobra II counter (78% efficiency). In all assays, total radioligand bound to the filter (total binding) was 20% or less of the total amount of radioligand added.
cAMP accumulation assays.
HEK293 cells were plated 24 h before assay on polylysine-coated 96-well tissue culture plates at a density of 10,000 cells/well. Immediately before assay cells were washed with 200 μl/well PBS. Subsequently 50 μl cAMP assay buffer was added to the wells (DMEM without phenol red supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, and 1 mM isobutylmethylxanthine). Twenty-five microliters of NBI-12i were then added, followed 2 min later by 25 μl -MSH. Cells were then incubated for 30 min at 37 C in 7% CO2. After cell lysis, cAMP was measured by chemiluminescent immunoassay (Applied Biosystems, Bedford, MA).
In vivo characterization
Animals.
C57BL/6J male mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were used for the feeding, metabolism, and cachexia studies. MC3-R knockout (MC3-RKO) mice and their wild-type (WT) controls were derived from the original C57BL/6Jx129 colonies (29, 30) maintained within the Vollum Institute that had been bred seven generations into the C57BL/6J strain and maintained as homozygous lines. MC4-RKO mice, described previously (29), were bred 10 generations into the same C57BL/6J strain. KO mice were raised group housed, weaned at 21 d, and allowed ad libitum access to powdered Laboratory Rodent Diet (Purina, St. Louis, MO).
Before the start of an experiment, mice were housed individually for at least 7 d. Additionally, for the behavioral experiments, they were handled daily for a minimum of 5 d. All animals used in these studies were between 5 and 7 wk of age at the start of the study. Previous studies have demonstrated that differences in food intake between WT and MC4-RKO animals are increased when the animals are on a chow of moderate fat content (31). Therefore, all animals were maintained on Purina 5015 chow (13% fat) for the behavioral studies. All studies were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animal and approved by the Animal Care and Use Committee of the Oregon Health Sciences.
NBI-12i preparation and administration.
NBI-12i was synthesized in the Medicinal Chemistry Department of Neurocrine Biosciences Inc. using the synthesis methods described by Tucci and colleagues1. In all studies, the compound was administered to the ip cavity. NBI-12i was dissolved in normal saline daily. Knockout mice and littermate controls had basal feeding monitored for 2 d, and then during each 12-h period after an ip saline injection to demonstrate that the observed effects were not due to differential stress responses observed in other models of melanocortin blockade (32). In the daytime (satiated) feeding studies, all compounds were injected ip at 0800 h, and food intake was measured at hourly intervals. For the nighttime studies, compounds were injected at lights out, and food intake was measured every 2 h overnight. All animals were weighed before compound injection, and the doses were normalized to individual animal body weight. In the tumor studies, the drug was injected twice daily at 0800 and 1600 h at a dose of 3 mg/kg. Food intake and body weight were measured daily.
Food intake measurement.
Animals were individually housed for a minimum of 1 wk before starting each experiment. Animals were habituated to eating powdered mouse chow from containers designed to minimize spill and contamination of the remaining food. Food intake was measured at hourly intervals or daily at the same time.
Indirect calorimetry.
Oxygen consumption (VO2) and carbon dioxide production (VCO2) were simultaneously determined by indirect calorimetry (Oxymax, Columbus Instruments, Columbus, OH) while animals were housed in separate chambers at 24 ± 1 C. Mice were first acclimatized to the chambers for 2 d. Measurements were recorded for 6–8 h during the middle of the light cycle (1100–1600 h). Samples were recorded every 3 min with the room air reference taken every 30 min and the air flow to chambers 500 ml/min. Total oxygen consumption was determined by averaging all of the samples recorded corresponding to periods of movement as well as inactivity. The respiratory quotient (RQ) was calculated as the molar ratio of VO2:VCO2.
Tumor inoculation.
Lewis lung carcinoma (LLC) cells were maintained as a primary culture in DMEM with 10% fetal bovine serum as recommended by the supplier (American Type Culture Collection, Manassas, VA). We have found that the anorexia and cachexia produced by this cell line is quite variable, so we first subcloned this cell line and established 10 separate cell lines that were screened for the degree of cachexia produced in experimental animals. The subclone line that produced the most consistent cachexia was then expanded, separated into aliquots, and stored in liquid nitrogen. For each study, a fresh sample of this subcloned cell line was removed from storage and cultured. LLC tumor cells were harvested during exponential growth of the culture, washed in Hanks’ balanced salt solution, and 1 x 106 cells were injected sc into the upper flank of the mice. Sham-injected animals received an implant of a similar amount of heat-killed tumor cells. In all cases, the time of appearance of a tumor mass was noted in the log, and all experimental animals were found to have a palpable tumor within 5 d of the start of the experiment. At the time the animals were killed, tumors were dissected away from surrounding tissue and weighed. Gross examination of organs did not reveal the presence of any observable metastasis.
Body composition.
Body composition was determined at the start and the end of the LLC experiment by dual-energy x-ray absorbtometry (DEXA, PIXImus mouse densitometer, Lunar Corp.) as previously described (25). The instrument was calibrated at the start of each recording session with a murine calibration standard. All animals were fasted for 12 h before DEXA analysis to minimize the effect of ingested food on the DEXA analysis. Animals were anesthetized before the first scan, and asphyxiated with CO2 before the tumor dissection and the final scan.
Statistical methods.
Radioligand binding data were fitted to a four parameter-logistic equation using XLfit (ID Business Solutions Ltd., Emeryville, CA) to provide fitted values of inhibition constant (Ki). Ligand concentration-dependence data from cAMP accumulation assays were fitted to a four parameter-logistic equation using Prism 3.0 (GraphPad Software, San Diego, CA) to determine EC50 and Emaximal effect. Antagonism of -MSH-stimulated cAMP accumulation was analyzed by the method of Arunlakshana and Schild (33).
Differences among groups in feeding and activity in all experiments were analyzed by two-way, repeated measures ANOVA with time and treatment as the measured variables. Post hoc effects were determined by Fisher’s least significant difference test. Final tumor and body weights, and body composition by DEXA were analyzed by Student’s t test when two groups were included, or one-way ANOVA with post hoc analysis when three or more groups were included. Data sets were analyzed for statistical significance using SigmaStat (SPSS, Inc., Chicago, IL).
Results
Receptor pharmacology
The affinity of NBI-12i for melanocortin receptor subtypes was determined using [125I]-NDP-MSH binding to membranes prepared from HEK293 cells expressing individual human or mouse receptors. As demonstrated by Tucci et al. (see footnote 1), NBI-12i was a selective inhibitor of binding to human MC4 receptors (Table 1; Fig. 1A), with a Ki value of 9.8 nM, having 245-, 122-, and 32-fold selectivity over human MC1, 3, and 5 receptors, respectively. The affinity of NBI-12i for mouse MC4 and MC3 receptors was similar to that of human (Table 1; Fig. 1A), with an mMC4:mMC3 selectivity ratio of 48. In an assay of MC4 receptor function, NBI-12i at concentrations up to 3 μM induced no significant accumulation of cAMP in HEK293 cells stably expressing hMC4, but antagonized -MSH-stimulated cAMP accumulation (Fig. 1B). Increasing concentrations of NBI-12i produced rightward shifts in the -MSH concentration-response curve, and Schild analysis (Fig. 1C) revealed a pA2 value of 7.22 ± 0.13 (60 nM) with a slope of 1.02 ± 0.12 (mean ± SEM, n = 3). An approximately 25% reduction in the maximal effect of -MSH was observed at the highest concentration of NBI-12i tested (3 μM). These data are consistent with the idea that NBI-12i acts as a competitive antagonist of hMC4 receptors.
TABLE 1. NBI-12i and -MSH affinity for human and mouse melanocortin receptor subtypes
FIG. 1. Receptor pharmacology of NBI-12i. A, Inhibition of [125I]NDP-MSH binding to melanocortin receptors by NBI-12i. Radioligand binding to the receptors in HEK293 cell membranes was measured, and the data analyzed, as described in Materials and Methods. Data are from representative experiments performed three times. B, Schild analysis of NBI-12i antagonism of -MSH-stimulated cAMP accumulation in HEK293 cells expressing the human MC4 receptor. The experiment was performed and data analyzed as described in Materials and Methods. -MSH concentration dependence for stimulating cAMP accumulation in the presence of a range of concentrations of NBI-12i. Data are from a representative experiment performed three times. C, Schild plot of antagonism by NBI-12i. Data are mean ± SEM of dose ratios from three independent experiments.
The wider receptor selectivity of NBI-12i was examined in a panel of radioligand binding assays for the following receptors, ion channels enzymes and transporters: adenosine (A1, A2a, A3); adrenergic (1, 2, ?1, ?2), angiotensin (AT1), benzodiazepine, bradykinin (B1); CGRP, cannabinoid (CB1, CB2), cholecystokinin (CCKA, CCKB), dopamine (D1, D2S, D3, D4.4), endothelin, -aminobutyric acid, glutamate (-amino-3-hydroxy-5-methyl-4-isoxazole propionate, N-methyl-D-aspartate, kainate), histamine (H1, H2, H3), imadazole (I1, I2), leukotriene (LTD4), monoamine oxidase (MAO-A, MAO-B), muscarinic (M1, M2, M3, M4, M5); neuropeptide Y, nicotinic, opiate and opiate receptor-like 1, purinergic (P2X, P2Y), serotonin, somatostatin, glucocorticoid, estrogen, progesterone, androgen, TRH, vasopressin (V1a, V2), norepinephrine, dopamine, serotonin, -aminobutyric acid, and choline transporters. In all of these assays, NBI-12i gave less than 50% inhibition at a concentration of 10 μM. Significant activity of NBI-12i was observed in radioligand binding assays for human neurokinin-1 (NK1) and -2 (NK2) receptors and the human ghrelin receptor. Concentration-response curves revealed that NBI-12i had Ki values of 480, 1700, and 110 nM for NK1, NK2 and ghrelin receptors, respectively, representing affinities 49-, 173-, and 11-fold less that that observed for NBI-12i at hMC4.
Light-phase feeding
A dose response analysis of the effect of NBI-12i on daytime food intake was performed. Mice were randomly assigned to groups (n = 5/group) and vehicle, 1, 3, or 6 mg/kg of NBI-12i (ip) was administered at 0700 h. Cumulative food intake was significantly increased by the 2 highest doses of NBI-12i (3 and 6 mg/kg) at 4 and 6 h after administration (Fig. 2; two-way mixed design ANOVA; dose, F3, 16 = 4.58, P < 0.05; time, F2, 32 = 82.50, P < 0.01; interaction, F6, 59 = 2.94, P < 0.05).
FIG. 2. Effect of NBI-12i on cumulative food intake. Groups of mice (n = 5/group) received 0, 1, 3, or 6 mg/kg of NBI-12i at 0700 h. The 3 and 6 mg/kg doses significantly increased feeding relative to vehicle-treated animals at 4 and 6 h after treatment (two-way ANOVA; dose, F3, 16 = 4.58, P < 0.05; time, F2, 32 = 82.50, P < 0.01; interaction, F6, 59 = 2.94, P < 0.05, * significantly different from vehicle control).
MC3-RKO and MC4-RKO mice
To test the receptor specificity of this compound in vivo, we examined the ability of NBI-12i to stimulate feeding in MC4-RKO mice. In an initial experiment, we tested groups of MC4-RKO mice and compared daytime feeding in response to 3 mg/kg NBI-12i or saline. In this case, we saw no stimulation of feeding by NBI-12i at any time point studied (2 h saline 0.34 ± 0.1, NBI-12i 0.34 ± 0.07; 2–4 h saline 0.09 ± 0.05, NBI-12i 0.086 ± 0.03; 4–6 h saline 0.11 ± 0.07, NBI-12i 0.09 ± 0.03; n = 10 per group, ANOVA P = 0.98). In a second study, we compared the responses of groups of MC4-RKO, MC3-RKO, and WT mice to injections of saline or NBI-12i. We observed a significant increase in feeding in both WT and MC3-RKO mice that was not present in MC4-RKO mice. In this case, the stimulation of feeding was still apparent between 4 and 6 h post injection (Fig. 3; two-way ANOVA group, F5, 135 = 19.03, P < 0.001; time, F2, 135 = 80.79, P < 0.001; interaction, F10, 152 = 1.93, P < 0.05, n = 7 per group for KO mice, n = 10 for WT mice).
FIG. 3. Effect of NBI-12i or vehicle (veh) in WT (A), MC3-RKO (B), and MC4-RKO (C) mice. NBI-12i (3 mg/kg) significantly increased light-phase food intake in both WT and MC3-RKO mice but not in MC4-RKO mice (n = 7 per group). The stimulation of feeding was still apparent between 4 and 6 h after injection (two-way ANOVA group, F5, 135 = 19.03, P < 0.001; time, F2, 135 = 80.79, P < 0.001; interaction, F10, 152 = 1.93, P < 0.05, n = 7 per group for KO mice, n = 10 for WT mice; *, significantly different from vehicle-control).
Metabolic effects
Previous investigators have demonstrated that blockade of the MC4-R is associated with a decrease in basal VO2, whereas melanocortin agonists are found to increase basal VO2 (34, 35, 36). To investigate the effects of NBI-12i on metabolic rate, we first habituated groups of C57BL/6J mice (n = 16/group) to indirect calorimeter cages for 2 d, with saline injections being given immediately before being placed in the chambers. The total VO2 for these 2 habituation days were averaged and represent baseline values for each animal. On the experimental day, animals were either injected with saline or with 3 mg/kg of NBI-12i and VO2 was recorded for the subsequent 7 h. We found that NBI-12i injected animals had lower total VO2 than those injected with saline both when represented as raw values (saline 3932 ± 85 vs. NBI-12i 3568 ± 96 ml/kg·h; n = 16, P < 0.01), and when represented as percentage of the average baseline for each animal (Fig. 4, saline –0.8 ± 0.6% vs. NBI-12i –9.1 ± 0.6%, P < 0.001). Consistent with the VO2 findings, energy expenditure was also significantly decreased (saline 0.56 ± 0.01 vs. NBI-12i 0.51 ± 0.01 kcal/h; n = 16, P < 0.01). Changes in VO2 were accompanied by changes in VCO2 such that the RQ did not change, indicating that there were no major alterations in the relative oxidation of fats and carbohydrates (RQ: saline 0.78 ± 0.01 vs. NBI-12i 0.79 ± 0.01;. n = 16, P = 0.71).
FIG. 4. Change in oxygen consumption from baseline value with NBI-12i injection. On the experimental day, animals (n = 16/group) were either injected with saline or with 3 mg/kg of NBI-12i and VO2 was recorded for the subsequent 7 h. NBI-12i-treated animals had lower total VO2 than those given a vehicle injection (P < 0.001). Data are represented as percentage of change from baseline (treatment-baseline/baseline x 100).
LLC cancer cachexia
We have previously demonstrated that cancer cachexia can be reversed and prevented with genetic and pharmacologic blockade of the MC4-R, with peptide antagonists administered intracerebroventricularly (25, 37). We sought to test the efficacy of NBI-12i in preventing the relative anorexia and loss of lean body mass that occurs with tumor growth in a murine cancer model. Mice were anesthetized, scanned with a DEXA scanner, then implanted with tumor cells. Food intake and body weight was measured daily beginning d 8 after tumor inoculation. At the end of the study, the animals were killed, tumors were dissected and weighed, and the carcass was subjected to DEXA scanning. In previous studies, we found that the tumor-bearing animals would begin to show relative anorexia starting approximately 2 d after the tumors became palpable under the skin, and that they would need to be killed approximately 1 wk after this due to tumor volume. Thus, on d 11, animals were randomly assigned to groups and drug treatment was started. Each animal received a total of 4 d of ip injection with either vehicle (n = 11) or 3 mg/kg NBI-12i (n = 15) twice per day before the animals were killed. Food intake of the vehicle-treated controls was decreased by approximately 50% compared with mice treated with NBI-12i by the end of the experiment (Fig. 5A; two-way mixed design ANOVA; dose, F1, 24 = 25.79, P < 0.001; day, F7, 168 = 4.502, P < 0.001; interaction, F7, 207 = 10.06, P < 0.001). The food intake of the drug-treated animals was significantly greater than that of the saline control for the final 3 d of the study (post hoc test, all P values < 0.01). The change in body weight, lean mass, and fat mass were analyzed in terms percent change from baseline and as a difference score (data not shown). Results were the same with both types of analyses. When compared with their starting weight, animals treated with NBI-12i gained significantly more weight than those treated with saline (Fig. 5B; saline 2.9 ± 1.8% vs. NBI-12i 11.5 ± 2.4%, P < 0.05), but the final tumor mass was not different between groups (data not shown; saline 1.4 ± 0.07 g vs. NBI-12i 1.5 ± 0.06 g, P = 0.4). DEXA analysis revealed that the saline control group gained significantly less lean mass than the NBI-12i-treated group (Fig. 5C; saline 3.5 ± 1.8% vs. NBI-12i 13 ± 2.6% P < 0.01), and accumulated less fat (Fig. 5D; saline 7.2 ± 4.1% vs. NBI-12i 19 ± 2.9%, P < 0.05).
FIG. 5. Food intake and body composition analysis in tumor-bearing animals. All mice had tumors implanted at d 0, and were given either vehicle (n = 11) or 3 mg/kg NBI-12i (n = 15) twice a day starting on d 10. A, Daily food intake was significantly greater in NBI-12i-treated mice on d 11–13 (two-way ANOVA; dose, F1, 24 = 25.79, P < 0.001; day, F7, 168 = 4.502, P < 0.001; interaction, F7, 207 = 10.06, P < 0.001; post hoc P values < 0.01 for d 13–15; *, significantly different from vehicle-control). B, The percent change in body weight was greater in NBI-12I-treated mice relative to controls (P < 0.05). C, The percent change in lean body mass by serial DEXA scan demonstrated a relative preservation of lean mass in the NBI-12i injected animals (P < 0.01). D, The percent change in body fat mass was greater with NBI-12i than with saline injection (P < 0.05).
Discussion
Cachexia is a common feature of a variety of diseases, and is often associated with poor clinical outcome (1, 2, 38, 39). The cardinal features of cachexia are relative anorexia, a lack of a protective decrease in basal metabolic rate with weight loss, and a high rate of loss of lean body mass. Drugs that are currently in use for cachexia have not been able to reverse all of these processes and therefore have been of limited clinical utility. Recently our group and others have demonstrated that blockade of the central melanocortin system can prevent cachexia in models of acute infection and inflammation as well as in chronic cancer and renal failure (22, 23, 24, 25, 40). The fact that melanocortin blockade reverses or prevents cachexia in diseases with very different etiologies indicates that MC4 antagonists are likely to block a key integrative site in this process and therefore be of widespread clinical utility.
The potential therapeutic utility of an MC4 antagonist prompted us to embark on a program to identify small molecule MC4 receptor antagonists (Ref. 41 and see Footnote 1). The desired features of such molecules are high affinity and selectivity for the MC4 receptor, antagonism of MC4-mediated responses in functional assays, good central nervous system penetration and desirable pharmacokinetic properties for systemic administration. NBI-12i meets all of these criteria, having nanomolar affinity for the MC4 receptor and 30- to 200-fold selectivity over other melanocortin receptor subtypes, and approximately 1000-fold selectivity over 57 other receptors, enzymes and transporters tested. The receptors with affinity for NBI-12i closest to that of MC4 were NK1 (49-fold), NK2 (173-fold) and ghrelin (11-fold). Given that NBI-12i had no effect in the MC4-R KO mouse, it is likely that this compound’s effect on food intake is due to its activity at the MC4-R and not the ghrelin receptor. If ghrelin activity did contribute to NBI-12i’s effects on feeding in vivo, increased food intake in the MC4-R KO mouse would be expected.
Previous studies of the effects of central melanocortin antagonism have primarily relied on intracerebroventricular injections of agouti-related peptide (AgRP), an endogenous mixed MC3-R/MC4-R antagonist. Our data differ from those produced with AgRP in several important respects. First, a single injection of AgRP produces a prolonged increase in food intake (up to a full week from a single injection), whereas short-term effects on feeding are more difficult to discern (42). In the present studies with NBI-12i administered peripherally, we observed significant increases in food intake for up to 6 h. Whereas these differences may simply be due to the half-life of these compounds in the brain, it is also possible that AgRP has other important properties that distinguish it from a pure MC4 antagonist. Indeed, AgRP has been shown to function as an inverse agonist at the MC4-R, and this property may be important for its long duration of action (43, 44). It is also true that AgRP is a potent antagonist of the MC3-R, and the fact that MC3/4-R double knockout mice are more obese than either single knockout strain may indicate that blockade of the MC3-R provides some synergism with blockade of the MC4-R (45). However, we have found that MC3-R KO mice are more prone to cachexia than WT mice, indicating that the ability of AgRP to block the MC3-R is not an important feature of its anticachexia properties (25). This idea is bolstered by our current data that clearly demonstrates that the MC4-R, but not the MC3-R, is necessary for the stimulatory effect on feeding of our antagonist compound. The fact that the duration of the feeding effect is not prolonged in the MC3-RKO mouse is further evidence that the long duration of action of AgRP is not due to its ability to simultaneously block the MC3-R and the MC4-R.
Another relevant physiological effect of AgRP is its ability to decrease basal metabolic rate (36). It is known that basal metabolic rate normalized to lean body mass is elevated in individuals suffering from a variety of chronic diseases, and this is likely to be an important contributor to the development of cachexia (46, 47). Thus, drugs that are most likely to be efficacious in reversing cachexia should be effective in decreasing basal metabolic rate to an extent similar to that found with AgRP administration. We have demonstrated that NBI-12i can effectively decrease basal metabolic rate in groups of normal animals. Furthermore, during the final night of our cancer study, saline-injected animals lost significantly more weight than animals injected with NBI-12i even though both groups were being fasted in preparation for the final DEXA scan. This implies that some of the protection against weight loss is likely to be due to decreasing energy expenditure in this model. Of course, our studies do not rule out the possibility that these data indicate that drug-treated animals drank more fluid or ate more nonfood items (e.g. bedding) during the final night of this study.
The most important proof of efficacy of our small molecule MC4 antagonist was provided by the studies of cancer cachexia. We have previously demonstrated that genetic and pharmacologic blockade of signaling through the MC4-R attenuates the anorexia, increase in basal metabolic rate, and loss of lean body mass that is normally found in experimental models of cachexia (25, 37). Recently, Vos and colleagues (26) have demonstrated that sc administration of a small-molecule melanocortin antagonist can prevent weight loss due to the growth of a CT-26 tumor in a xenograft mouse model. In this study, there were no data regarding the effect on food intake, body composition, or metabolic rate, and no studies were performed to demonstrate receptor specificity in vivo. Nonetheless, this study again provides evidence that the development of potent melanocortin antagonists with high specificity and central nervous system penetration is technically feasible and that these compounds have the potential to provide effective therapy for cachexia in a wide variety of disease states.
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Address all correspondence and requests for reprints to: Daniel L. Marks M.D., Ph.D., Department of Pediatrics, Mailcode CDRCP, 707 Southwest Gaines Road, Portland, Oregon 97239. E-mail: marksd@ohsu.edu.
Abstract
Cachexia is metabolic disorder characterized by anorexia, an increased metabolic rate, and loss of lean body mass. It is a relatively common disorder, and is a pathological feature of diseases such as cancer, HIV infection, and renal failure. Recent studies have demonstrated that cachexia brought about by a variety of illnesses can be attenuated or reversed by blocking activation of the melanocortin 4 subtype receptor (MC4-R) within the central nervous system. Although the potential use of central MC4-R antagonists for the treatment of cachexia was supported by these studies, utility was limited by the need to deliver these agents intracerebroventricularly. In the current study, we present a series of experiments demonstrating that peripheral administration of a small molecule MC4-R antagonist can effectively stimulate daytime (satiated) food intake as well as decrease basal metabolic rate in normal animals. Furthermore, this compound attenuated cachexia and preserved lean body mass in a murine cancer model. These data clearly demonstrate the potential of small molecule MC4-R antagonists in the treatment of cachexia and underscore the importance of melanocortin signaling in the development of this metabolic disorder.
Introduction
INVOLUNTARY WEIGHT LOSS is a feature of many acute and chronic diseases. Unlike simple starvation, weight loss during illness is not associated with a significant degree of protective metabolic or behavioral responses, and it leads to enhanced loss of lean body mass. This type of weight loss, known as cachexia, is found in patients with a variety of diseases including cancer, renal failure, heart failure, and chronic infections (1). The severity of cachexia in these illnesses is often the primary determining factor in both quality of life and in eventual mortality (1, 2). Unfortunately, despite our increased understanding of this process, there is currently no safe and effective treatment for this condition. Extensive trials with antiinflammatory agents have shown modest benefit, and significant gastrointestinal and renal side effects are common (3). Perhaps the most promising agents in early trials were the progestational agents (e.g. megesterol acetate), but more recent trials have shown that the modest weight gain produced by these agents is primarily due to gains in body fat and water retention, with little beneficial effects on lean body tissues (4). GH has also received attention as a potential anticachexia agent, but its usefulness is limited by cost, difficulty of delivery, worsened insulin resistance, potential to promote the growth of tumors in individuals with cancer, and GH resistance in most forms of cachexia (5, 6). Thus, there is a critical need for the development of new agents with efficacy in treating cachexia brought about by a variety of diseases.
There is a growing body of evidence to indicate that cachexia in disorders as different as HIV disease and congestive heart failure may have a common mechanism that involves the action of elevated levels of circulating cytokines (7, 8, 9, 10, 11). This is also true in chronic renal failure, where circulating concentrations of cytokines such as IL-1, IL-6, and TNF- are increased in patients with uremia and correlate with the degree of cachexia in these individuals (12, 13). Collectively, the existing animal and human data strongly suggest that increases in the level of circulating inflammatory cytokines can produce all of the major features of cachexia, including anorexia, increased metabolic rate, and loss of lean body mass. The association between elevated circulating cytokines and cachexia has led to the proposal of numerous hypotheses regarding the mechanism underlying this process. At this point, most authors reason that one important mechanism involves the action of cytokines released during inflammation and malignancy on the central nervous system. Ultimately, these cytokines are thought to alter the release and function of a number of key neurotransmitters, thereby altering appetite, metabolic rate, and nutrient partitioning (1, 14, 15, 16, 17).
The hypothalamic arcuate nucleus is known to be critical for the control of body weight and is responsive to numerous circulating compounds, including cytokines (18, 19). Proopiomelanocortin (POMC) is a propeptide precursor that is produced in neurons found in the hypothalamic arcuate nucleus (20). POMC neurons are thought to provide an important tonic inhibition of food intake and energy storage, primarily via production and release of -MSH from the POMC precursor. -MSH binds to central melanocortin receptors (particulary the type 4 melanocortin receptor, MC4-R) where it acts to inhibit food intake (21). Recently, our own lab and others (22, 23, 24, 25) have demonstrated that cachexia in acute and chronic disease models in rodents can be reversed by genetic or pharmacologic blockade of central melanocortin signaling via the MC4-R. Thus, antagonists of central MC4-R are plausible candidates for novel therapeutics for cachexia.
We describe here the characteristics of NBI-12i as a potent, selective, and bioavailable small molecule MC4-R antagonist. We have examined the effects of peripheral administration of NBI-12i on food intake in normal mice, receptor knockout mice, and for its ability to attenuate cachexia in a murine cancer model.
Materials and Methods
In vitro characterization
Cell culture.
Human embryonic kidney (HEK) 293 cells expressing human MC1, MC3, MC4. and MC5 receptors, and mouse MC3 and MC4 receptors, were obtained as previously described (27). All of these cell lines were cultured in DMEM, supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 0.2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively), and 200 μg/ml G418. Cells were grown in a humidified atmosphere of 7% CO2 at 37 C.
Preparation of cell membranes.
P2 membrane fractions of cells were prepared using a high-pressure nitrogen cell and differential centrifugation as previously described (28) The protein concentration in the membrane pellet was determined using the Coomassie method (Pierce, Rockford, IL), using BSA as the standard. Membranes were stored at –80 C before use.
Radioligand binding assays.
Binding assays were set up in low-binding 96-well plates (no. 3605; Corning, Palo Alto, CA). The assay buffer was 25 mM HEPES, 1.5 mM CaCl2, 1 mM MgSO4, 100 mM NaCl (pH 7). The following were added sequentially to the plates: 75 μl [125I]NDP-MSH [specific activity 2200 Ci/mmol, PerkinElmer Life Sciences (Boston, MA)], 75 μl unlabeled ligand, and 50 μl membrane suspension. The final concentration of [125I]NDP-MSH used was 200 pM. The assay was incubated for 90 min at room temperature, then bound and free radioligand were separated by rapid filtration as previously described (28). Glass fiber grade C filters were pretreated for 20 min with 0.1% polyethylenimine. Radioactivity on filters (Auger electrons) was counted using a Topcount NXT (Packard, Meriden, CT) at 35% efficiency, and the total amount of radioligand added to the assay was measured using a Packard Cobra II counter (78% efficiency). In all assays, total radioligand bound to the filter (total binding) was 20% or less of the total amount of radioligand added.
cAMP accumulation assays.
HEK293 cells were plated 24 h before assay on polylysine-coated 96-well tissue culture plates at a density of 10,000 cells/well. Immediately before assay cells were washed with 200 μl/well PBS. Subsequently 50 μl cAMP assay buffer was added to the wells (DMEM without phenol red supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, and 1 mM isobutylmethylxanthine). Twenty-five microliters of NBI-12i were then added, followed 2 min later by 25 μl -MSH. Cells were then incubated for 30 min at 37 C in 7% CO2. After cell lysis, cAMP was measured by chemiluminescent immunoassay (Applied Biosystems, Bedford, MA).
In vivo characterization
Animals.
C57BL/6J male mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were used for the feeding, metabolism, and cachexia studies. MC3-R knockout (MC3-RKO) mice and their wild-type (WT) controls were derived from the original C57BL/6Jx129 colonies (29, 30) maintained within the Vollum Institute that had been bred seven generations into the C57BL/6J strain and maintained as homozygous lines. MC4-RKO mice, described previously (29), were bred 10 generations into the same C57BL/6J strain. KO mice were raised group housed, weaned at 21 d, and allowed ad libitum access to powdered Laboratory Rodent Diet (Purina, St. Louis, MO).
Before the start of an experiment, mice were housed individually for at least 7 d. Additionally, for the behavioral experiments, they were handled daily for a minimum of 5 d. All animals used in these studies were between 5 and 7 wk of age at the start of the study. Previous studies have demonstrated that differences in food intake between WT and MC4-RKO animals are increased when the animals are on a chow of moderate fat content (31). Therefore, all animals were maintained on Purina 5015 chow (13% fat) for the behavioral studies. All studies were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animal and approved by the Animal Care and Use Committee of the Oregon Health Sciences.
NBI-12i preparation and administration.
NBI-12i was synthesized in the Medicinal Chemistry Department of Neurocrine Biosciences Inc. using the synthesis methods described by Tucci and colleagues1. In all studies, the compound was administered to the ip cavity. NBI-12i was dissolved in normal saline daily. Knockout mice and littermate controls had basal feeding monitored for 2 d, and then during each 12-h period after an ip saline injection to demonstrate that the observed effects were not due to differential stress responses observed in other models of melanocortin blockade (32). In the daytime (satiated) feeding studies, all compounds were injected ip at 0800 h, and food intake was measured at hourly intervals. For the nighttime studies, compounds were injected at lights out, and food intake was measured every 2 h overnight. All animals were weighed before compound injection, and the doses were normalized to individual animal body weight. In the tumor studies, the drug was injected twice daily at 0800 and 1600 h at a dose of 3 mg/kg. Food intake and body weight were measured daily.
Food intake measurement.
Animals were individually housed for a minimum of 1 wk before starting each experiment. Animals were habituated to eating powdered mouse chow from containers designed to minimize spill and contamination of the remaining food. Food intake was measured at hourly intervals or daily at the same time.
Indirect calorimetry.
Oxygen consumption (VO2) and carbon dioxide production (VCO2) were simultaneously determined by indirect calorimetry (Oxymax, Columbus Instruments, Columbus, OH) while animals were housed in separate chambers at 24 ± 1 C. Mice were first acclimatized to the chambers for 2 d. Measurements were recorded for 6–8 h during the middle of the light cycle (1100–1600 h). Samples were recorded every 3 min with the room air reference taken every 30 min and the air flow to chambers 500 ml/min. Total oxygen consumption was determined by averaging all of the samples recorded corresponding to periods of movement as well as inactivity. The respiratory quotient (RQ) was calculated as the molar ratio of VO2:VCO2.
Tumor inoculation.
Lewis lung carcinoma (LLC) cells were maintained as a primary culture in DMEM with 10% fetal bovine serum as recommended by the supplier (American Type Culture Collection, Manassas, VA). We have found that the anorexia and cachexia produced by this cell line is quite variable, so we first subcloned this cell line and established 10 separate cell lines that were screened for the degree of cachexia produced in experimental animals. The subclone line that produced the most consistent cachexia was then expanded, separated into aliquots, and stored in liquid nitrogen. For each study, a fresh sample of this subcloned cell line was removed from storage and cultured. LLC tumor cells were harvested during exponential growth of the culture, washed in Hanks’ balanced salt solution, and 1 x 106 cells were injected sc into the upper flank of the mice. Sham-injected animals received an implant of a similar amount of heat-killed tumor cells. In all cases, the time of appearance of a tumor mass was noted in the log, and all experimental animals were found to have a palpable tumor within 5 d of the start of the experiment. At the time the animals were killed, tumors were dissected away from surrounding tissue and weighed. Gross examination of organs did not reveal the presence of any observable metastasis.
Body composition.
Body composition was determined at the start and the end of the LLC experiment by dual-energy x-ray absorbtometry (DEXA, PIXImus mouse densitometer, Lunar Corp.) as previously described (25). The instrument was calibrated at the start of each recording session with a murine calibration standard. All animals were fasted for 12 h before DEXA analysis to minimize the effect of ingested food on the DEXA analysis. Animals were anesthetized before the first scan, and asphyxiated with CO2 before the tumor dissection and the final scan.
Statistical methods.
Radioligand binding data were fitted to a four parameter-logistic equation using XLfit (ID Business Solutions Ltd., Emeryville, CA) to provide fitted values of inhibition constant (Ki). Ligand concentration-dependence data from cAMP accumulation assays were fitted to a four parameter-logistic equation using Prism 3.0 (GraphPad Software, San Diego, CA) to determine EC50 and Emaximal effect. Antagonism of -MSH-stimulated cAMP accumulation was analyzed by the method of Arunlakshana and Schild (33).
Differences among groups in feeding and activity in all experiments were analyzed by two-way, repeated measures ANOVA with time and treatment as the measured variables. Post hoc effects were determined by Fisher’s least significant difference test. Final tumor and body weights, and body composition by DEXA were analyzed by Student’s t test when two groups were included, or one-way ANOVA with post hoc analysis when three or more groups were included. Data sets were analyzed for statistical significance using SigmaStat (SPSS, Inc., Chicago, IL).
Results
Receptor pharmacology
The affinity of NBI-12i for melanocortin receptor subtypes was determined using [125I]-NDP-MSH binding to membranes prepared from HEK293 cells expressing individual human or mouse receptors. As demonstrated by Tucci et al. (see footnote 1), NBI-12i was a selective inhibitor of binding to human MC4 receptors (Table 1; Fig. 1A), with a Ki value of 9.8 nM, having 245-, 122-, and 32-fold selectivity over human MC1, 3, and 5 receptors, respectively. The affinity of NBI-12i for mouse MC4 and MC3 receptors was similar to that of human (Table 1; Fig. 1A), with an mMC4:mMC3 selectivity ratio of 48. In an assay of MC4 receptor function, NBI-12i at concentrations up to 3 μM induced no significant accumulation of cAMP in HEK293 cells stably expressing hMC4, but antagonized -MSH-stimulated cAMP accumulation (Fig. 1B). Increasing concentrations of NBI-12i produced rightward shifts in the -MSH concentration-response curve, and Schild analysis (Fig. 1C) revealed a pA2 value of 7.22 ± 0.13 (60 nM) with a slope of 1.02 ± 0.12 (mean ± SEM, n = 3). An approximately 25% reduction in the maximal effect of -MSH was observed at the highest concentration of NBI-12i tested (3 μM). These data are consistent with the idea that NBI-12i acts as a competitive antagonist of hMC4 receptors.
TABLE 1. NBI-12i and -MSH affinity for human and mouse melanocortin receptor subtypes
FIG. 1. Receptor pharmacology of NBI-12i. A, Inhibition of [125I]NDP-MSH binding to melanocortin receptors by NBI-12i. Radioligand binding to the receptors in HEK293 cell membranes was measured, and the data analyzed, as described in Materials and Methods. Data are from representative experiments performed three times. B, Schild analysis of NBI-12i antagonism of -MSH-stimulated cAMP accumulation in HEK293 cells expressing the human MC4 receptor. The experiment was performed and data analyzed as described in Materials and Methods. -MSH concentration dependence for stimulating cAMP accumulation in the presence of a range of concentrations of NBI-12i. Data are from a representative experiment performed three times. C, Schild plot of antagonism by NBI-12i. Data are mean ± SEM of dose ratios from three independent experiments.
The wider receptor selectivity of NBI-12i was examined in a panel of radioligand binding assays for the following receptors, ion channels enzymes and transporters: adenosine (A1, A2a, A3); adrenergic (1, 2, ?1, ?2), angiotensin (AT1), benzodiazepine, bradykinin (B1); CGRP, cannabinoid (CB1, CB2), cholecystokinin (CCKA, CCKB), dopamine (D1, D2S, D3, D4.4), endothelin, -aminobutyric acid, glutamate (-amino-3-hydroxy-5-methyl-4-isoxazole propionate, N-methyl-D-aspartate, kainate), histamine (H1, H2, H3), imadazole (I1, I2), leukotriene (LTD4), monoamine oxidase (MAO-A, MAO-B), muscarinic (M1, M2, M3, M4, M5); neuropeptide Y, nicotinic, opiate and opiate receptor-like 1, purinergic (P2X, P2Y), serotonin, somatostatin, glucocorticoid, estrogen, progesterone, androgen, TRH, vasopressin (V1a, V2), norepinephrine, dopamine, serotonin, -aminobutyric acid, and choline transporters. In all of these assays, NBI-12i gave less than 50% inhibition at a concentration of 10 μM. Significant activity of NBI-12i was observed in radioligand binding assays for human neurokinin-1 (NK1) and -2 (NK2) receptors and the human ghrelin receptor. Concentration-response curves revealed that NBI-12i had Ki values of 480, 1700, and 110 nM for NK1, NK2 and ghrelin receptors, respectively, representing affinities 49-, 173-, and 11-fold less that that observed for NBI-12i at hMC4.
Light-phase feeding
A dose response analysis of the effect of NBI-12i on daytime food intake was performed. Mice were randomly assigned to groups (n = 5/group) and vehicle, 1, 3, or 6 mg/kg of NBI-12i (ip) was administered at 0700 h. Cumulative food intake was significantly increased by the 2 highest doses of NBI-12i (3 and 6 mg/kg) at 4 and 6 h after administration (Fig. 2; two-way mixed design ANOVA; dose, F3, 16 = 4.58, P < 0.05; time, F2, 32 = 82.50, P < 0.01; interaction, F6, 59 = 2.94, P < 0.05).
FIG. 2. Effect of NBI-12i on cumulative food intake. Groups of mice (n = 5/group) received 0, 1, 3, or 6 mg/kg of NBI-12i at 0700 h. The 3 and 6 mg/kg doses significantly increased feeding relative to vehicle-treated animals at 4 and 6 h after treatment (two-way ANOVA; dose, F3, 16 = 4.58, P < 0.05; time, F2, 32 = 82.50, P < 0.01; interaction, F6, 59 = 2.94, P < 0.05, * significantly different from vehicle control).
MC3-RKO and MC4-RKO mice
To test the receptor specificity of this compound in vivo, we examined the ability of NBI-12i to stimulate feeding in MC4-RKO mice. In an initial experiment, we tested groups of MC4-RKO mice and compared daytime feeding in response to 3 mg/kg NBI-12i or saline. In this case, we saw no stimulation of feeding by NBI-12i at any time point studied (2 h saline 0.34 ± 0.1, NBI-12i 0.34 ± 0.07; 2–4 h saline 0.09 ± 0.05, NBI-12i 0.086 ± 0.03; 4–6 h saline 0.11 ± 0.07, NBI-12i 0.09 ± 0.03; n = 10 per group, ANOVA P = 0.98). In a second study, we compared the responses of groups of MC4-RKO, MC3-RKO, and WT mice to injections of saline or NBI-12i. We observed a significant increase in feeding in both WT and MC3-RKO mice that was not present in MC4-RKO mice. In this case, the stimulation of feeding was still apparent between 4 and 6 h post injection (Fig. 3; two-way ANOVA group, F5, 135 = 19.03, P < 0.001; time, F2, 135 = 80.79, P < 0.001; interaction, F10, 152 = 1.93, P < 0.05, n = 7 per group for KO mice, n = 10 for WT mice).
FIG. 3. Effect of NBI-12i or vehicle (veh) in WT (A), MC3-RKO (B), and MC4-RKO (C) mice. NBI-12i (3 mg/kg) significantly increased light-phase food intake in both WT and MC3-RKO mice but not in MC4-RKO mice (n = 7 per group). The stimulation of feeding was still apparent between 4 and 6 h after injection (two-way ANOVA group, F5, 135 = 19.03, P < 0.001; time, F2, 135 = 80.79, P < 0.001; interaction, F10, 152 = 1.93, P < 0.05, n = 7 per group for KO mice, n = 10 for WT mice; *, significantly different from vehicle-control).
Metabolic effects
Previous investigators have demonstrated that blockade of the MC4-R is associated with a decrease in basal VO2, whereas melanocortin agonists are found to increase basal VO2 (34, 35, 36). To investigate the effects of NBI-12i on metabolic rate, we first habituated groups of C57BL/6J mice (n = 16/group) to indirect calorimeter cages for 2 d, with saline injections being given immediately before being placed in the chambers. The total VO2 for these 2 habituation days were averaged and represent baseline values for each animal. On the experimental day, animals were either injected with saline or with 3 mg/kg of NBI-12i and VO2 was recorded for the subsequent 7 h. We found that NBI-12i injected animals had lower total VO2 than those injected with saline both when represented as raw values (saline 3932 ± 85 vs. NBI-12i 3568 ± 96 ml/kg·h; n = 16, P < 0.01), and when represented as percentage of the average baseline for each animal (Fig. 4, saline –0.8 ± 0.6% vs. NBI-12i –9.1 ± 0.6%, P < 0.001). Consistent with the VO2 findings, energy expenditure was also significantly decreased (saline 0.56 ± 0.01 vs. NBI-12i 0.51 ± 0.01 kcal/h; n = 16, P < 0.01). Changes in VO2 were accompanied by changes in VCO2 such that the RQ did not change, indicating that there were no major alterations in the relative oxidation of fats and carbohydrates (RQ: saline 0.78 ± 0.01 vs. NBI-12i 0.79 ± 0.01;. n = 16, P = 0.71).
FIG. 4. Change in oxygen consumption from baseline value with NBI-12i injection. On the experimental day, animals (n = 16/group) were either injected with saline or with 3 mg/kg of NBI-12i and VO2 was recorded for the subsequent 7 h. NBI-12i-treated animals had lower total VO2 than those given a vehicle injection (P < 0.001). Data are represented as percentage of change from baseline (treatment-baseline/baseline x 100).
LLC cancer cachexia
We have previously demonstrated that cancer cachexia can be reversed and prevented with genetic and pharmacologic blockade of the MC4-R, with peptide antagonists administered intracerebroventricularly (25, 37). We sought to test the efficacy of NBI-12i in preventing the relative anorexia and loss of lean body mass that occurs with tumor growth in a murine cancer model. Mice were anesthetized, scanned with a DEXA scanner, then implanted with tumor cells. Food intake and body weight was measured daily beginning d 8 after tumor inoculation. At the end of the study, the animals were killed, tumors were dissected and weighed, and the carcass was subjected to DEXA scanning. In previous studies, we found that the tumor-bearing animals would begin to show relative anorexia starting approximately 2 d after the tumors became palpable under the skin, and that they would need to be killed approximately 1 wk after this due to tumor volume. Thus, on d 11, animals were randomly assigned to groups and drug treatment was started. Each animal received a total of 4 d of ip injection with either vehicle (n = 11) or 3 mg/kg NBI-12i (n = 15) twice per day before the animals were killed. Food intake of the vehicle-treated controls was decreased by approximately 50% compared with mice treated with NBI-12i by the end of the experiment (Fig. 5A; two-way mixed design ANOVA; dose, F1, 24 = 25.79, P < 0.001; day, F7, 168 = 4.502, P < 0.001; interaction, F7, 207 = 10.06, P < 0.001). The food intake of the drug-treated animals was significantly greater than that of the saline control for the final 3 d of the study (post hoc test, all P values < 0.01). The change in body weight, lean mass, and fat mass were analyzed in terms percent change from baseline and as a difference score (data not shown). Results were the same with both types of analyses. When compared with their starting weight, animals treated with NBI-12i gained significantly more weight than those treated with saline (Fig. 5B; saline 2.9 ± 1.8% vs. NBI-12i 11.5 ± 2.4%, P < 0.05), but the final tumor mass was not different between groups (data not shown; saline 1.4 ± 0.07 g vs. NBI-12i 1.5 ± 0.06 g, P = 0.4). DEXA analysis revealed that the saline control group gained significantly less lean mass than the NBI-12i-treated group (Fig. 5C; saline 3.5 ± 1.8% vs. NBI-12i 13 ± 2.6% P < 0.01), and accumulated less fat (Fig. 5D; saline 7.2 ± 4.1% vs. NBI-12i 19 ± 2.9%, P < 0.05).
FIG. 5. Food intake and body composition analysis in tumor-bearing animals. All mice had tumors implanted at d 0, and were given either vehicle (n = 11) or 3 mg/kg NBI-12i (n = 15) twice a day starting on d 10. A, Daily food intake was significantly greater in NBI-12i-treated mice on d 11–13 (two-way ANOVA; dose, F1, 24 = 25.79, P < 0.001; day, F7, 168 = 4.502, P < 0.001; interaction, F7, 207 = 10.06, P < 0.001; post hoc P values < 0.01 for d 13–15; *, significantly different from vehicle-control). B, The percent change in body weight was greater in NBI-12I-treated mice relative to controls (P < 0.05). C, The percent change in lean body mass by serial DEXA scan demonstrated a relative preservation of lean mass in the NBI-12i injected animals (P < 0.01). D, The percent change in body fat mass was greater with NBI-12i than with saline injection (P < 0.05).
Discussion
Cachexia is a common feature of a variety of diseases, and is often associated with poor clinical outcome (1, 2, 38, 39). The cardinal features of cachexia are relative anorexia, a lack of a protective decrease in basal metabolic rate with weight loss, and a high rate of loss of lean body mass. Drugs that are currently in use for cachexia have not been able to reverse all of these processes and therefore have been of limited clinical utility. Recently our group and others have demonstrated that blockade of the central melanocortin system can prevent cachexia in models of acute infection and inflammation as well as in chronic cancer and renal failure (22, 23, 24, 25, 40). The fact that melanocortin blockade reverses or prevents cachexia in diseases with very different etiologies indicates that MC4 antagonists are likely to block a key integrative site in this process and therefore be of widespread clinical utility.
The potential therapeutic utility of an MC4 antagonist prompted us to embark on a program to identify small molecule MC4 receptor antagonists (Ref. 41 and see Footnote 1). The desired features of such molecules are high affinity and selectivity for the MC4 receptor, antagonism of MC4-mediated responses in functional assays, good central nervous system penetration and desirable pharmacokinetic properties for systemic administration. NBI-12i meets all of these criteria, having nanomolar affinity for the MC4 receptor and 30- to 200-fold selectivity over other melanocortin receptor subtypes, and approximately 1000-fold selectivity over 57 other receptors, enzymes and transporters tested. The receptors with affinity for NBI-12i closest to that of MC4 were NK1 (49-fold), NK2 (173-fold) and ghrelin (11-fold). Given that NBI-12i had no effect in the MC4-R KO mouse, it is likely that this compound’s effect on food intake is due to its activity at the MC4-R and not the ghrelin receptor. If ghrelin activity did contribute to NBI-12i’s effects on feeding in vivo, increased food intake in the MC4-R KO mouse would be expected.
Previous studies of the effects of central melanocortin antagonism have primarily relied on intracerebroventricular injections of agouti-related peptide (AgRP), an endogenous mixed MC3-R/MC4-R antagonist. Our data differ from those produced with AgRP in several important respects. First, a single injection of AgRP produces a prolonged increase in food intake (up to a full week from a single injection), whereas short-term effects on feeding are more difficult to discern (42). In the present studies with NBI-12i administered peripherally, we observed significant increases in food intake for up to 6 h. Whereas these differences may simply be due to the half-life of these compounds in the brain, it is also possible that AgRP has other important properties that distinguish it from a pure MC4 antagonist. Indeed, AgRP has been shown to function as an inverse agonist at the MC4-R, and this property may be important for its long duration of action (43, 44). It is also true that AgRP is a potent antagonist of the MC3-R, and the fact that MC3/4-R double knockout mice are more obese than either single knockout strain may indicate that blockade of the MC3-R provides some synergism with blockade of the MC4-R (45). However, we have found that MC3-R KO mice are more prone to cachexia than WT mice, indicating that the ability of AgRP to block the MC3-R is not an important feature of its anticachexia properties (25). This idea is bolstered by our current data that clearly demonstrates that the MC4-R, but not the MC3-R, is necessary for the stimulatory effect on feeding of our antagonist compound. The fact that the duration of the feeding effect is not prolonged in the MC3-RKO mouse is further evidence that the long duration of action of AgRP is not due to its ability to simultaneously block the MC3-R and the MC4-R.
Another relevant physiological effect of AgRP is its ability to decrease basal metabolic rate (36). It is known that basal metabolic rate normalized to lean body mass is elevated in individuals suffering from a variety of chronic diseases, and this is likely to be an important contributor to the development of cachexia (46, 47). Thus, drugs that are most likely to be efficacious in reversing cachexia should be effective in decreasing basal metabolic rate to an extent similar to that found with AgRP administration. We have demonstrated that NBI-12i can effectively decrease basal metabolic rate in groups of normal animals. Furthermore, during the final night of our cancer study, saline-injected animals lost significantly more weight than animals injected with NBI-12i even though both groups were being fasted in preparation for the final DEXA scan. This implies that some of the protection against weight loss is likely to be due to decreasing energy expenditure in this model. Of course, our studies do not rule out the possibility that these data indicate that drug-treated animals drank more fluid or ate more nonfood items (e.g. bedding) during the final night of this study.
The most important proof of efficacy of our small molecule MC4 antagonist was provided by the studies of cancer cachexia. We have previously demonstrated that genetic and pharmacologic blockade of signaling through the MC4-R attenuates the anorexia, increase in basal metabolic rate, and loss of lean body mass that is normally found in experimental models of cachexia (25, 37). Recently, Vos and colleagues (26) have demonstrated that sc administration of a small-molecule melanocortin antagonist can prevent weight loss due to the growth of a CT-26 tumor in a xenograft mouse model. In this study, there were no data regarding the effect on food intake, body composition, or metabolic rate, and no studies were performed to demonstrate receptor specificity in vivo. Nonetheless, this study again provides evidence that the development of potent melanocortin antagonists with high specificity and central nervous system penetration is technically feasible and that these compounds have the potential to provide effective therapy for cachexia in a wide variety of disease states.
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