QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.097725v1 317/2/771    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nicholson, J. R. Articles by Hofbauer, K. G. Search for Related Content PubMed PubMed Citation Articles by Nicholson, J. R. Articles by Hofbauer, K. G.

BEHAVIORAL PHARMACOLOGY

Peripheral Administration of a Melanocortin 4-Receptor Inverse Agonist Prevents Loss of Lean Body Mass in Tumor-Bearing Mice

Applied Pharmacology (J.R.N., C.S., K.G.H.) and Biophysical Chemistry (G.K.), Biozentrum, University of Basel, Basel, Switzerland; and Santhera Pharmaceuticals, Liestal, Switzerland (F.S., P.W.)

Received October 27, 2005; accepted January 23, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Cachexia affects many different chronically ill patient populations, including those with cancer. It results in loss of body weight, particularly of lean body mass (LBM), and is estimated to be responsible for over 20% of all cancer-related deaths. Currently, available drugs are ineffective, and new therapies are urgently needed. Melanocortin 4-receptor (MC4-R) blockade has been shown recently to be effective in preventing cancer cachexia in rodent models. In the present study, we have tested a MC4-R blocker, ML00253764 [2-{2-[2-(5-bromo-2-methoxyphenyl)-ethyl]-3-fluorophenyl}-4,5-dihydro-1H-imidazolium hydrochloride] (Vos et al., 2004), in vitro and in vivo. In membranes of human embryonic kidney 293 cells expressing human MC4-R, ML00253764 displaced [Nle⁴, D-Phe⁷]--melanocyte-stimulating hormone binding with an IC₅₀ of 0.32 µM. At concentrations above 1 µM, ML00253764 decreased cAMP accumulation (maximal reduction of -20%) indicative of inverse agonist activity. ML00253764 was administered twice daily (15 mg/kg s.c.) for 13 days to C57BL6 mice bearing s.c. Lewis lung carcinoma tumors. Food intake and body weight were measured, and body composition was assessed using magnetic resonance relaxometry. ML00253764 stimulated light-phase food intake relative to vehicle-treated controls (p < 0.05), although no effect was observed on 24-h food intake. During the 21 days of the experiment, the LBM of tumor vehicle-treated mice decreased (p < 0.05). In contrast, the tumor-bearing mice treated with ML00253764 maintained their LBM. These data support the view that MC4-R blockade may be a suitable approach for the treatment of cancer cachexia and that MC4-R inverse agonists may have potential as drug candidates.

Although the etiologies of the various diseases associated with cachexia are clearly different, a common element in all forms of cachexia is the patient's defense response. Both preclinical and clinical evidence has demonstrated elevated levels of proinflammatory cytokines in different forms of cachexia. Cytokines are known to act in the central nervous system to alter the functioning of feeding circuitry and thereby to influence appetite and metabolic rate. One central feeding circuit known to be affected by cytokines is the leptin/melanocortin 4-receptor (MC4-R) pathway. Indeed, it has been demonstrated that cytokine-induced anorexia may be prevented by both pharmacological and genetic blockade of central MC4-R, indicating that cytokines in the brain lead to activation of MC4-R, which results in a decrease in food intake (Huang et al., 1999; Lawrence and Rothwell, 2001; Marks et al., 2001). Furthermore, it has been postulated that chronic elevation of cytokines in various disease states results in cachexia via prolonged overactivation of central MC4-R (Marks et al., 2001).

The central melanocortin system has been recognized as an important regulator of energy balance for several years (for review, see Cone, 2005). Activation of hypothalamic MC4-R by the pro-opiomelanocortin-derived endogenous agonist -melanocyte-stimulating hormone (-MSH) decreases food intake and leads to an increase in energy expenditure, whereas blockade of MC4-R by the endogenous MC4-R inverse agonist agouti-related protein (AgRP) increases food intake, decreases energy expenditure, and leads to weight gain. The physiological importance of balance within the central melanocortin system has been demonstrated in mouse models of MC4-R knockout (Huszar et al., 1997) and AgRP overexpression (Ollmann et al., 1997) where hyperphagia and obesity are the most obvious aspects of the phenotypes observed. Furthermore, humans who have mutations in MC4-R exhibit profound hyperphagia and obesity as a consequence of reduced MC4-R signaling (Farooqi et al., 2003). However, the physiological importance of the MC4-R extends beyond the regulation of food intake, and a further consequence of blockade of MC4-R in both rodents and humans is increased linear growth, including LBM (Huszar et al., 1997; Farooqi et al., 2003). Overall, this suggests that selective MC4-R blockade could be suitable approach for the treatment of cachexia. Indeed, MC4-R knockout mice have been shown to be protected from tumor-induced (Marks et al., 2003), lipopolysaccharide-induced (Marks et al., 2001), and uremia-associated (Cheung et al., 2005) cachexia. In addition, central administration of AgRP to mice (Marks et al., 2001) and the MC3/4-R antagonist SHU-9119 to rats has been shown to protect against cancer-induced anorexia (Wisse et al., 2001). Finally, in a recent study by Markison et al. (2005), a low-mol. wt., selective, MC4-R antagonist was administered peripherally to mice and was shown to protect against tumor-induced anorexia and to increase LBM relative to vehicle-treated controls.

In the present study, we have investigated the effect of chronic peripheral administration of a MC4-R ligand, ML00253764 (Vos et al., 2004), in a murine model of cancer cachexia. This compound has been previously shown to increase body weight in CT-26 tumor-bearing BALB/c mice (Vos et al., 2004), and in the present study, we have extended these findings by investigating the in vitro characteristics of this compound and its in vivo effects on food intake and body composition in C57BL6 mice bearing s.c. Lewis lung carcinoma tumors. Body composition was determined using a traditional chemical extraction method and the more recently established method, magnetic resonance relaxometry (MRR) (Künnecke et al., 2004). We have found ML00253764 to be an inverse agonist at MC4-R in vitro. Inverse agonists act not only by antagonizing the effect of agonists but also by reducing constitutive activity of receptors. Consequently, they can reduce receptor activity in the absence of agonists and display increased efficacy in comparison with antagonists. When administered to mice, ML00253764 increased light-phase food intake and prevented tumor-induced loss of LBM. The data presented support the view that MC4-R blockade may be a suitable therapeutic approach for the treatment of cachexia and suggest that MC4-R inverse agonists may have potential as drug candidates.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
In Vitro Studies
Preparation of Membranes Containing Melanocortin Receptors. HEK-293 cells expressing human melanocortin receptors were suspended in ice-cold buffer (50 mM HEPES-NaOH, pH 7.4, 250 mM sucrose, and complete protease inhibitors in analytical H₂O) and passed through a 25-gauge syringe needle 10 times at a flow rate of 40 ml/min. Lysis of the cells was monitored by microscopy. Debris was removed from the lysate by a short centrifugation step (2500g, 10 min, 4°C). The pellet was discarded, and the supernatant was centrifuged again in a high-speed centrifuge (47,000g, 90 min, 4°C). The resulting pellet was resuspended in ice-cold buffer followed by three passages through a 25-gauge syringe needle. Aliquots of the preparation were snap-frozen in dry ice and stored at -80°C until use.

Measurement of MC4-R Binding. Binding of ligands to human MC4-R, MC3-R, and MC5-R was measured using a homogenous, nonradioactive competition assay. In brief, suitable dilutions of test ligands were prepared in dimethyl sulfoxide (DMSO), and 1 µl/well was transferred into a black 384-well plate with a nonbinding surface (number 3654; Corning Inc., Corning, NY). Twenty-nine microliters of 0.77 nM Cy3B-NDP--MSH fluorescent tracer (custom synthesis by Thermo Electron, Ulm, Germany) in assay buffer (50 mM HEPES-NaOH, 2.5 mM CaCl₂, 0.1% bovine serum albumin, and complete protease inhibitors in H₂O) and 10 µl of MC-receptor membrane preparation containing 1.6 to 6 µg of membrane protein (equivalent to 5.2 fmol receptor/well) were added. The assay was placed for 60 min at 24°C, overnight at 4°C, and 3.5 h at 24°C before reading fluorescence polarization (FP) in a FARCyte (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) or TECAN Ultra (Tecan, Männedorf, Switzerland) plate reader using an FP filter set with 535-nm excitation/590-nm emission wavelengths, 40-µs integration, and 0-µs lag times.

Measurement of the MC4-R-Induced Functional Activity (cAMP Production). cAMP levels were measured in a homogenous, nonradioactive cAMP agonist assay using membrane preparations instead of whole cells. Assay conditions (Allen et al., 2002) were as follows. Suitable dilutions of test ligands were prepared in DMSO, and 1 µl/well was transferred into a black 384-well plate with a nonbinding surface (Corning number 3654). Per well, 1.8 µg of MC4-R membrane protein diluted in 9 µl of membrane buffer (55 mM HEPES-NaOH, pH 7.4, 11 mM MgCl₂, 110 mM NaCl, and EDTA-free complete protease inhibitors in analytical H₂O) was added, and the plate was placed on ice immediately. After a preincubation of 20 min on ice, 10 µl of stimulation buffer (50 mM HEPES-NaOH, pH 7.4, 10 mM MgCl₂, 100 mM NaCl, 100 µM ATP, 2 µM GTP, and 200 µM isobutyl-methyl-xanthine in H₂O) containing 0.33 µl of anti-cAMP antibody (FPA202002KT assay kit; PerkinElmer Life Sciences, Boston, MA) was added, and the assay was transferred from ice to 37°C. After 45 min, the reaction was stopped by transferring the assay to ice and by the addition of 20 µl of ice-cold detection buffer (FP1087c, provided with the FPA202002 kit) containing 3.3 nM fluorescein-cAMP (Perkin Elmer FPA202002KT assay kit or BLG-F002; BioLog, Bremen, Germany). After 2 h on ice, the assay was allowed to warm up to 24°C for 10 to 15 min. Immediately afterward, FP was read in a FARCyte (Amersham Biosciences UK, Ltd.) or TECAN Ultra (Tecan) plate reader using an FP filter set with 485-nm excitation/535-nm emission wavelengths, 60-µs integration, and 0-µs lag times.

Data Processing of in Vitro Results. FP raw data were obtained using the XFLUOR4 plug-in (Tecan) for Excel (Microsoft, Zurich, Switzerland). The raw data were processed in custom Excel worksheets using the XLfit4 curve-fitting package (IDBS, Guildford, UK) for calculation of the result charts.

In Vitro Reagents. Reagents were purchased from Fluka Chemie GmbH/Sigma-Aldrich Corporation (Buchs, Switzerland; sucrose, CaCl₂, MgCl₂, H₂O, bovine serum albumin, ATP, and GTP), Acros Organics (Geel, Belgium; DMSO, 3-isobutyl-1-methylxanthine), VWR International AG Life Science (Lucerne, Switzerland; HEPES), and Roche Diagnostics (Basel, Switzerland; complete protease inhibitor tablets).

In Vivo Studies
Animals. Male C57BL6 mice (20 g) were obtained from RCC (Füllinsdorf, Switzerland) and were individually housed for at least 7 days before the start of experiments under conditions of controlled temperature (21-22°C) and a 12/12-h light/dark cycle (lights on from 6:00 AM to 6:00 PM). The mice were placed on a standard ad libitum chow diet (NAFAG 3432; 3.0 kcal/g, 61.6% of total calories from carbohydrate, 24.8% of total calories from protein, and 13.6% of total calories from fat) and had free access to tap water. All experiments were performed in accordance with the Swiss regulations for animal experimentation.

Tumor Cell Culture and Implantation. Lewis lung carcinoma (LLC) cells were purchased from American Type Culture Collection (Molsheim, France) and were maintained as a primary culture as recommended by the supplier. Cells were harvested during the exponential growth phase and were suspended in phosphate-buffered saline for injection. For the implantation of tumor cells, mice were momentarily anesthetized with isoflurane (4% with medicinal O₂ as the carrier gas) and received a s.c. injection of 1 x 10⁶ cells in 100-µl volume in the upper flank. Sham control mice underwent the same procedure but received 100 µl of phosphate-buffered saline.

Compound Administration. ML00253764 was synthesized at Santhera Pharmaceuticals (Liestal, Switzerland). The compound was weighed out and dissolved in polyethylene glycol 200/saline (1:10) just before each application and was injected s.c. in a volume of 10 ml/kg. The dose of 15 mg/kg was selected based on the results of the study by Vos et al. (2004). Applications were b.i.d. during the early light phase (2-3 h after lights on) and during the last hour before the onset of the dark cycle from day 8 postimplantation of the LLC cells until the end of the study (day 21).

Measurement of Light-Phase Food Intake. All measurements of food intake were conducted in the home cage. At the start of the study, enough chow was placed into each food hopper for the duration of the study, and this was weighed and returned to the hopper twice daily, at the time of each injection, to determine light phase and 24-h food intake.

Assessment of Tumor Size. Quantitative assessment of tumor size was carried out upon completion of the study by weighing the tumor that had been dissected from each animal.

Measurement of Body Composition. Body composition was measured in conscious mice by MRR during the light phase just before tumor inoculation (day 1) and 21 days postimplantation of the tumor cells. The second measurement was taken post mortem in mice that had been killed by CO₂ asphyxiation and had had their s.c. tumors removed. Sham mice underwent the same procedure, including opening of the skin. Care was taken to ensure that the same time ensued between death and the second MRR measurement for all animals.

MRR method. NMR relaxometry measurements were carried out on a Bruker Biospec 70/20 (7 T, 300 MHz) using a commercial whole-body ¹H resonator (Bruker, Newark, DE). Whole-body transversal relaxation was used for body-composition analysis of conscious mice. The method used has been described previously (Künnecke et al., 2004). Few changes were necessary to transfer the method to the spectrometer used in the present study. At the beginning of each measurement series, the magnetic field was homogenized by using the in-built Bruker autoshim procedure on the first mouse. If necessary, the shim was improved manually until the half-line width of the water proton line was below 250 Hz. For each measurement sequence, the probe was tuned and matched, and the pulse power was individually adjusted using the inbuilt pulse power calibration routine. A Carr-Purcell-Meiboom-Gill multispin echo sequence was used. The initial 90° excitation was performed by a rectangular 150-µs pulse. The following 256 refocusing 180° rectangular pulses had a duration of 300 µs and were each separated by an echo time of 3 ms. Two averages were taken for one sequence with a repetition time of 10s. Three sequences were averaged for each mouse.

Proton relaxations were obtained by picking the maximal echo amplitude of each echo signal of the Carr-Purcell-Meiboom-Gill signal. Body composition was calculated by processing the time domain of proton relaxation by an inverse Laplace transformation as provided by CONTIN software (Provencher, 1982). The area of the two separated peaks was assumed to be proportional to the number of protons of tissue water and of fat. Conversion to absolute weight (in grams) was performed using agarose phantoms (Künnecke et al., 2004) whose transversal relaxation times (T₂) were adjusted to 31 and 200 ms for LBM and fat, respectively.

Chemical analysis of body composition. After the second MRR measurement, body composition was assessed by chemical extraction as described by Markewicz et al. (1993). The abdominal cavity of each mouse was opened, the bladder was emptied, and the gastrointestinal tract was removed. The gastrointestinal tract content was pushed out of the tract, which was then returned to the carcass of each mouse. Care was taken not to lose blood during this procedure. The carcasses were then weighed to establish the wet weight of each animal. Mice were placed into preweighed metal containers, covered with aluminum foil, and placed into an oven at 65°C for one week. After 1 week, three daily weight measurements were taken, and when they gave consistent values, the carcasses were considered to be completely dry. The final dry measurement taken was considered the dry weight of the mice, and the water mass of each animal was calculated by subtracting wet weight from the dry weight. Mouse carcasses were then wrapped in fat-free paper and exposed to chloroform extraction of fat using standard Soxhlet's apparatus. Following fat extraction, carcasses were placed in a hood overnight to allow for the complete evaporation of chloroform and were then reweighed to establish fat-free dry mass. Subtraction of the fat-free dry mass from the dry mass gave a measure of fat mass for each mouse and the sum of the fat-free dry mass, and the calculated water mass was considered to be a measure of lean body mass.

Statistical Methods. Light-phase food intake in mice was analyzed using the unpaired two-tailed Student's t test. Analysis of body composition data was done using two-way repeated measures ANOVA with time and treatment as the measured variables. Post hoc analysis was carried out using the Student-Newman-Keuls method. Statistical tests were performed using SigmaStat and Excel software. A statistical comparison of the MRR and chemical extraction methods was conducted according to the method of Bland and Altman (1986).

	Results

Top Abstract Materials and Methods Results Discussion References

 
In Vitro Pharmacology
In a nonradioactive assay using HEK-293 cells, which expressed hMC4-R, ML00253764 (Fig. 1) displaced NDP--MSH binding with a mean IC₅₀ of 0.32 µM. With hMC3-R and hMC5-R, IC₅₀ values of 0.81 and 2.12 µM were obtained, indicating approximately 2.5-fold selectivity for MC4-R over MC3-R and approximately 7-fold over MC5-R (Fig. 2a). In the cAMP functional activity assay on membranes expressing human MC4-R, ML00253764 decreased cAMP accumulation at concentrations above 1 µM with a maximal decrease of -20%. AgRP(83-132) decreased cAMP accumulation in this assay at concentrations above 1 nM with a maximal decrease of -40% (Fig. 2b). On membrane preparations from cells expressing human MC3- or MC5-R, ML00253764 did not show any induction of cAMP activity at concentrations up to 100 µM.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Chemical structure of ML00253764 (Vos et al., 2004).

View larger version (11K): [in this window] [in a new window]   Fig. 2. Binding and functional activity data for ML00253764. a, concentration-dependent displacement of NDP--MSH binding by ML00253764 in HEK-293 cells expressing human MC3-R, MC4-R, and MC5-R. b, functional activity of MC4-R as assessed by cAMP production in a cell membrane preparation. Data are expressed as mean ± S.E.M. from triplicate observations.

Food Intake
Mice were treated with ML00253764 from day 8 postimplantation of the tumor cells until the end of the study. Cumulative 13-day light-phase food intake was significantly increased by ML00253764 treatment in both sham and tumor-bearing mice (p < 0.05; Fig. 3a). However, there was no effect of ML00253764 on cumulative 13-day 24-h food intake, either in the sham or the tumor-treated groups (p = 0.80 and 0.08, respectively; Fig. 3b).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Cumulative light phase and 24-h food intake in sham control and tumor-bearing mice. Mice were injected s.c. b.i.d. with 15 mg/kg ML00253764 during the early light phase and just before the onset of the dark phase, from day 8 postimplantation of the tumor cells until the termination of the study (day 21). Cumulative light phase food intake (a) and cumulative 24-h food intake (b) for the 13-day treatment period are shown. Data are presented as mean ± S.E.M., and statistical analysis was carried out using the unpaired Student's t test; *, p < 0.05.

Body Composition as Assessed by MRR
Fat Mass. Sham control mice treated with vehicle and ML00253764 gained fat mass (FM) during the course of the study (p < 0.001; Fig. 4a) (+28.4 ± 6.9 and + 27.5 ± 6.9% in sham-vehicle- and sham-ML00253764-treated mice, respectively). The extent of this increase was not different between the vehicle- and ML00253764-treated groups (p = 0.55). Tumor vehicle-treated mice also gained FM during the course of the experiment (+21.5 ± 8.7%; p < 0.01), an effect that was significantly different (p < 0.001) from that observed in the tumor-ML00253764-treated group whose FM did not change significantly (-3.3 ± 5.0%, p = 0.72; Fig. 4b).

View larger version (43K): [in this window] [in a new window]   Fig. 4. Body-composition analysis in sham control and tumor-bearing mice at the start (day 1) and end (day 21) of the study. Sham control and mice bearing s.c. Lewis lung carcinoma tumors were treated with vehicle or ML00253764 (15 mg/kg b.i.d. s.c. for 13 days). Fat content (a and b) and LBM (c and d) were measured using MRR in sham control and tumor-bearing mice with and without ML00253764 treatment. Data are presented as mean ± S.E.M. Statistical analysis was carried out using two-way repeated measures ANOVA with Student-Newman-Keuls post hoc analysis; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Body Composition as Assessed by Chemical Extraction and Comparison with MRR
Figure 5 shows the differences between the values obtained by MRR and chemical extraction for LBM (Fig. 5a) and FM (Fig. 5b). For LBM measurements, the mean difference between the two methods was -1.99 g, with a 95% confidence interval of -2.43 to -1.55 g. The limits of agreement for lean body mass were -4.59 to +0.61 g. The mean difference between the fat measurements was 0.34 g with a 95% confidence interval of 0.28 to 0.40 g. The limits of agreement were -0.08 to +0.76 g. According to the method of Bland and Altman (1986), the two methods provided comparable results.

View larger version (20K): [in this window] [in a new window]   Fig. 5. A statistical comparison of the two methods of body-composition analysis using the method of Bland and Altman (1986) for LBM (a) and fat mass (b). Large dashed lines represent the mean differences between the methods and small dashed lines, mean ± 2 S.D.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we have tested the effect of chronic peripheral application of a MC4-R inverse agonist on food intake and body composition in a murine model of tumor-induced cachexia. We report that chronic application of ML00253764 stimulates light-phase food intake and prevents the loss of LBM induced by s.c. LLC tumor burden.

In our in vitro membrane assay, we have observed a comparable binding affinity of ML00253764 for MC4-R to that reported by Vos et al. (2004). ML00253764 was also found to decrease cAMP induction, indicative of inverse agonism, although this effect was found to be only 50% of that observed for AgRP(83-132). Constitutive activity of MC4-R has been reported in vitro (Nijenhuis et al., 2001), and its physiological importance has been confirmed recently in humans (Srinivasan et al., 2004). Because of the inverse agonist activity of ML00253764, the positive effects on LBM thus described could be the result of a blockade of endogenous agonist-induced MC4-R activity as well as a reduction in intrinsic MC4-R activity.

We report that ML00253764 displays only 7- and 2.5-fold differences in affinity for MC4-R over MC5-R and MC3-R, respectively. Like MC4-R, MC3-Rs are abundant in rat brain hypothalamus, and although they seem not to be directly involved in food intake (Kask et al., 2000), they are thought to play a role in energy partitioning (Butler et al., 2000). It has also been proposed that MC3-R may function as inhibitory autoreceptors on pro-opiomelanocortin neurons of the arcuate nucleus of the hypothalamus (Bagnol et al., 1999). Inhibitory autoreceptors are located presynaptically and function to negatively modify neurotransmitter release. Activation of such inhibitory autoreceptors would lead to a decrease in MC4-R activity, whereas blockade would lead to increased MC4-R activity. Consequently, blockade of MC3-R would be expected to lead to enhanced symptoms of cachexia, and this has been demonstrated in animal models (Marks et al., 2003). Because of the lack of selectivity of ML00253764 for MC4-R, the involvement of MC3-R in the in vivo results thus described cannot be excluded, although in view of the positive results thus described, we speculate that the involvement of MC3-R is likely to have been minimal. Of the other melanocortin receptor subtypes with possible involvement in the results described, the low binding affinity at MC5-R (IC₅₀ of 2 µM) and at MC1-R (K_i 65 µM; T. J. Vos, personal communication) make involvement of these receptors unlikely.

In our in vivo studies, although ML00253764 stimulated light-phase food intake, it did not affect 24-h food intake. This effect was observed in sham control mice, as well as in tumor-bearing mice, and is likely to be due to pharmacokinetic properties of ML00253764. Vos et al. (2004) have studied the brain concentration versus time profile of ML00253764 after s.c. administration and report that, at 30 mg/kg, ML00253764 achieved brain concentrations in excess of its functional MC4-R IC₅₀ for 6 h. Presumably, this time profile would be reduced at the dose used in the present study (15 mg/kg) and is a likely explanation for the lack of cumulative effect on 24-h food intake and body weight that we observed.

For the determination of body composition, MRR was used in addition to a traditional chemical extraction method, and our analysis using the statistical method of Bland and Altman (1986) confirmed the similarity of the results obtained using these two methods, with the limits of agreement being within acceptable boundaries. The MRR method has been adapted recently to provide quantitative body-composition analysis in rodents (Künnecke et al., 2004) and provides numerous advantages over other methods of body-composition analysis, including increased precision and speed (Taicher et al., 2003). Furthermore, MRR may be performed in conscious mice with no need for anesthesia. Anesthesia disrupts diurnal rhythms and results in decreased food intake, which may lead to changes in body composition; therefore, the lack of requirement for anesthesia may be considered a great advantage. In addition, using MRR, sequential measurements of the same animal are possible, although in the present study, due to the complicating presence of a growing tumor, only two measurements were conducted at the start and end of the study, with the latter being conducted in mice post mortem with the tumors removed. In other animal models of cachexia where tumor growth is not a complicating factor, it could be of interest to take several MRR measurements of body composition per mouse during the course of the experiment. Such assessment could provide important information regarding the progression of the disease and the optimal time to begin drug treatment. It is noteworthy that MRR does not measure bone mass or the contents of the gastrointestinal tract; therefore, the sum of LBM and FM as measured by MRR would not be expected to equal total body weight.

MRR analysis indicated that ML00253764 administration did not affect body composition in sham control mice; both sham control mice treated with vehicle and with ML00253764 gained fat during the 3-week experimental period with no change in LBM and no differences between the experimental groups. In contrast, differences in body composition were observed between the tumor-bearing groups. The tumor-vehicle mice lost LBM (indicative of a cachexic state) while displaying an increase in FM. The tumor-ML00253764 treatment group, however, was protected from this loss of LBM (indicating protection from cachexia), with no change in their FM. Because cumulative energy intake was the same in the two tumor-bearing groups, this suggests that metabolic changes were induced in the tumor-bearing mice as a consequence of ML00253764 treatment, which may have affected the manner in which energy was used and stored. Consequently, in the tumor-ML00253764-treated mice, LBM was maintained rather than lost and FM was maintained rather than increased. The data also suggest that treatment of cachexia using MC4-R blockade need not increase overall energy consumption to be effective.

The difference between the effect of ML00253764 on body composition in the sham and the tumor-bearing mice suggests that, despite the absence of anorexia, an imbalance existed in the tumor-bearing mice, which was partially reversed by ML00253764. Such an imbalance might have been brought about by increased levels of cytokines and other inflammatory mediators that are likely to be present in this model (Marks and Cone, 2003; Argiles et al., 2005).

It has been postulated that cachexia due to chronic overactivation of MC4-R is common to many forms of cachexia (Marks et al., 2001). Therefore, in terms of pharmacotherapy, the MC4-R may be a suitable target because it constitutes a final common pathway in the etiology of this disorder. Preclinical evidence supports this hypothesis, as MC4-R blockade recently has been shown to prevent cachexia occurring from a number of different causes. Cytokine (Huang et al., 1999; Lawrence and Rothwell, 2001; Marks et al., 2001), uremia (Cheung et al., 2005), and various forms of tumor-induced cachexia in rats and mice (Marks et al., 2001; Wisse et al., 2001; Markison et al., 2005) have been shown to be prevented by both pharmacological and genetic blockade of MC4-R.

In summary, we report that the loss of LBM that occurs in tumor-induced cachexia in mice may be prevented by chronic dosing of an MC3/4-R inverse agonist. This finding supports the view that MC4-R blockade may be a suitable approach for the treatment of cachexia.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.097725.

ABBREVIATIONS: LBM, lean body mass; MC4-R, melanocortin 4-receptor; -MSH, -melanocyte-stimulating hormone; AgRP, agouti-related protein; HEK, human embryonic kidney; ML00253764, 2-{2-[2-(5-bromo-2-methoxyphenyl)-ethyl]-3-fluorophenyl}-4,5-dihydro-1H-imidazolium hydrochloride; MRR, magnetic resonance relaxometry; DMSO, dimethyl sulfoxide; FP, fluorescence polarization; LLC, Lewis lung carcinoma; FM, fat mass; NDP, [Nle⁴, D-Phe⁷].

Address correspondence to: Dr. Karl G. Hofbauer, Biozentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland. E-mail: karl.hofbauer{at}unibas.ch

	References

Top Abstract Materials and Methods Results Discussion References

 

Allen M, Hall D, Collins B, and Moore K (2002) A homogeneous high throughput nonradioactive method for measurement of functional activity of G_s-coupled receptors in membranes. J Biomol Screen 7: 35-44.[Abstract]

Argiles JM, Busquets S, and Lopez-Soriano FJ (2005) The pivotal role of cytokines in muscle wasting during cancer. Int J Biochem Cell Biol 37: 1609-1619.[CrossRef][Medline]

Bagnol D, Lu X-Y, Kaelin CB, Day HEW, Ollmann M, Gantz I, Akil H, Barsh GS, and Watson SJ (1999) Anatomy of an endogenous antagonist: relationship between agouti-related protein and proopiomelanocortin in brain. J Neurosci 19: RC26.[Abstract/Free Full Text]

Bland JM and Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307-310.[CrossRef][Medline]

Bruera E (1998) Pharmacological treatment of cachexia: any progress? Support Cancer Care 6: 109-113.

Butler AA, Kesterson RA, Khong K, Cullen MJ, Pelleymounter MA, Dekoning J, Baetscher M, and Cone RD (2000) A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse. Endocrinology 141: 3518-3521.[Abstract/Free Full Text]

Cheung W, Yu PX, Little BM, Cone RD, Marks DL, and Mak RH (2005) Role of leptin and melanocortin signaling in uremia-associated cachexia. J Clin Investig 115: 1659-1665.[CrossRef][Medline]

Cone RD (2005) Anatomy and regulation of the central melanocortin system. Nat Neurosci 8: 571-578.[CrossRef][Medline]

Farooqi IS, Keogh JM, Yeo GSH, Lank EJ, Cheetham T, and O'Rahilly S (2003) Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene. N Engl J Med 348: 1085-1095.[Abstract/Free Full Text]

Hofbauer KG, Inui A, Anker SD, and Nicholson JR (2006) Perspectives and outlook, in Pharmacotherapy of Cachexia (Hofbauer KG, Anker SD, Inui A, and Nicholson JR eds) CRC Press, Inc., Boca Raton, FL.

Huang QH, Hruby VJ, and Tatro JB (1999) Role of central melanocortins in endotoxin-induced anorexia. Am J Physiol 276: R864-R871.

Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, et al. (1997) Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88: 131-141.[CrossRef][Medline]

Kask A, Rago L, Wikberg JE, and Schioth HB (2000) Differential effects of melanocortin peptides on ingestive behaviour in rats: evidence against the involvement of MC(3) receptor in the regulation of food intake. Neurosci Lett 283: 1-4.[CrossRef][Medline]

Künnecke B, Verry P, Benardeau A, and von Kienlin M (2004) Quantitative body composition analysis in awake mice and rats by magnetic resonance relaxometry. Obes Res 12: 1604-1615.[Medline]

Lawrence CB and Rothwell NJ (2001) Anorexic but not pyrogenic actions of interleukin-1 are modulated by central melanocortin-3/4 receptors in the rat. J Neuroendocrinol 13: 490-495.[CrossRef][Medline]

Markewicz B, Kuhmichel G, and Schmidt I (1993) Onset of excess fat deposition in Zucker rats with and without decreased thermogenesis. Am J Physiol 265: E478-E486.

Markison S, Foster AC, Chen C, Brookhart GB, Hesse A, Hoare SR, Fleck BA, Brown BT, and Marks DL (2005) The regulation of feeding and metabolic rate and the prevention of murine cancer cachexia with a small-molecule melanocortin-4 receptor antagonist. Endocrinology 146: 2766-2773.[Abstract/Free Full Text]

Marks DL, Butler AA, Turner R, Brookhart G, and Cone RD (2003) Differential role of melanocortin receptor subtypes in cachexia. Endocrinology 144: 1513-1523.[Abstract/Free Full Text]

Marks DL and Cone RD (2003) The role of the melanocortin-3 receptor in cachexia. Ann NY Acad Sci 994: 258-266.[Medline]

Marks DL, Ling N, and Cone RD (2001) Role of the central melanocortin system in cachexia. Cancer Res 61: 1432-1438.[Abstract/Free Full Text]

Nijenhuis WA, Oosterom J, and Adan RA (2001) AgRP(83-132) acts as an inverse agonist on the human-melanocortin-4 receptor. Mol Endocrinol 15: 164-171.[Abstract/Free Full Text]

Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I, and Barsh GS (1997) Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science (Wash DC) 278: 135-138.[Abstract/Free Full Text]

Provencher SW (1982) Contin: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Comput Phys Commun 27: 229-242.

Srinivasan S, Lubrano-Berthelier C, Govaerts C, Picard F, Santiago P, Conklin BR, and Vaisse C (2004) Constitutive activity of the melanocortin-4 receptor is maintained by its N-terminal domain and plays a role in energy homeostasis in humans. J Clin Investig 114: 1158-1164.[CrossRef][Medline]

Taicher GZ, Tinsley FC, Reiderman A, and Heiman ML (2003) Quantitative magnetic resonance (QMR) method for bone and whole-body-composition analysis. Anal Bioanal Chem 377: 990-1002.[CrossRef][Medline]

Vos TJ, Caracoti A, Che JL, Dai M, Farrer CA, Forsyth NE, Drabic SV, Horlick RA, Lamppu D, Yowe DL, et al. (2004) Identification of 2-[2-[2-(5-bromo-2-methoxyphenyl)-ethyl]-3-fluorophenyl]-4,5-dihydro-1H-imidazole (ML00253764), a small molecule melanocortin 4 receptor antagonist that effectively reduces tumor-induced weight loss in a mouse model. J Med Chem 47: 1602-1604.[CrossRef][Medline]

Wisse BE, Frayo RS, Schwartz MW, and Cummings DE (2001) Reversal of cancer anorexia by blockade of central melanocortin receptors in rats. Endocrinology 142: 3292-3301.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.097725v1 317/2/771    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nicholson, J. R. Articles by Hofbauer, K. G. Search for Related Content PubMed PubMed Citation Articles by Nicholson, J. R. Articles by Hofbauer, K. G.